IL10 reverse monomers

Abstract

The present disclosure relates to engineered polypeptides comprising a molecular component of IL-10 capable of modulating signal transduction associated with IL-10 and methods of use thereof. In some aspects, the polypeptides of the present disclosure exhibit cell type bias activity. In some aspects, the polypeptides of the present disclosure substantially retain the anti-inflammatory properties of IL-10, but significantly reduce the pro-inflammatory properties of IL-10. The present disclosure provides methods of use of engineered polypeptides in the treatment of disease.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 505,651, filed June 1, 2023, the entire contents of which are incorporated herein by reference for all purposes. Background Technology

[0003] Cytokine and growth factor ligands typically transmit signals through the polymerization of cell surface receptor subunits. In some cases, cytokines, acting as multispecific (e.g., bispecific or trispecific) ligands, promote the binding of extracellular domains of receptor subunits, thereby bringing the intracellular domains of receptor subunits closer together and facilitating intracellular signal transduction. The nature of cytokine ligands and their interactions with the extracellular domains of receptor subunits determine which receptor subunits and how they bind to form ligand-receptor complexes, and the intracellular signal transduction characteristics of such complexes.

[0004] Some cytokine receptor subunits possess a Janus kinase (JAK) binding domain within their intracellular domain. The JAK binding domain is typically located in the box1 / box region of the intracellular domain of the cytokine receptor subunit, near the inner surface of the cell membrane. Intracellular Janus kinases bind to the JAK binding domain and phosphorylate it. Four Janus kinases have been identified in mammalian cells: JAK1, JAK2, JAK3, and TYK2. wait (1995) Nature 377(6550):591-4, 1995; O'Shea and Plenge (2012) Immunity 36(4):542-50. Janus kinase promotes transphosphorylation of the JAK-binding domain when the intracellular domains of cytokine receptor subunits containing the JAK-binding domain come close to each other. JAK phosphorylation induces a conformational change in JAK, enabling it to further phosphorylate other intracellular proteins. The resulting phosphorylation cascade activates a variety of intracellular factors that transduce intracellular signals associated with cytokine ligand-activated receptors. In some cases, JAK-phosphorylated intracellular proteins are members of the signal transduction and transcription activator (STAT) protein family. To date, seven members of the mammalian STAT family have been identified: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. Delgoffe, wait, (2011)Curr Opin Immunol. 23(5):632-8; Levy and Darnell (2002) Nat Rev Mol Cell Biol. 3(9):651-62; and Murray, (2007) J Immunol. 178(5):2623-9. The selective interaction between activated JAK and STAT proteins is collectively referred to as the JAK / STAT pathway, which involves a variety of intracellular responses observed after cytokine binding. Such intracellular responses triggered by the binding of cytokines to their receptors are generally referred to as downstream signaling.

[0005] Human interleukin-10 (hIL10), also known as human cytokine synthesis inhibitor (CSIF), belongs to type 2 cytokines. This class of cytokines includes IL-19, IL-20, IL-22, IL-24 (Mda-7), and IL-26, interferons (IFN-α, -β, -γ, -δ, -ε, -κ, -Ω, and -τ), and interferon-like molecules (e.g., Limitin, IL-28A, IL-28B, and IL-29). hIL-10 is a non-covalent homodimer composed of two hIL-10 monomeric polypeptides. Each hIL-10 monomeric polypeptide is a 160-amino acid polypeptide containing two intramolecular disulfide bonds. Each hIL10 monomer is expressed as a precursor protein composed of 178 amino acids, of which the first 18 amino acids contain a signal peptide. Although hIL10 is mainly expressed by macrophages, it has also been detected in activated T cells, B cells, mast cells, NK cells, dendritic cells, eosinophils, neutrophils, and monocytes.

[0006] Human IL10 exerts its effects on cells through interaction with the hIL10 receptor (hIL10R). hIL10R is a type II cytokine receptor comprising hIL10Rα and hIL10Rβ subunits, also known as hIL10R1 and hIL10R2, respectively. The hIL10Rα receptor subunit is a "proprietary" or "exclusive" subunit specific to the hIL10 receptor. In contrast, the hIL10Rβ subunit is shared with other cytokine receptors, including the IL22, IL26, IL28, and interferon λL1 (IFNλ1) receptor complex. Activation of hIL10R is characterized by the binding of each hIL10 monomer to one hIL10Rα subunit and one hIL10Rβ subunit of hIL10R. Each monomer of the dimeric hIL10 cytokine binds to one hIL10Rα subunit and one hIL10Rβ subunit to form a hexameric ligand / receptor hIL10R complex consisting of two hIL10 monomers, two hIL10Rα subunits, and two hIL10Rβ subunits.

[0007] The hIL10Rα receptor subunit is a transmembrane protein, a 578-amino acid precursor protein containing a 21-amino acid signaling sequence at the N-terminus. The mature, typical hIL10Ra receptor subunit has a 557-amino acid polypeptide sequence as shown below: HGTELPSPPSVWFEAEFFHHILHWTPIPNQSESTCYEVALLRYGIESWNSISNCSQTLSYDLTAVTLDLYHSNGYRARVRAVDGSRHSNWTVTNTRFSVDEVTLTVGSVNLEIHNGFILGKIQLPRPKMAPANDTYESI FSHFREYEIAIRKVPGNFTFTHKKVKHENFSLLTSGEVGEFCVQVKPSVASRSNKGMWSKEECISLTRQYFTVTNVIIFFAFVLLLSGALAYCLALQLYVRRRKKLPSVLLFKKPSPFIFISQRSPETQDTIHPLDEE AFLKVSPELKNLDLHGSTDSGFGSTKPSLQTEEPQFLLPDPHPQADRTLGNREPPVLGDSCSGSSNSTDSGICLQEPSLSPSTGPTWEQQVGSNSRGQDDSGIDLVQNSEGRAGDTQGGSALGHHSPPEPEVPGEEDP AAVAFQGYLRQTRCAEEKATKTGCLEEESPLTDGLGPKFGRCLVDEAGLHPPALAKGYLKQDPLEMTLASSGAPTGQWNQPTEEWSLLALSSCSDLGISDWSFAHDLAPLGCVAAPGGLLGSFNSDLVTLPLISSLQSSE (SEQ ID NO:24) (UniProt reference number Q13651). Residues 22-235 of SEQ ID NO:24 (amino acids 1-214 of mature hIL10Rβ protein) correspond to the extracellular domain (ECD), residues 236-256 of SEQ ID NO:24 (amino acids 215-235 of mature hIL10Rβ protein) correspond to the transmembrane domain (TM), and residues 257-578 of SEQ ID NO:24 (amino acids 236-557 of mature hIL10Rβ protein) correspond to the intracellular domain (ICD).

[0008] The human IL10Rβ (hIL10Rβ) receptor subunit is a transmembrane protein, expressed as a 325-amino acid precursor protein containing a 19-amino acid N-terminal signal. The mature, typical hIL10Rβ receptor subunit has an amino acid sequence of 306 amino acids, as shown below: MVPPPENVRMNSVNFKNILQWESPAFAKGNLTFTAQYLSYRIFQDKCMNTTLTECDFSSLSKYGDHTLRVRAEFADEHSDWVNITFCPVDDTIIGPPGMQVEVLADSLHMRFLAPKIENEYETWTMKNVYNSWTYNVQYWKNGTDEKFQITPQ YDFEVLRNLEPWTTYCVQVRGFLPDRNKAGEWSEPVCEQTTHDETVPSWMVAVILMASVFMVCLALLGCFALLWCVYKKTKYAFSPRNSLPQHLKEFLGHPHHNTLLFFSFPLSDENDVFDKLSVIAEDSESGKQNPGDSCSLGTPPGQGPQS (SEQ ID NO:25) (UniProt reference number Q08334). Amino acids 20-220 (amino acids 1-201 of mature hIL10Rβ protein) correspond to the extracellular domain, amino acids 221-242 (amino acids 202-223 of mature hIL10Rβ protein) correspond to the transmembrane domain of 22 amino acids, and amino acids 243-325 (amino acids 224-306 of mature hIL10Rβ protein) correspond to the intracellular domain.

[0009] The mouse IL10Rβ (mIL10Rβ) receptor subunit is expressed as a 349-amino acid precursor protein containing a 19-amino acid N-terminal signal sequence. The mature, typical hIL10Rβ receptor subunit is a 330-amino acid polypeptide with the sequence shown below: MIPPPEKVRMNSVNFKNILQWEVPAFPKTNLFTTAQYESYRSFQDHCKRTASTQCDFSHLSKYGDYTVRVRAELADEHSEWVNVTFCPVEDTIIGPPEMQIESLAESLHLRFSAPQIENEPETWTLKNIYDSWAYRVQYWKNGTNEKFQVVSPYDSEVLRNLEPW TTYCIQVQGFLLDQNRTGEWSEPICERTGNDEITPSWIVAIILIVSVLVVFLFLLGCFVVLWLIYKKTKHTFRSGTSLPQHLKEFLGHPHHSTFLLFSFPPPEEAEVFDKLSIISEESEGSKQSPEDNCASEPPSDPGPRELESKDEAPSPPHDDPKLLTSTSEV (SEQ ID NO:26) The entry number for mIL10Rβ (mIL10R2) in the UniProtKB database is Q61190. Amino acids 20-220 (amino acids 1-201 in mature proteins) correspond to the extracellular domain, amino acids 221-241 (amino acids 202-222 in mature proteins) correspond to the 21-amino acid transmembrane domain, and amino acids 242-349 (amino acids 223-330 in mature proteins) correspond to the intracellular domain.

[0010] The interaction between IL10 and its receptor and receptor subunits has been studied and described in the scientific literature. wait Structural information is provided regarding the soluble IL-10Rβ receptor chain and the IL-10 / sIL-10Rα / sIL-10Rβ ternary complex, as well as information on residues involved in ligand-receptor and receptor-receptor interactions. (Pletnev) wait (2005) BMC Structural Biology 5:10. While the interaction between hIL-10 and the hIL-10Rα receptor subunit is a specific high-affinity interaction, the binding of hIL-10 to hIL-10Rβ is a relatively low-affinity interaction. It has been reported that the interaction between hIL10 and hIL10Rα induces conformational changes in hIL10 and / or hIL10Rα, thereby promoting the binding of the [hIL10:hIL10Rα] complex to hIL10Rβ. The formation of the ternary [hIL10:hIL10Rα:hIL10Rβ] complex is considered a limiting factor in initiating hIL10 signaling.

[0011] The interaction between IL-10 and IL10R influences the activation of JAK1 (which binds to hIL10Rα) and Tyk2 (which binds to hIL10Rβ), and induces the activation of STAT1, STAT3, and STAT5 in some cells. STAT3 is directly recruited to the hIL-10 / hIL10R complex via one of two tyrosine residues in the hIL10Rα cytoplasmic domain. These two tyrosine residues undergo phosphorylation in response to hIL-10 and are essential for IL-10 signaling. Homodimerization of STAT3 leads to its release from the receptor and translocation of the phosphorylated STAT homodimer into the nucleus, where it binds to STAT3-binding elements in many gene promoters, including the IL10 promoter, which is positively regulated by STAT3. The intracellular domain of the hIL10 receptor possesses sequences associated with its anti-inflammatory activity, while other STAT3-activated cytokine receptors do not have these sequences. Riley, et al. (1999) Journal of Biological Chemistry 274(23):15967-16664.

[0012] The expression of hIL10Rα and IL10Rβ receptor subunits varies with cell type and cell activation state. The activation state of cells expressing hIL10Rα can lead to significant differences in its expression levels. Hematopoietic cells constitutively express low levels of hIL10Rα, and this expression is typically significantly upregulated by various stimuli. Unlike hIL10Rα, which is primarily expressed on hematopoietic cells, the IL10Rβ receptor subunit is universally expressed. Although some cell types express hIL10Rβ at varying levels, the expression level of hIL10Rβ in a given cell type is generally less affected by cell activation state than that of hIL10Rα.

[0013] hIL-10 is associated with multiple functions and exhibits dual immunosuppressive and immunostimulatory activities through interactions with T cells, B cells, macrophages, and antigen-presenting cells (APCs). The immunosuppressive activity of hIL-10 has been well-established. hIL-10 is associated with the inhibition of expression of IL-1α, IL-1β, IL-6, IL-8, TNF-α, GM-CSF, and G-CSF in activated monocytes and activated macrophages, as well as the inhibition of NK cell production of the pro-inflammatory cytokine interferon-γ (IFN-γ). However, hIL-10 also exhibits immunostimulatory effects, stimulating CD8+ T cells to produce the pro-inflammatory cytokine IFN-γ. The immunostimulatory and immunosuppressive properties of hIL-10 have proven to be a challenge for its clinical application in the treatment of human diseases.

[0014] Saxton waitThe interaction between IL10 and the IL10Rβ subunit is described, along with the amino acid residues involved in IL10-IL10Rβ binding. It is noted that modifications to these residues may produce IL10 variants that retain the immunosuppressive function of IL10 on myeloid cells but reduce its pro-inflammatory activity on CD8+ T cells. (Saxton) wait (2021) Science 371(6535), eabc8433. Summary of the Invention

[0015] This disclosure provides compositions for modulating interleukin-10 (IL10)-mediated signal transduction. Specifically, the invention discloses inverse monomers and dimers thereof as partial agonists of STAT3-mediated signal transduction (“STAT3 signaling”). In some embodiments, the inverse monomers and dimers thereof described herein activate STAT3 signaling in some cell types and result in attenuation of STAT3 signaling in other cell types. In some embodiments, the inverse monomers and dimers thereof described herein activate STAT3 signaling in myeloid cells and result in attenuation of STAT3 signaling in lymphocytes. In some embodiments, the inverse monomers and dimers thereof described herein exhibit preferential activation of STAT3 signaling in myeloid cells compared to wild-type (wt)hIL-10, relative to lymphocytes.

[0016] On the one hand, a polypeptide is provided, comprising the amino acid sequence shown in Formula 1: [A]-L1x-[B]-L2-[C]-L3-[D]-L4y-[E]-L5-[F] (1) in: x and y are independently selected from 0 (not present) or 1 (present); [A] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence SKAVEQVKNAFNKL (SEQ ID NO:1); x = 0 (no connector, L1 is missing); or x = 1, and L1 contains a polypeptide linker consisting of 1 to 5 amino acids; [B] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence “EKGIYKAMSEFDIFINYIEAYMTMKIR” (SEQ ID NO:2); L2 contains a linker consisting of 10 to 25 amino acids; [C] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence “NLPNMLRDLRDAFSRVKTFFQMKD” (SEQ ID NO:3); L3 contains a linker consisting of 4 to 11 amino acids; [D] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence “KESLLEDFKG” (SEQ ID NO:4); y = 0 (L4 is missing), or y = 1, and L4 contains a polypeptide linker consisting of 1 to 5 amino acids; [E] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence “LGCQALSEMIQFYLEEVMPQAEN” (SEQ ID NO:5); L5 contains a linker consisting of 1 to 7 amino acids; and [F] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence “IKAHVNSLGENLKTLLRLRLRRC” (SEQ ID NO:6).

[0017] In some embodiments, L1, L2, L3, L4, and / or L5 comprise a GS linker. In some embodiments, L1 is the amino acid glutamine (Q). In some embodiments, L2 is a polypeptide having the amino acid sequence NSPGQGTQSENSCTHFPG (SEQ ID NO:7) or NTSPGQGTQSENSCTHFPG (SEQ ID NO:23). In some embodiments, L3 is a polypeptide having the amino acid sequence QLDNLLL (SEQ ID NO:8). In some embodiments, L4 is the amino acid glycine (“G”) or contains the amino acid sequence GY. In some embodiments, L5 is a polypeptide having the amino acid sequence QDPD (SEQ ID NO:9). In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:10 or SEQ ID NO:11.

[0018] In some embodiments, the polypeptide comprises an amino acid sequence: or In some embodiments, the polypeptide comprises an amino acid sequence: KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNTSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCEN (SEQ ID NO:11), or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:10 or SEQ ID NO:11.

[0019] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the hIL10 reverse monomer selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.

[0020] In some embodiments, a polypeptide comprising formula (2) is provided: [Single Unit 1] - Connector x -[Single 2] (2) In this formula, monomer 1 and monomer 2 are polypeptides of formula 1. Monomer 1 and monomer 2 may be the same or different, and x = 0 (linker does not exist) or 1 (linker exists).

[0021] In some embodiments, the polypeptide of formula (2) comprises, in the following order from amino to carboxyl groups: (a) Monomer 1, which comprises: (i) A first polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 of human IL-10 (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector xWhere x = 0 (connector does not exist) or 1 (connector exists); and (iii) A second polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-160 of human IL-10 (SEQ ID NO:20); amino acid residues 2-160 of human IL-10 (SEQ ID NO:36); amino acid residues 3-160 of human IL-10 (SEQ ID NO:37); amino acid residues 4-160 of human IL-10 (SEQ ID NO:38); amino acid residues 5-160 of human IL-10 (SEQ ID NO:39); amino acid residues 6-160 of human IL-10 (SEQ ID NO:40); amino acid residues 7-160 of human IL-10 (SEQ ID NO:41); amino acid residues 8 ...7-160 of human IL (SEQ ID NO:42); amino acid residues 9-160 of human IL-10 (SEQ ID NO:43); amino acid residues 10-160 of human IL-10 (SEQ ID NO:44); and amino acid residues 11-160 of human IL-10 (SEQ ID NO:45). (b) Second connector y Where y = 0 (joint does not exist) or 1 (joint exists); and (c) Monomer 2, comprising a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-116 (SEQ ID NO:15), amino acid residues 2-116 (SEQ ID NO:16), amino acid residues 3-116 (SEQ ID NO:17), amino acid residues 4-116 (SEQ ID NO:18), amino acid residues 5-116 (SEQ ID NO:19), amino acid residues 6-116 (SEQ ID NO:29), amino acid residues 7-116 (SEQ ID NO:30), amino acid residues 8-116 (SEQ ID NO:20), amino acid residues 9 ... SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID NO:32), amino acid residues 10-116 of human IL-10 (SEQ ID NO:33), and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34).

[0022] In some embodiments, the polypeptide of formula (2) comprises, in the following order from amino to carboxyl groups: (a) Monomer 1, which comprises: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 of mature human IL-10 (SEQ ID NO:20) (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector x ; (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 of human IL-10 (SEQ ID NO:15). (b) Second connector y ; (c) Monomer 2, which comprises: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 of mature human IL-10 (SEQ ID NO:20) (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) Third connector z ;and (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 of human IL-10 (SEQ ID NO:15).

[0023] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the hIL10 reverse monomer selected from the group consisting of SEQ ID NO: 46-51.

[0024] In some embodiments, the polypeptide exhibits cell type-biased activity relative to wild-type IL-10 derived from the reverse IL-10 monomer. In some embodiments, the polypeptide (a) exhibits a significant level of at least one anti-inflammatory property of wild-type IL-10; and (b) exhibits a significantly reduced level of at least one pro-inflammatory property of wild-type IL-10. In some embodiments, at least one anti-inflammatory property is selected from the group consisting of inhibition of expression or secretion of IL-1β, TNF-α, or IL-6 in myeloid cells. In some embodiments, at least one pro-inflammatory property is selected from the group consisting of inhibition of expression or secretion of IFNγ, granzyme A, or granzyme B in T cells. In some embodiments, at least one anti-inflammatory property is selected from the group consisting of inhibition of expression or secretion of IL1β, TNF-α, or IL-6 in myeloid cells; and at least one pro-inflammatory property is selected from the group consisting of inhibition of expression or secretion of IFNγ, granzyme A, or granzyme B in T cells.

[0025] In some embodiments, cell type-biased activity is the production of phosphorylated STAT3. In some embodiments, the peptide's pSTAT3 E max pSTAT3 E greater than wild-type hIL10 in myeloid cells max 20%, 30%, 40%, 50%, 60% or 70%.

[0026] In some implementations, pSTAT3 E in lymphocytes max pSTAT3 E is smaller than wild-type hIL10 in lymphocytes max70%, 60%, 50%, 40% or 30%.

[0027] In some embodiments, (a) the pSTAT3 E of the polypeptide max pSTAT3E is greater than wild-type hIL10 in myeloid cells max 20%, 30%, 40%, 50%, 60% or 70%; and (b) pSTAT3 E in lymphocytes max pSTAT3 E is smaller than wild-type hIL10 in lymphocytes max 70%, 60%, 50%, 40% or 30%.

[0028] In some embodiments, a polypeptide comprising formula (2) is provided: [Single Unit 1] - Connector x -[Single 2] (2) In this embodiment, monomer 1 and monomer 2 each independently contain a polypeptide selected from the polypeptide of claim 1, and x = 0 (linker not present) or 1 (linker present).

[0029] In some embodiments, monomer 1 and monomer 2 each independently comprise a polypeptide containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.

[0030] In some embodiments, monomer 1 and monomer 2 each comprise a polypeptide containing the amino acid sequence of SEQ ID NO:10.

[0031] In some embodiments, monomer 1 and monomer 2 each comprise a polypeptide containing the amino acid sequence of SEQ ID NO:11.

[0032] In some implementations, x is 1 (a connector exists), and the connector includes a GS connector.

[0033] In some implementations, x is 0 (the connector does not exist).

[0034] In some embodiments, the polypeptide of formula (2) comprises, in the following order from amino to carboxyl groups: (a) Monomer 1, which comprises: (i) A first polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 of human IL-10 (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector x Where x = 0 (connector does not exist) or 1 (connector exists); and (iii) A second polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-160 of human IL-10 (SEQ ID NO:20); amino acid residues 2-160 of human IL-10 (SEQ ID NO:36); amino acid residues 3-160 of human IL-10 (SEQ ID NO:37); amino acid residues 4-160 of human IL-10 (SEQ ID NO:38); amino acid residues 5-160 of human IL-10 (SEQ ID NO:39); amino acid residues 6-160 of human IL-10 (SEQ ID NO:40); amino acid residues 7-160 of human IL-10 (SEQ ID NO:41); amino acid residues 8 ...7-160 of human IL (SEQ ID NO:42); amino acid residues 9-160 of human IL-10 (SEQ ID NO:43); amino acid residues 10-160 of human IL-10 (SEQ ID NO:44); and amino acid residues 11-160 of human IL-10 (SEQ ID NO:45). (b) Second connector y Where y = 0 (joint does not exist) or 1 (joint exists); and (c) Monomer 2, comprising a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-116 (SEQ ID NO:15), amino acid residues 2-116 (SEQ ID NO:16), amino acid residues 3-116 (SEQ ID NO:17), amino acid residues 4-116 (SEQ ID NO:18), amino acid residues 5-116 (SEQ ID NO:19), amino acid residues 6-116 (SEQ ID NO:29), amino acid residues 7-116 (SEQ ID NO:30), amino acid residues 8-116 (SEQ ID NO:20), amino acid residues 9 ... SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID NO:32), amino acid residues 10-116 of human IL-10 (SEQ ID NO:33), and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34).

[0035] In some embodiments, the polypeptide of formula (2) comprises, in the following order from amino to carboxyl groups: (a) Monomer 1, which comprises: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 of mature human IL-10 (SEQ ID NO:20) (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector x , where x = 0 (connector does not exist) or 1 (connector exists); (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 of human IL-10 (SEQ ID NO:15). (b) Second connector y , where y = 0 (connector does not exist) or 1 (connector exists); (c) Monomer 2, which comprises: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 of mature human IL-10 (SEQ ID NO:20) (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) Third connector z Where z = 0 (connector does not exist) or 1 (connector exists); and (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 of human IL-10 (SEQ ID NO:15).

[0036] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the hIL10 reverse monomer selected from the group consisting of SEQ ID NO: 46-51.

[0037] In some embodiments, the peptide is PEGylated. In some embodiments, the PEG molecule is linear or branched, with a molecular weight of about 10 kDa to about 80 kDa. In some embodiments, the PEG molecule is a 40 kDa branched PEG molecule containing two 20 kDa arms. In some embodiments, the PEG molecule is covalently linked to the N-terminus of the peptide.

[0038] In some embodiments, the polypeptide exhibits cell type-biased activity relative to the wild-type IL-10 monomer of the derived polypeptide. In some embodiments, the polypeptide (a) exhibits a significant level of at least one anti-inflammatory property of wild-type IL-10; and (b) exhibits a significantly reduced level of at least one pro-inflammatory property of wild-type IL-10. In some embodiments, at least one anti-inflammatory property is selected from the group consisting of inhibition of expression or secretion of IL1β, TNFα, or IL6 in myeloid cells. In some embodiments, at least one pro-inflammatory property is selected from the group consisting of inhibition of expression or secretion of IFNγ, granzyme A, or granzyme B in T cells. In some embodiments, at least one anti-inflammatory property is selected from the group consisting of inhibition of expression or secretion of IL1β, TNFα, or IL6 in myeloid cells; and at least one pro-inflammatory property is selected from the group consisting of inhibition of expression or secretion of IFNγ, granzyme A, or granzyme B in T cells.

[0039] In some embodiments, cell type-biased activity is the production of phosphorylated STAT3. In some embodiments, the peptide's pSTAT3 E maxpSTAT3 E greater than wild-type hIL10 in myeloid cells max 20%, 30%, 40%, 50%, 60%, or 70%. In some embodiments, pSTAT3 E in lymphocytes... max pSTAT3 E is smaller than wild-type hIL10 in lymphocytes max 70%, 60%, 50%, 40%, or 30%. In some embodiments, (a) the pSTAT3 E of the peptide. max pSTAT3 E greater than wild-type hIL10 in myeloid cells max 20%, 30%, 40%, 50%, 60% or 70%; and (b) pSTAT3 E in lymphocytes max pSTAT3 E is smaller than wild-type hIL10 in lymphocytes max 70%, 60%, 50%, 40% or 30%.

[0040] Nucleic acid sequences encoding the polypeptides described above or elsewhere in this document are also provided.

[0041] A recombinant vector is also provided, comprising a nucleic acid sequence as described above or elsewhere herein, the nucleic acid sequence being operatively linked to one or more expression control sequences.

[0042] In addition, a recombinant cell transformed with a recombinant vector as described above or elsewhere herein is also provided.

[0043] A method for preparing the polypeptide as described above or in other parts of this document is also provided, the method comprising the steps of: (a) culturing a host cell as described above or in other parts of this document under conditions suitable for polypeptide expression; and (b) recovering the polypeptide from the host cell culture. In some embodiments, the host cell is a mammalian host cell. In some embodiments, the host cell is a bacterial cell.

[0044] A composition is also provided comprising a polypeptide as described above or in other parts of this document, a nucleic acid sequence as described above or in other parts of this document, or a recombinant vector as described above or in other parts of this document, and one or more pharmaceutically acceptable salts, excipients, and / or diluents.

[0045] A method of treating a mammalian subject suffering from a disease, disorder, or condition is also provided, the method comprising administering to the subject a therapeutically effective amount of the polypeptide or composition described above or in other parts of this document as described above or in other parts of this document. In some embodiments, the disease, disorder, or condition is an autoimmune disease, disorder, or condition. In one embodiment, the autoimmune disease, disorder, or condition is selected from the group consisting of: ulcerative colitis, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, adjacent autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis. Wet arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative enthesoid arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic flare-up rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (seronegative enthesoid arthropathy syndrome). In some embodiments, the disease, disorder, or symptom is cancer associated with chronic inflammation. In some embodiments, the treatment method prevents the progression of the disease, disorder, or symptom. In some embodiments, the treatment method improves one or more symptoms of the disease, disorder, or symptom.

[0046] A method is also provided for preventing a mammalian subject at risk of developing a disease, disorder, or condition from contracting said disease, disorder, or condition, the method comprising administering to the subject a preventatively effective amount of the composition described above or in other parts of this document before the onset of symptoms of the disease, disorder, or condition described above or in other parts of this document, said disease, disorder, or condition being cancer associated with chronic inflammation. Attached Figure Description

[0047] Figure 1 Thermal stability data relating to the IL10 reverse monomer of this disclosure are shown, with detailed descriptions in the embodiments. The data indicate that the hIL10 reverse monomer has higher thermal stability than hIL10.

[0048] Figure 2AThe results of flow cytometry assays were shown, evaluating the intracellular STAT3 levels (y-axis) in human mononuclear cells exposed to different concentrations (x-axis) of wild-type human IL-10 and reverse hIL-10 monomers. For details, please refer to the examples. Figure 2B The results of flow cytometry assays were shown, evaluating the intracellular STAT3 levels (y-axis) in primary human CD8 T cells exposed to different concentrations (x-axis) of wild-type human IL-10 and reverse hIL-10 monomers. See the examples for details.

[0049] Figure 3 The results of the human monocyte viability assay are shown, and the secretion of IL-1β in human monocytes exposed to different concentrations (x-axis) of wild-type hIL10 (h_SM0043_AA) and the reverse monomer of this disclosure (h_DR1061_AA) is detected (y-axis), as detailed in the examples.

[0050] Figures 4A-4C The image shows the IFNγ response in activated human CD8+ T cells to different concentrations (x-axis) of wild-type hIL10 (h_SM0043_AA) and the reverse monomer of this disclosure (h_DR1061_AA). Figure 4A Granzyme A ( Figure 4B ) and granzyme B ( Figure 4C The secretion level (y-axis) is described in detail in the examples.

[0051] Figure 5A The results of flow cytometry assays were shown, which evaluated the intracellular STAT3 levels (y-axis) in mouse myeloid cells exposed to different concentrations (x-axis) of wild-type mouse IL-10 (m_DR756_AA) and reverse mIL-10 monomer (m_WC161_AA), as detailed in the examples. Figure 5B The results of flow cytometry assays were shown, which evaluated the intracellular STAT3 levels (y-axis) in mouse CD8 T cells exposed to different concentrations (x-axis) of wild-type mouse IL-10 (m_DR756_AA) and reverse mIL-10 monomer (m_WC161_AA). For details, please refer to the examples.

[0052] Figure 6A The experimental results show the evaluation of IL-6 secretion (y-axis) in mouse spleen cells in response to different concentrations (x-axis) of wild-type mouse IL-10 (m_DR756_AA) and reverse mIL-10 monomer (m_WC161_AA), and are described in detail in the examples.

[0053] Figure 6BThe results show the experimental results of evaluating TNFα secretion (y-axis) in mouse spleen cells in response to different concentrations (x-axis) of wild-type mouse IL-10 (m_DR756_AA) and reverse mIL-10 monomer (m_WC161_AA). For detailed description, please refer to the examples, TNF-α assay of mouse spleen cells.

[0054] Figure 7 The experimental results show the evaluation of granzyme B (y-axis) produced in activated mouse CD8+ T cells in response to different concentrations (x-axis) of wild-type mouse IL10 (m_DR756_AA) and reverse mIL10 monomer (m_WC161_AA), as detailed in the examples.

[0055] Figure 8 The results of the experiment on the survival of activated CD8+ mouse T cells (y-axis) produced in response to different concentrations (x-axis) of wild-type mouse IL10 (m_DR756_AA) and reverse mIL10 monomer (m_WC161_AA) in activated CD8+ mouse T cells are shown. For details, please refer to the examples.

[0056] Figure 9 The results of pharmacokinetic studies were presented, evaluating the changes in serum stability (y-axis) over time (x-axis) of PEGylated mouse reverse IL10 monomer (WC161) at different dose levels relative to PEGylated wild-type mouse IL10 control (DR756), as detailed in the examples.

[0057] Figure 10 The results show the experimental results evaluating the expression level of CD64 on the cell surface of human monocytes (y-axis) after treatment with different concentrations (0.1 pM-100 nM) of IL10 protein at 37°C for 48 hours. For details, please refer to the examples.

[0058] Figure 11 The comparison of reverse monomers of the present disclosure in humans (DR1060_aa / 1-171; SEQ ID NO:27) and mice (WC161_aa / 1-172; SEQ ID NO:28) is shown.

[0059] Figure 12A shows the results of a flow cytometry assay that assessed the intracellular STAT3 levels (y-axis) in human mononuclear cells exposed to different concentrations (x-axis) of wild-type human IL10 (h_DR757_AA) and reverse hIL10 monomers (h_DR1060_AA and h_DR1061_AA), as detailed in the examples.

[0060] Figure 12BThe results of flow cytometry assays were shown, evaluating the intracellular STAT3 levels (y-axis) in primary human CD8 T cells exposed to different concentrations (x-axis) of wild-type human IL10 (h_DR757_AA) and reverse hIL10 monomers (h_DR1060_AA and h_DR1061_AA), as detailed in the examples.

[0061] Figure 13 The data shown illustrates the change in mouse body weight (y-axis) over time (x-axis) in a DSS model of ulcerative colitis, detailed in the examples. The top figure reflects simple body weight measurements, while the bottom figure provides animal body weight relative to their initial body weight at the start of the study.

[0062] Figure 14 The data shown (y-axis) pertain to the weight of mice treated with the test reagents (x-axis) in the ulcerative colitis DSS model at the end of the study (D15), as detailed in the examples.

[0063] Figure 15 The data (y-axis) show the colon length of mice treated with the test reagent (x-axis) in the ulcerative colitis DSS model at the end of the study (D15), as detailed in the examples.

[0064] Figure 16 The data (y-axis) show the hematocrit levels of mice treated with the test reagents in the ulcerative colitis DSS model assessed on study day 4 (D4), as detailed in the examples.

[0065] Figure 17 The percentage of peritoneal CD163+ macrophages in mice treated with the test reagent (x-axis) in a DSS model of ulcerative colitis is shown (y-axis). For details, please refer to the examples.

[0066] Figure 18 The percentage data (y-axis) of peritoneal CD64 macrophages in mice treated with the test reagents (x-axis) in a DSS model of ulcerative colitis are shown. For details, please refer to the examples.

[0067] Figure 19 Data on the changes in serum cytokine levels (y-axis) over time (x-axis) in a mouse DSS model of ulcerative colitis are shown. For a detailed description, please refer to the examples.

[0068] Figure 20 Data on the changes in serum cytokine levels (y-axis) over time (x-axis) in a mouse DSS model of ulcerative colitis are shown. For a detailed description, please refer to the examples.

[0069] Figure 21The data (y-axis) show the percentage of epithelial damage in mice treated with the test reagent in the D15-measured ulcerative colitis DSS model. For a detailed description, please refer to the examples.

[0070] Figure 22 The left figure shows data (y-axis) on the CD11b cell levels in the intestinal mucosa of mice treated with the test reagent in a D15-measured ulcerative colitis DSS model. For details, please refer to the examples. Figure 22 The right figure shows data (y-axis) on the level of Th17 cells in the intestinal mucosa of mice treated with the test reagent in the D15 ulcerative colitis DSS model. For details, please refer to the examples.

[0071] Figure 23 The results of a pharmacokinetic study conducted in mice are shown, evaluating the changes in serum concentration (y-axis) of the test reagent over time (x-axis) after administration of different levels of the test reagent, as detailed in the examples.

[0072] Figure 24 The data show the STAT3 induction levels (y-axis) in CD4 T cells (top), CD8 T cells (middle), and B cells (bottom) after administration of different levels of the test reagent. For detailed descriptions, please refer to the examples. Detailed Implementation

[0073] For ease of understanding of this disclosure, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be interpreted based on the knowledge of someone skilled in the art.

[0074] Before describing the methods and compositions herein, it should be understood that the invention is not limited to the specific methods or compositions described. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting.

[0075] Where numerical ranges are provided, unless the context explicitly specifies otherwise, it should be understood that all intermediate values ​​(accurate to one-tenth of a unit) between the upper and lower limits of the range are also explicitly disclosed. This invention also includes any set value or intermediate value within the set range and smaller ranges between any other set value or intermediate value within the set range. The upper and lower limits of these smaller ranges may be independently included in the range, or may not be included in the range; if a smaller range includes one, two, or none of the two ranges, then any smaller range is also included in the scope of this invention without violating any limitations explicitly excluded from the range. If the range includes one or two limitations, then the range excluding one or two included limitations is also included in the scope of this invention.

[0076] Unless otherwise defined, the technical and scientific terms used herein should be interpreted as those commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention, some potential and preferred methods and materials are described herein. All publications, patents, published patent applications, GenBank accession numbers, and UniProt index numbers mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0077] It should be noted that, unless the context clearly specifies otherwise, the singular forms "a," "an," and "the" used herein and in the appended claims all include plural references. Thus, for example, "a cell" includes a plurality of such cells; "a peptide" includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0078] The publications discussed herein refer only to their disclosures prior to the filing date of this application. Nothing herein should be construed as an admission that the invention does not precede these publications by virtue of a prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may require separate verification.

[0079] Unless otherwise stated, parts are by weight, molecular weight is by weight-average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric pressure. Use standard abbreviations, including: bp = base pair; kb = kilobase; pl = picolitrogen; s or sec = second; min = minute; h or hr = hour; AA or aa = amino acid; kb = kilobase; nt = nucleotide; pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = millimole; M = mole; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = no statistical significance; PBS = phosphate buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA =Mouse serum albumin; DMEM = Darwin's modified Ehrlich medium; EDTA = ethylenediaminetetraacetic acid.

[0080] It should be understood that amino acids are referred to in this disclosure based on single-letter or three-letter codes.

[0081] The standard methods of molecular biology are described in the scientific literature (see, for example, Sambrook and Russell (2001), Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; and Ausubel, et al. (2001), Current Protocols in Molecular Biology, Volumes 1-4, John Wiley and Sons, Inc., New York, NY, which describe cloning and site-directed mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)). Scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, fusion protein production, and protein glycosylation (see, for example, Coligan, et al. (2000), Current Protocols in Protein Science, Volumes 1-2, John Wiley & Sons, NY).

[0082] Nomenclature for amino acid substitutions and deletions

[0083] This disclosure provides variant peptides comprising amino acid substitutions relative to the wild-type or parental peptide. The following nomenclature is used herein to indicate substitutions, deletions, or insertions. Residues herein are designated by a single-letter or three-letter amino acid code of an amino acid found naturally in the wild-type molecule. In this disclosure, the amino acid residue numbering of the reverse monomer of the human IL10 peptide refers to the residue numbering provided in SEQ ID NO:10. For hIL10 mutants, substitutions herein are indicated by a single-letter amino acid code followed by the position of the wild-type hIL10 (SEQ ID NO:10) amino acid, followed by the single-letter amino acid code of the substituted amino acid. For example, the “D25K” modification in the hIL10 mutant indicates that the aspartic (D) residue at position 25 of (SEQ ID NO:10) is replaced by a lysine (K) residue. Deletion of amino acid residues is indicated by “des” or the symbol “Δ”, followed by the amino acid residue and its position.

[0084] Immunoglobulin, Upper Hinge, and Fc Residue Numbering: Several numbering rules exist for numbering immunoglobulin amino acid residues, including Kabat numbering, Chothia numbering, EU numbering, and IMGT numbering rules. In the context of this disclosure, the amino acid residues of immunoglobulin molecules, including their domains, including the upper hinge and Fc domains (including the lower hinge, CH2, and CH3 domains), are numbered according to the EU numbering rule. Those skilled in the art will readily understand how the EU numbering rule used herein can be translated into Kabat numbering, Chothia numbering, or IMGT numbering rules. Dondelinger, et al. (2018) understand the importance and significance of antibody numbering and antigen-binding surface / residue definition ( Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition Frontiers in Immunology, Volume 9, Article No. 2278.

[0085] definition

[0086] Unless otherwise stated, the following terms are intended to have the following meanings. Other terms are defined throughout this specification.

[0087] about: The term "approximately" refers to plus or minus 10% of the values ​​described herein, such as plus or minus 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The term "approximately" also applies to all ranges of values ​​described herein. All values ​​described herein should be understood as being modified by the term "approximately," whether or not the term "approximately" is explicitly stated for a given value.

[0088] Activation / Activation: As used herein, the term “activation / activation” refers to a receptor or receptor complex to reflect a biological effect that arises directly and / or through participation in a multi-component signaling cascade, a biological effect that is a ligand-binding response produced by the binding of an agonist ligand to the receptor. The terms “activated” or “activated” can also be used to refer to a cellular state presented after exposure to an activator.

[0089] activeAs used herein, the term “activity” relative to a molecule is used to describe the properties or biological or chemical characteristics of a molecule in a test system (e.g., an assay) (e.g., the degree to which the molecule binds to other molecules), the effect of a reagent on cells (e.g., cell activation / activation), or the physical properties of a material or cell (e.g., changes in cell membrane potential). Examples of such biological functions include, but are not limited to, the catalytic activity of a bioagent, its ability to stimulate intracellular signal transduction, gene expression, cell proliferation, and its ability to regulate immunological activities (e.g., inflammatory responses). “Activity” is typically expressed as the level of biological activity per unit of the test reagent, such as [catalytic activity] / [mg protein], [immunological activity] / [mg protein], International Unit (IU) activity, [STAT3 phosphorylation] / [mg protein], [proliferation] / [mg protein], plaque-forming units (pfu), etc. As used herein, the term “proliferative activity” refers to the activity of a reagent that promotes cell proliferation and / or replication.

[0090] Administration / Given: The terms “administer / given” and “application” are used interchangeably herein to refer to the act of contacting a subject, including, under in vitro, in vivo, or ex vivo conditions, contacting the subject’s cells, tissues, organs, or biological fluids with a reagent (e.g., a reverse monomer, a nucleic acid encoding a reverse monomer, a vector containing a nucleic acid encoding a reverse monomer (e.g., an expression vector), or an engineered cell expressing a reverse monomer) or a pharmaceutical preparation containing one or more of the above reagents. Administration / given can be achieved by any of a variety of methods recognized in the art, including but not limited to local administration, oral administration, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal administration, transmucosal administration, iontophoresis administration, intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhalation (e.g., respiratory inhalers, including dry powder inhalers), intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), etc. The term "administration" includes contacting a reagent with cells, tissues, or organs, as well as contacting a reagent with body fluids, wherein the body fluids contact cells, tissues, or organs.

[0091] Affinity As used herein, the term "affinity" refers to the degree to which a first molecule (e.g., a ligand) specifically binds to a second molecule (e.g., a receptor) in order to balance the dissociation constant (K). D The rate constant (K) is measured as the dissociation rate constant between the molecule and its target. off ) and the association rate constant between the molecule and its target (K on The ratio of ).

[0092] agonists:As used herein, the term "agonist" refers to a first agent that specifically binds to a second agent ("target") and interacts with the target to induce or promote an increase in target activation. In some cases, an agonist is an activator of a receptor protein that modulates cellular activity, enhances activation, sensitizes cells to activation by a second agent, or upregulates the expression of one or more genes, proteins, ligands, receptors, or biological pathways, thereby modulating cellular activity, including but not limited to cell activation and / or proliferation or cell cycle. In some embodiments, an agonist is an agent that binds to a receptor and alters the state of the receptor, thereby producing a biological response that mimics the action of the receptor's endogenous ligand. In some embodiments, an agonist is a modified form of a homoligand that binds to the homoligand's homoreceptor and alters the state of the homoreceptor in a biological response that mimics the biological effects of the interaction between the naturally occurring homoligand and its homoreceptor. The term "agonist" includes partial agonists, complete agonists, and superagonists. An agonist may be described as a "complete agonist" when it causes substantially all biological responses (i.e., responses associated with naturally occurring ligand / receptor binding interactions). "Partial agonist" refers to an agonist that produces a response less than that of the endogenous agonist of the target receptor, and therefore its maximum activity is less than 100% of that of the target natural ligand. In some embodiments, when evaluated at similar concentrations in comparable assays, the superagonist exhibits a response less than 100% but greater than 10%, or greater than 20%, or greater than 30%, or greater than 40%, or greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90% of the evaluable quantitative or qualitative parameters of the endogenous agonist of the target receptor. "Superagonist" is an agonist whose maximum response to the target receptor is greater than that of the endogenous agonist, and therefore its maximum activity exceeds 100% of that of the target natural ligand. In some embodiments, when evaluated at similar concentrations in comparable assays, the superagonist's response is greater than 110%, 120%, 130%, 140%, 150%, 160%, or 170% of the evaluable quantitative or qualitative parameters of the endogenous agonist of the target receptor. It should be noted that the biological effects of partial agonists, full agonists, or superagonists not only differ in degree but may also differ in nature from the biological effects of endogenous agonists of the target receptor.

[0093] Antagonist As used herein, the terms “antagonist” or “inhibitor” refer to a molecule that has one or more effects in contrast to an agonist. Antagonists can prevent, reduce, inhibit, or neutralize the activity of an agonist, and they can also prevent, inhibit, or reduce the constitutive activity of a target (such as a target receptor), even without an identified agonist. Inhibitors are molecules that reduce, block, prevent, delay activation, inactivate, desensitize, or downregulate, for example, genes, proteins, ligands, receptors, biological pathways (including immune checkpoint pathways), or cellular molecules.

[0094] biological samples The term “biological sample” or “sample” as used herein refers to a sample obtained from (or derived from) an object. For example, a biological sample includes material selected from the group consisting of: body fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluids (such as vitreous fluid, aqueous humor), lymph, lymph node tissue, spleen tissue, bone marrow, tumor tissue, including portions of one or more enriched or cell type-specific enriched immunoglobulins from these tissues.

[0095] Quite As used herein, the term "comparable" describes the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements are considered "comparable" when neither a first measurement of the evaluable quantitative parameter nor a second measurement of the same parameter deviates from a range that a person skilled in the art would consider not to present a statistically significant difference between the two results. In some cases, a measurement may be considered "comparable" if one measurement deviates from another measurement by less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 7%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%. In a particular embodiment, a measurement is considered comparable to a reference standard if it deviates from a reference standard by less than 15%, or less than 10%, or less than 5%.

[0096] Conservative amino acid substitution: As used herein, the term “conservative amino acid substitution” refers to the substitution of a given amino acid with a different amino acid that has similar biochemical properties (e.g., charge, hydrophobicity, and size). The following groups of amino acids are generally considered to be conserved amino acids to each other: (1) hydrophobic amino acids: alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, and glycine; (2) polar amino acids: glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, and cysteine; (3) basic amino acids: lysine and arginine; and (4) acidic amino acids: aspartic acid and glutamic acid.

[0097] "Corresponding to" or "corresponding to" as used herein, in the context of an amino acid or nucleotide in a polypeptide or polynucleotide, refers to the equivalent position (e.g., amino acid or nucleotide) of a reference polypeptide or polynucleotide sequence when the reference sequence is aligned with a second sequence to maximize the percentage of sequence similarity. For example, "the amino acid position corresponding to a specific IL10 polypeptide amino acid position [X]" means, based on the alignment result, the equivalent position in other IL10 polypeptides (including structural homologs and variants) or IL10 from different species (e.g., human IL10 and mouse IL10). This corresponding position can be based on a reference sequence, a wild-type sequence, or a parental sequence; for example, for an hIL10 mutant, the reference sequence could be the amino acid sequence of wild-type hIL10 (SEQ ID NO: 10).

[0098] Origin / Derived from As used herein, in the context of polypeptide or polynucleotide variants or mutants, the term "derived from / derived from" is intended to indicate that the sequence of the polypeptide or polynucleotide variant or mutant is based on a reference polypeptide (e.g., the amino acid sequence of wild-type hIL10 (SEQ ID NO:10)) or a polynucleotide sequence (e.g., cDNA encoding wild-type hIL10). The term "derived from / derived from" should not be construed as limiting the source or method of preparation of the polypeptide or polynucleotide variant or mutant.

[0099] Effective concentration (EC) The term “effective concentration” or its abbreviation “EC” as used herein is used interchangeably to indicate that the concentration of the reagent is sufficient to cause a change in a given parameter in the test system. The abbreviation “E” refers to the magnitude of a given biological effect observed in the test system when the test system is exposed to the test reagent. The abbreviation “EC” is used when the magnitude of the reaction is expressed as a factor of the concentration (“C”) of the test reagent. In the context of biological systems, the term Emax refers to the maximum magnitude of a particular biological effect observed at a saturated concentration of the test reagent. When the abbreviation EC with a subscript is provided (e.g., EC…),… 40 EC 50 When referring to concentrations such as Emax, the subscript indicates the percentage of the biological response observed at that concentration. For example, in a testing system, a test reagent that, in response to this test reagent, has a concentration sufficient to cause a reduction of a measurable biological parameter by 30% of the maximum level of that measurable biological parameter is referred to as the "EC" of the test reagent. 30 Similarly, the term "EC" 100 "Effective concentration" is used to indicate the effective concentration of a reagent, which results in a maximum (100%) response to the measurable parameter. Similarly, the term EC... 50(Used in pharmacokinetics) refers to the concentration of a reagent sufficient to cause a half-maximum (approximately 50%) change in a measurable parameter. The term "saturation concentration" refers to the maximum possible amount of a test reagent that can dissolve in a standard volume of a specific solvent (e.g., water) under standard temperature and pressure conditions. In pharmacokinetics, the saturation concentration of a drug is often used to express the concentration of drug sufficient to occupy all available receptors, while EC... 50 It is the drug concentration at which the half-maximal effect is achieved.

[0100] Enriched (of) As used herein, the term “enrichment” means the non-natural manipulation of a sample such that the concentration of a target substance (e.g., a molecule or cell) is: (a) higher than the concentration of the substance in the starting sample (e.g., a biological sample, such as a sample in which the molecule is naturally present or in which it is present after administration) (e.g., at least 3, at least 5, at least 10, at least 50, at least 100, or at least 1000 times higher); or (b) higher than the concentration in the environment in which the molecule was produced (e.g., recombinantly modified bacteria or mammalian cells).

[0101] extracellular domain As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to cell surface proteins located outside the cell membrane. For example Cell surface proteins are part of cell surface receptors. Cell surface proteins can be transmembrane proteins, cell surface proteins, or membrane-associated proteins. Cell surface proteins can also be multichannel transmembrane proteins with multiple discontinuous extracellular domains.

[0102] Fusion protein The term "fusion protein" refers to a polypeptide comprising a different protein or a synthetic sequence-derived amino acid sequence that provides different functions. Examples of fusion proteins include, but are not limited to, polypeptides comprising a protein and at least one additional functional domain, said additional functional / structural domain including, but not limited to, immunogenic domains (e.g., diphtheria toxin or tetanus toxin), expression-promoting polypeptide domains (e.g., signal peptides), sequences that promote isolation and / or purification (e.g., chelate peptides), targeting polypeptide domains (e.g., single-domain antibodies), proteins with unique additional functions (e.g., complementary functions, especially complementary therapeutic functions), and polypeptide domains that promote prolonged duration of action in vivo (e.g., albumin). The domains of a fusion protein can be further linked by a peptide linker. The functional / structural domains of a fusion protein can be provided in any N-terminal to C-terminal order. Fusion proteins are typically produced by translating a single recombinant DNA sequence, such that the protein and the additional functional / structural domain of the fusion protein are covalently linked by peptide bonds.

[0103] identityFor polypeptide or DNA sequences, the term "identity" as used herein refers to the subunit sequence identity between two molecules. Two molecules are identical at that position when the subunit position is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide). Fusion proteins are typically produced by translating a single recombinant DNA sequence, such that the protein and additional functional / structural domains of the fusion protein are covalently linked by peptide bonds. Sequence alignment is usually performed to obtain a highest-order match. If necessary, identity can be calculated using published techniques and widely used computer programs, such as Altschul. wait (1990) J. Mol. Biol 215: 403-410 and Altschul wait (1977) Nucleic Acids Res. The BLAST 2.0 algorithm described in 25:3389-3402. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. These HSPs match or satisfy a threshold score "T" when compared to words of the same length in the database sequence. T is called the adjacent word score threshold. wait (Same as above). These initial adjacent word hits are used as seeds to initiate the search in order to find longer HSPs containing them. The word hit is then extended in both directions along each sequence until the cumulative alignment score is increased. For nucleotide sequences, the cumulative score is calculated using parameters "M" (reward score for matching residues; always >0) and "N" (penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of the word hit in each direction is stopped if: (a) the cumulative alignment score decreases by X from its maximum gain; the cumulative score becomes zero or below due to the accumulation of one or more negative score residue alignments; or (b) the end of either sequence is reached. The BLAST algorithm parameters "W", "T", and "X" determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) functions similarly but uses the following default values: word length ("W") 28, expected value ("E") 10, M=1, N=-2, and compares two strands. For the amino acid sequence, the BLASTP program uses the following default values: word length (W) of 3, expected value (E) of 10, and BLOSUM62 score matrix (see Henikoff and Henikoff, (1989) PNAS(USA)89:10915-10919).

[0104] With an amount sufficient to produce a response / relief The phrase "an amount sufficient to produce a response" as used herein refers to an amount of test reagent sufficient to provide a detectable change in the level of a measured indicator before (e.g., at baseline levels) and after application of the test reagent to the test system. In some embodiments, the test system is a cell, tissue, or organism. In some embodiments, the test system is an in vitro test system, such as a fluorescence assay. In some embodiments, the test system is an in vivo system involving the measurement of changes in the levels of a parameter of a cell, tissue, or organism reflecting biological function before and after the application of a test reagent to the cell, tissue, or organism. In some embodiments, the indicator reflects the biological function or developmental state of the cells being assessed in the assay in response to the administration of a certain amount of test reagent. In some embodiments, the test system involves the measurement of changes in the levels of an indicator of a cell, tissue, or organism reflecting biological function before and after the application of one or more test reagents to the cell, tissue, or organism. The term "an amount sufficient to produce a response" can be an amount sufficient to be therapeutically effective, but may also be more or less than a therapeutically effective amount.

[0105] Needs treatment The term "in need of treatment" as used in this article refers to a physician's or other caregiver's judgment about a subject and whether the subject needs or is likely to benefit from treatment. This judgment is based on a number of factors within the physician's or caregiver's area of ​​expertise.

[0106] Preventable The term "in need of prevention" as used in this article refers to a physician's or other caregiver's judgment that the subject needs or is likely to benefit from preventive care. This judgment is based on a number of factors within the physician's or caregiver's area of ​​expertise.

[0107] Inhibitors As used herein, the term "inhibitor" refers to a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes, antagonizes, or downregulates genes, proteins, ligands, receptors, or cells. 。 Inhibitors can also be defined as molecules that reduce, block, or inactivate the constitutive activity of cells or organisms.

[0108] Intracellular domain: As used herein, the term “intracellular domain” or its abbreviation “ICD” refers to a portion of a cell surface protein (e.g., a cell surface receptor) located within the cell membrane. An ICD may comprise the entire intracellular portion of a transmembrane protein or a membrane-associated protein, or an intracellular protein. Cell surface proteins can be multichannel transmembrane proteins with multiple discontinuous intracellular domains.

[0109] SeparateAs used herein, the term “isolated” refers to the target molecule, or, if the molecule is naturally occurring, to an environment different from its natural environment. “Isolated” is intended to include molecules present in samples significantly enriched with the target molecule, and / or samples in which the target molecule has been partially or substantially purified. If the molecule is not naturally occurring, “isolated” means that the molecule has been isolated from its synthetic environment.

[0110] ligands As used herein, the term "ligand" refers to a molecule that specifically binds to a receptor and causes changes in the receptor, thereby altering receptor activity or evoking a measurable response in cells expressing that receptor. In one embodiment, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term "ligand" includes both natural and synthetic ligands. "Ligand" also includes peptide mimics of small molecules, cytokines, and antibodies. A complex of a ligand and a receptor is referred to as a "ligand-receptor complex." A ligand may include a domain of a multiprotein or fusion protein (e.g., a domain of an antibody / ligand fusion protein).

[0111] Connector: As used herein, the term "connector" refers to a molecule used to connect first and second heterologous molecules. In some embodiments, the connector may be a chemical connector. In some embodiments, the connector is a "peptide connector," which refers to a polypeptide (e.g., a fusion protein) used to connect functional subunits of a polypeptide composed of multiple functional domains or subunits.

[0112] Modified: As used herein, the term "modified" refers to a molecule, such as a polypeptide, whose structure has been altered relative to its unmodified parent molecule. Modified polypeptides typically retain one or more activities or functions of the unmodified parent molecule. For example, the reverse monomer of this disclosure can activate IL10 signaling in cells expressing the IL10 receptor as a homodimeric part, and its properties may be improved relative to the unmodified polypeptide. The term "modification" includes amino acid substitutions not present in the reverse conformation of the parent or wild-type IL10 polypeptide or the wild-type monomeric polypeptide, and includes variants and mutants of the IL10 reverse monomeric polypeptide.

[0113] adjust As used herein, the terms “modulation” and “modulation” refer to the ability of a test reagent to elicit a response in a system (including biological systems or biochemical pathways), which can be positive or negative, direct or indirect. The term “modulator” includes agonists (including partial agonists, full agonists, and superagonists), inhibitors, and antagonists.

[0114] Nucleic acidAs used herein, the terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably to refer to a polymer of nucleotides (including deoxyribonucleotides or ribonucleotides) of any length. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), guide RNA (gRNA), complementary DNA (cDNA), recombinant polynucleotides, recombinant viral or non-viral vectors, recombinant viral or non-viral expression vectors, hybridization probes, PCR primers, etc.

[0115] One or more amino acids are substituted As used herein, the term "one or more amino acid substitutions" refers to a single amino acid substitution relative to a reference sequence, or one, two, three, four, five, or more amino acid substitutions. In some embodiments, the reference sequence is the wild-type hIL10 monomer or reverse hIL10 of this disclosure.

[0116] Operable (ground) connection The term "operable linkage" in this paper refers to a relationship between a first-component molecule and a second-component molecule (typically a polypeptide or nucleic acid), arranged in a construct such that the construct retains the function of at least one component molecule, even though this operable linkage may result in positive or negative regulation of the activity of the components within the construct. For example, the operable linkage of a polyethylene glycol (PEG) molecule to a wild-type protein may result in a decrease in the biological activity of that protein in the construct relative to the wild-type molecule, but the two are still considered operably linked. When the term "operable linkage" is used to describe a relationship between multiple nucleic acid sequences encoding different functions, these multiple nucleic acid sequences are combined into a single nucleic acid molecule, for example, when the nucleic acid molecule is introduced into a cell using recombinant technology, and the nucleic acid can influence the transcription and / or translation of one or more nucleic acid sequences, and express one or more proteins intracellularly. For example, if it leads to the expression of a preprotein, the nucleic acid sequence encoding the signal sequence can be considered operationally linked to the DNA encoding the polypeptide, thereby the signal peptide promoting polypeptide secretion; if it affects the transcription of the sequence, the promoter or enhancer is considered operationally linked to the coding sequence; or if it is localized to promote translation, the ribosome binding site is considered operationally linked to the coding sequence. Generally, in the context of nucleic acid molecules, the term "operationally linked" means that the linked nucleic acid sequences are contiguous; in the context of secretory leaders or associated subdomains of molecules, they are contiguous and in the reading segment. However, some genetic elements, such as enhancers, can function at a distance and do not require the sequences interacting with them to be contiguous, but can still be considered operationally linked.

[0117] Parental polypeptidesAs used herein, the terms "parental polypeptide" or "parental protein" are used interchangeably to indicate the source of a second polypeptide (e.g., a derivative, mutant protein, or variant) that is modified relative to the first "parental" polypeptide. In some cases, the parental polypeptide is the wild-type or naturally occurring form of the protein. In other cases, the parental polypeptide may be modified to form a further modified naturally occurring protein. The term parental polypeptide may also be used interchangeably with "reference polypeptide".

[0118] Partial agonists As used herein, the term "partial agonist" refers to a molecule that specifically binds to and activates a given receptor, but only partially activates the receptor as opposed to a full agonist. Partial agonists can exhibit both agonist and antagonist effects. For example, when a full agonist and a partial agonist are present simultaneously, the partial agonist may act as a competitive inhibitor of the full agonist by competing for the receptor binding site, resulting in a net reduction in receptor activation relative to the state in which the receptor is in contact with the full agonist in the absence of the partial agonist. In the absence or depletion of endogenous ligand levels in the subject, partial agonists can be used to activate the receptor, thereby producing the desired submaximal response in the subject. In the presence of an excess of endogenous ligands, partial agonists can be used to reduce receptor overstimulation. The maximum response (Emax) produced by a partial agonist is called its intrinsic activity and is expressed as a percentage, where a full agonist produces a 100% response. When evaluated at similar concentrations in a given detection system, the activity of a partial agonist may be greater than 10% but less than 100%, or greater than 20% but less than 100%, or greater than 30% but less than 100%, or greater than 40% but less than 100%, or greater than 50% but less than 100%, or greater than 60% but less than 100%, or greater than 70% but less than 100%, or greater than 80% but less than 100%, or greater than 90% but less than 100% of the activity of a fully agonist peptide ligand.

[0119] 100% identity: As used herein, the term "percentage (%) sequence identity" in the context of nucleic acids or peptides refers to a sequence that has at least 50% sequence identity with a reference sequence. Additionally, percentage sequence identity can be any integer between 50% and 100%. In some embodiments, the sequence, as determined by BLAST using the standard parameters described below, has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence.

[0120] For sequence comparison, a sequence is typically used as a reference sequence to be compared with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and the coordinates of the subsequences are specified if necessary, along with the sequence algorithm program parameters. Default program parameters can be used, or additional parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence similarity between the test sequence and the reference sequence based on the program parameters.

[0121] The comparison window includes a reference to a segment of any one of a plurality of consecutive positions, such as a segment of at least 10 residues. In some embodiments, the comparison window has 10 to 600 residues, such as about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, wherein the sequence can be compared with a reference sequence of the same number of consecutive positions after optimal alignment of two sequences.

[0122] The BLAST and BLAST 2.0 algorithms are suitable for determining the percentage of sequence identity and the percentage of sequence similarity, respectively, and are described in Altschul. wait (1990) J. Mol. Biol 215: 403-410 and Altschul wait (1977) Nucleic Acids Res.25: 3389-3402. The software for BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. These HSPs match or satisfy a threshold score T when aligned with words of the same length in the database sequence. T is called the adjacent word score threshold (Altschul et al., ibid.). These initial adjacent word hits are used as seeds to initiate the search for longer HSPs containing them. The word hits are then extended in both directions along each sequence until the cumulative alignment score increases. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a score matrix. The extension of a word match in any direction is halted if: the cumulative alignment score decreases by X from its maximum attainable value; the cumulative score becomes zero or below due to the accumulation of one or more negatively scored residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The default values ​​used in the BLASTN program (for nucleotide sequences) are: word length (W) 28, expected value (E) 10, M=1, N=-2, and comparison of two strands. For amino acid sequences, the default values ​​used in the BLASTP program are: word length (W) 3, expected value (E) 10, and a BLOSUM62 score matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0123] The BLAST algorithm also performs statistical analysis on the similarity between two sequences (see, for example, Karlin and Altschul). Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One similarity measure provided by the BLAST algorithm is the minimum probability sum (P(N)), which indicates the probability that a match will occasionally occur between two nucleotide or amino acid sequences. For example, if the minimum probability sum in the comparison of the test amino acid sequence with the reference amino acid sequence is less than about 0.01, more preferably less than about 10, then the similarity is considered to be significantly higher. -5 And the optimal value is less than about 10. -20 If the amino acid sequence is similar to the reference sequence, then it is considered that the amino acid sequence is similar to the reference sequence.

[0124] polypeptideAs used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acids of any length. A polypeptide may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified polypeptide backbone. The term “polypeptide” includes fusion proteins. The term “polypeptide” also includes fusion proteins of a first polypeptide and a second heterologous polypeptide carrier protein (e.g., human serum albumin (HSA)).

[0125] Prevention / Prevention The terms "prevention" and "avoidance" as used in this article refer to actions initiated before the onset of a disease, condition, symptom, or similar illness, resulting in the temporary or permanent prevention, mitigation, suppression, or reduction of the subject's risk of developing such a disease, condition, symptom, or similar illness (e.g., determined by the absence of clinical symptoms), or postponement of its onset. Actions to prevent a subject's disease, condition, or symptom typically apply to situations where the subject is susceptible to a disease, condition, or symptom due to genetic, experiential, or environmental factors that contribute to its development.

[0126] receptor As used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds a ligand, the binding of which results in an alteration of at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane-associated protein comprising an extracellular domain (ECD) and a membrane-associated domain, the latter used to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD), which are linked by a transmembrane domain commonly referred to as a transmembrane domain (TM). Binding of a homologous ligand to the receptor causes a change in the receptor, resulting in a measurable biological effect. In some cases, when the receptor is a transmembrane polypeptide comprising an ECD, a TM, and an ICD, binding of the ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to ligand binding to the ECD. In some embodiments, the receptor is a component of a multi-component complex that, when assembled, leads to intracellular signaling. For example, a ligand can bind to a cell surface receptor that is not associated with any intracellular signaling on its own, but upon ligand binding promotes the formation of heteromeric (including heterodimers, heterotrimers, etc.) or homomeric (including homodimers, homotrimers, homotetramers, etc.) complexes, leading to measurable biological effects in the cell, such as activation of intracellular signaling cascades (e.g., the Jak / STAT pathway). In some embodiments, the receptor is a transmembrane single-chain polypeptide comprising ECD, TM, and ICD domains, wherein the ECD, TM, and ICD domains are derived from the same or different naturally occurring receptor variants or their synthetic functional equivalents.

[0127] Reorganization As used herein, the term “recombinant” is used as an adjective to refer to the method of modifying polypeptides, nucleic acids, or cells using recombinant DNA technology. “Recombinant protein” refers to a protein produced using recombinant DNA technology, usually abbreviated with a lowercase “r” before the protein name to indicate the method of production (e.g., recombinant human growth hormone is usually abbreviated as “rhGH”). Similarly, if cells are modified by incorporating (e.g., transfection, transduction, infection) exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, granules, etc.) using recombinant DNA technology, the cells are called “recombinant cells.” The techniques and protocols used for recombinant DNA technology are well-known in the art and can be found, for example, in Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY), and other standard molecular biology laboratory manuals.

[0128] Response / Responsiveness / Relief The term "response / relief" refers to, for example, the response of cells, tissues, organs, or organisms, including quantitative or qualitative changes in assessable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, rate of energy consumption, differentiation level, or state) that are associated with activation, stimulation, or treatment, or with exposure to exogenous agents or internal mechanisms (such as genetic programming). In some cases, the terms "activation," "stimulation," etc., refer to cell activation regulated by internal mechanisms and external or environmental factors; while the terms "inhibition," "downregulation," etc., refer to the opposite effect. "Reaction / response / relief" can be assessed in vitro, for example, using assay systems, flow cytometry, surface plasmon resonance, enzyme activity, mass spectrometry, amino acid, or protein sequencing techniques. "Reaction / response / relief" can be assessed quantitatively in vivo, qualitatively by assessing objective physiological parameters such as body temperature, weight, tumor volume, blood pressure, X-ray, or other imaging techniques, or by reporting changes in subjective feelings (happiness, depression, anxiety, or pain). In some embodiments, the activation level of the T-cell response assay reagent administered can be determined by flow cytometry, such as by measuring the levels of one or more STATs (e.g., STAT1, STAT3, or STAT5) according to methods known in the art.

[0129] Significantly reduced binding / significantly reduced binding:As used herein, the term "exhibits significantly reduced binding" is used to indicate that a variant of a first molecule (e.g., a ligand or antibody) exhibits a significantly reduced affinity for a second molecule (e.g., a receptor or antigen) relative to the parental form of the first molecule. For an antibody variant, an antibody variant "exhibits significantly reduced binding" if its affinity for the antigen or for the native form of the receptor is less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of that of its parental antibody. For a variant ligand or ligand mutant, a variant ligand or mutant "exhibits significantly reduced binding" if its affinity for the receptor is less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of that of its parental ligand. Similarly, for variant receptors, if the affinity of the variant receptor for the ligand is less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5%, then the variant receptor “exhibits significantly reduced binding.”

[0130] Specific binding:As used herein, the term "specific binding" refers to the degree of affinity / affinity exhibited by the first molecule relative to the second molecule. In the context of binding pairs (e.g., ligand / receptor, antibody / antigen), the first molecule of a binding pair is referred to as the second molecule of a specifically binding pair when it does not bind in significant amounts to other components present in the sample. The first molecule of a binding pair is referred to as the second molecule of a specifically binding pair when its affinity for the second molecule is at least 2 times, at least 5 times, at least 10 times, at least 20 times, or at least 100 times greater than its affinity for other components present in the sample. Specific binding can be assessed using techniques known in the art, including but not limited to competitive ELISA, radioligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting procedures, and competitive binding assays); non-radioligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); liquid-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR) and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, bead-on-bead ligand binding assays, column-on-column ligand binding assays, and filtration assays) and surface plasmon resonance assays (see, for example, Drescher et al. (2009), Methods Mol Biol 493:323-343, using commercially available instruments such as Biacore 8K, Biacore 8K+, Biacore S200, Biacore T200 (Stopfan, 100 Results Way, Marlborough MA 01752).

[0131] object The terms “recipient,” “individual,” “object,” and “patient” are used interchangeably herein and refer to any mammalian object requiring diagnosis, treatment, or therapy. For therapeutic purposes, “mammal” means any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sporting, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0132] Basically pureThe term "substantially pure" as used herein means that the components of the composition constitute more than about 50%, or more than about 60%, or more than about 70%, or more than about 80%, or more than about 90%, or more than about 95% of the total content of the composition. "Substantially pure" means that the proteins constitute more than about 50%, or more than about 60%, or more than about 70%, or more than about 80%, or more than about 90%, or more than about 95% of the total content of the composition.

[0133] Suffering from As used herein, the term “having” refers to a physician’s judgment of a subject based on objective or subjective information recognized in the art for identifying whether a subject has a disease, disorder, or symptom (including but not limited to X-rays, CT scans, routine laboratory diagnostic tests (e.g., blood cell counts), genomic data, protein expression data, and immunohistochemistry), that the subject needs or will benefit from treatment.

[0134] T cells: As used herein, the term "T-cell" or "T cell" in its conventional sense refers to lymphocytes differentiated in the thymus. In some embodiments, the term "T cell" includes, but is not limited to, primary CD8+ cells. + T cells, cytotoxic CD8 + T cells, primary CD4 + T cells, helper T cells, for example, T H 1. T H 2. T H 9. T H 11. T H 22. T FH Regulatory T cells, for example, T cells R 1. Tregs, induced Tregs; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs), and engineered variants of these T cells, including but not limited to CAR-T cells, recombinant modified TILs, and TCR engineered cells.

[0135] End / End As used in the context of polypeptide structure, "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the polar amino and carboxyl terms of the polypeptide, respectively. The terms "N-terminal" and "C-terminal" refer to the relative positions of the amino acid residues in the polypeptide sequence toward the N-terminus and C-terminus, respectively, and may include N-terminal and C-terminal residues, respectively. "Nearest N-terminus" refers to the position of the first amino acid residue relative to the second amino acid residue in a continuous polypeptide sequence, with the first amino acid being closer to the N-terminus of the polypeptide. "Nearest C-terminus" refers to the position of the first amino acid residue relative to the second amino acid residue in a continuous polypeptide sequence, with the first amino acid being closer to the C-terminus of the polypeptide.

[0136] Therapeutic effective dose As used herein, the phrase "therapeutic effective amount" refers to the amount of reagent administered to a subject, alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a series of doses, to produce any quantifiable or qualitative positive effect on any symptom, aspect, or characteristic of a disease, condition, or ailment. Therapeutic effective amounts can be determined by measuring the associated physiological effects and can be adjusted in conjunction with the dosing regimen and diagnostic analysis of the subject's condition. The assessment parameters used to determine the therapeutic effective amount of a reagent are determined by a physician using recognized diagnostic criteria, including but not limited to indicators such as age, weight, sex, general health status, ECOG score, observable physiological parameters, blood concentrations, blood pressure, electrocardiogram, computed tomography, X-ray, etc. Alternatively or additionally, other parameters commonly used in the clinical setting may be monitored to determine whether a therapeutically effective dose of the drug has been administered to the subject, such as normalization of body temperature, heart rate, blood chemistry, blood pressure, cholesterol levels, or any symptoms, aspects, or characteristics of the disease, disorder, or condition; biomarkers (e.g., inflammatory cytokines, IFN-γ, granzymes, etc.); reduction in serum tumor markers; improvement in condition; prolonged survival; prolonged progression-free survival; prolonged time to progression; prolonged time to treatment failure; prolonged event-free survival; prolonged time to next treatment; improved objective response rate; prolonged duration of response; reduced tumor burden; complete remission; partial remission; stable condition; and indicators relied upon by clinicians when assessing the subject's response to the drug administration. In one embodiment, a therapeutically effective dose refers to a dose of the drug, when used alone or in combination with another drug, that improves any quantity or quality of symptoms, aspects, or characteristics of the disease, disorder, or condition, and does not cause irreversible serious adverse events during administration to a mammalian subject.

[0137] treat The terms “treatment,” “curing,” and “management” refer to actions taken in response to a diagnosis of a disease, disorder, or symptom or its symptoms (e.g., contacting the subject with a pharmaceutical composition comprising a reverse monomer and / or its dimer, alone or in combination with an adjuvant), aimed at temporarily or permanently eliminating, reducing, suppressing, alleviating, or improving at least one of: (a) the underlying cause of the disease, disorder, or symptom troubling the subject; and / or (b) at least one symptom associated with such disease, disorder, or symptom. In some embodiments, treatment includes actions taken on a subject suffering from a disease, wherein such actions result in suppression of the subject’s disease (e.g., preventing the development of the disease, disorder, or symptom or improving one or more symptoms associated therewith). In some embodiments, the term “treatment” refers to actions that slow the progression of a disease, disorder, or symptom from its current state to a more harmful state.

[0138] variants The terms "variant," "protein variant," "variant protein," or "variant polypeptide" are used interchangeably herein to refer to a polypeptide that differs from a parent polypeptide due to at least one amino acid modification, substitution, or deletion. The parent polypeptide can be a naturally occurring or wild-type (WT) polypeptide or a modified form (e.g., a reverse form) of a WT polypeptide. The term "variant polypeptide" can refer to the polypeptide itself, a composition comprising the polypeptide, or the nucleic acid sequence encoding it. In some embodiments, the variant polypeptide comprises, relative to the parent polypeptide, about 1 to about 10, or about 1 to about 8, or about 1 to about 7, or about 1 to about 5, or about 1 to about 4, or about 1 to about 3, or 1 to 2 amino acid modifications, substitutions, or deletions, or a single amino acid modification, substitution, or deletion. The variant may have at least about 99% identity with the parent polypeptide from which it is derived, or at least about 98% identity, or at least about 97% identity, or at least about 95% identity, or at least about 90% identity. The term "variant" also includes nucleic acid molecules that encode proteins or peptides with altered or modified amino acid sequences compared to their parent peptides.

[0139] wild type: "Wild-type," "WT," or "natural" in this article refers to an amino acid or nucleotide sequence that exists in nature, including allelic variations. Wild-type proteins, peptides, antibodies, immunoglobulins, IgG, etc., have amino acid or nucleotide sequences that have not been artificially modified.

[0140] It should be understood that the various embodiments described separately herein for clarity and brevity can be combined without limitation. Therefore, this disclosure includes one or more or all combinations of the embodiments described herein as if each combination were individually and explicitly disclosed. This also applies to any and all sub-combinations of the embodiments disclosed herein, such that this disclosure includes one or more or all sub-combinations of the embodiments described herein as if each and every sub-combination were individually and explicitly disclosed.

[0141] reverse monomer

[0142] This disclosure provides inverse monomers of IL-10, including inverse monomers of hIL-10. In some embodiments, the IL-10 inverse monomer is derived from the sequence of a mature IL-10 (human or mouse) monomer. A typical 160-amino acid sequence (corresponding to amino acids 19-178 of the precursor protein) of a mature (“wild-type” human IL-10 monomer (UniProt reference number P22301) with the signal sequence removed has the following amino acid sequence: SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 20).

[0143] The typical amino acid sequence (corresponding to amino acids 19-178 of the precursor protein) of mature mouse IL10 protein (UniProt reference number P18893) without the signal sequence is as follows: SRGQYSREDN NCTHFPVGQS HMLLELRTAF SQVKTFFQTK DQLDNILLTD SLMQDFKGYLGCQALSEMIQ FYLVEVMPQA EKHGPEIKEH LNSLGEKLKT LRMRLRRCHR FLPCENKSKA VEQVKSDFNKLQDQGVYKAM NEFDIFINCI EAYMMIKMKS (SEQ ID NO:141).

[0144] Based on its crystal structure, wild-type IL10 contains six (6) α-helix domains. See PDB DOI: 10.2210 / pdb2ILK / pdb. The amino acid sequences of the helices and their positions relative to SEQ ID NO:11 are shown in Table 1 below:

[0145] The terms "inverse monomer of IL10" and "IL10 inverse monomer" are used interchangeably to refer to an IL10 variant comprising a partial sequence derived from wild-type IL10 rearranged relative to the N-terminal to C-terminal sequence present in the natural IL10 monomer. For example, in some embodiments, one or more helices of IL10 are rearranged relative to their sequence arrangement in wild-type IL10. In some embodiments of the inverse monomer of this disclosure, a polypeptide sequence comprising helices 5 and 6 is located at the N-terminus of a polypeptide sequence comprising helices 1-4. In one embodiment, the inverse IL10 monomer comprises a polypeptide having helices 5 and 6 located at the N-terminus of a polypeptide comprising helices 1, 2, 3, and 4 in an amino-to-carboxyl sequence (i.e., helices 5, 6, 1, 2, 3, and 4 in an amino-to-carboxyl sequence). In some embodiments, the individual helices are linked by one or more separate amino acid or polypeptide linkers. In some embodiments, the linker may comprise a polypeptide derived from the wild-type IL10 sequence. In some embodiments, the linker comprises a heterologous or synthetic amino acid or polypeptide sequence. In some embodiments, the reverse monomer may also comprise amino acid sequences located at the N-terminus of the first helix (e.g., helix 5) and the C-terminus of the last helix (e.g., helix 4). Other N-terminal and C-terminal amino acid sequences may comprise native or wild-type IL10 sequences, which are typically located at the N-terminus or C-terminus of a particular helix.

[0146] In some embodiments, the reverse monomeric polypeptide comprises the amino acid sequence shown in Formula 1: [A]-L1 x -[B]-L2-[C]-L3-[D]-L4 y -[E]-L5-[F] (1) in: x and y are independently chosen from 0 (not present) or 1 (present), and L1 to L5 refer to connectors 1 to 5 respectively.

[0147] In some embodiments, [A] corresponds to helix 5 of wild-type IL10 and amino acid residues 118-131 of hIL10 (as per SEQ ID NO:11). In some embodiments, [A] comprises the amino acid sequence SKAVEQVKNAFNKL (SEQ ID NO:1). In some embodiments, [A] comprises a variant of SEQ ID NO:1 having one or two amino acid substitutions relative to SEQ ID NO:1. In some embodiments, the variant sequence substantially retains the helical structure of [A] compared to the wild-type helix.

[0148] In some embodiments, x = 0 (no linker, i.e., L1 is absent) or x = 1 (L1 is present), and L1 comprises a polypeptide linker of any suitable length, such as about 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 amino acids. In some embodiments, L1 comprises 1, 2, 3, 4, or 5 amino acids. In some embodiments, L1 comprises the GS linker described herein. In some embodiments, L1 is the amino acid glutamine (Q).

[0149] In some embodiments, [B] corresponds to helix 6 of wild-type IL10 and amino acid residues 133-159 of hIL10 (according to SEQ ID NO:11). In some embodiments, [B] comprises the amino acid sequence EKGIYKAMSEFDIFINYIEAYMTMKIR (SEQ ID NO:2). In some embodiments, [B] comprises a variant of SEQ ID NO:2 having one or two amino acid substitutions relative to SEQ ID NO:2. In some embodiments, the variant sequence substantially preserves the helical structure of [B] compared to the wild-type helix.

[0150] In some embodiments, L2 includes a connector of any suitable length, for example, approximately 1 to 40, 1 to 30, 1 to 25, 1 to 20, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 25, or 10 to 20 amino acids. In one embodiment, L2 contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, L2 includes the GS connector described herein. In some embodiments, L2 is a polypeptide containing the amino acid sequence NSPGQGTQSENSCTHFPG (SEQ ID NO: 7). In some embodiments, L2 is a polypeptide containing the amino acid sequence NTSPGQGTQSENSCTHFPG (SEQ ID NO: 23).

[0151] In some embodiments, [C] corresponds to helix 1 of wild-type IL10 and amino acid residues 18-41 of hIL10 (as per SEQ ID NO:11). In some embodiments, [C] comprises the amino acid sequence NLPNMLRDLRDAFSRVKTFFQMKD (SEQ ID NO:3). In some embodiments, [C] comprises a variant of SEQ ID NO:3 having one or two amino acid substitutions relative to SEQ ID NO:3. In some embodiments, the variant sequence substantially retains the helical structure of [C] compared to the wild-type helix.

[0152] In some embodiments, L3 comprises a linker of any suitable length, for example, approximately 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 10, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 10, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids. In some embodiments, L3 comprises 4, 5, 6, 7, 8, 9, 10, or 11 amino acids. In some embodiments, L3 comprises the GS linker described herein. In some embodiments, L3 is a polypeptide comprising the amino acid sequence QLDNLLL (SEQ ID NO: 8).

[0153] In some embodiments, [D] corresponds to helix 2 of wild-type IL10 and amino acid residues 49-58 of hIL10 (according to SEQ ID NO:11). In some embodiments, [C] comprises the amino acid sequence KESLLEDFKG (SEQ ID NO:4). In some embodiments, [D] comprises a variant of SEQ ID NO:4 having one or two amino acid substitutions relative to SEQ ID NO:4. In some embodiments, the variant sequence substantially retains the helical structure of [D] compared to the wild-type helix.

[0154] In some embodiments, y = 0 (L4 is absent), or y = 1 and L4 contains a polypeptide linker. The length of the L4 linker can be any suitable length, for example, about 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or about 1 to 5 amino acids. In some embodiments, L4 contains 1, 2, 3, 4, or 5 amino acids. In some embodiments, L4 contains the GS linker described herein. In some embodiments, L4 is the amino acid glycine (G). In some embodiments, L4 contains the amino acid sequence "GY".

[0155] In some embodiments, [E] corresponds to helix 3 of wild-type IL10 and amino acid residues 60-82 of hIL10 (according to SEQ ID NO:11). In some embodiments, [E] comprises the amino acid sequence LGCQALSEMIQFYLEEVMPQAEN (SEQ ID NO:5). In some embodiments, [E] comprises a variant of SEQ ID NO:5 having one or two amino acid substitutions relative to SEQ ID NO:5. In some embodiments, the variant sequence substantially preserves the helical structure of [E] compared to the wild-type helix.

[0156] In some embodiments, L5 comprises a linker. The linker L5 can be of any suitable length, for example, about 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or about 1 to 5 amino acids. In some embodiments, L5 comprises 1, 2, 3, 4, 5, 6, or 7 amino acids. In some embodiments, L5 comprises the GS linker described herein. In some embodiments, L5 is a polypeptide comprising the amino acid sequence QDPD (SEQ ID NO: 9).

[0157] In some embodiments, [F] corresponds to helix 3 of wild-type IL10 and amino acid residues 87-108 of hIL10 (according to SEQ ID NO:11). In some embodiments, [F] comprises the amino acid sequence IKAHVNSLGENLKTLRLRLRRC (SEQ ID NO:6). In some embodiments, [F] comprises a variant of SEQ ID NO:6 having one or two amino acid substitutions relative to SEQ ID NO:6. In some embodiments, the variant sequence substantially preserves the helical structure of [F] compared to the wild-type helix.

[0158] In some implementations, the structural domains of Equation 1 are shown in Table 2.

[0159]

[0160] Each of the above adapters can be independently selected as wild-type, i.e., the amino acid sequence of the wild-type hIL10 sequence connecting the two current helices. Alternatively, one or more adapters can be variants of the wild-type adapter sequence, i.e., having one, two, or more amino acid substitutions, deletions, or insertions compared to the wild-type adapter sequence. In some embodiments, L1 is the amino acid glutamine (Q). In some embodiments, L2 is a polypeptide containing the amino acid sequence NSPGQGTQSENSCTHFPG (SEQ ID NO: 7). In some embodiments, L2 is a polypeptide containing the amino acid sequence NTSPGQGTQSENSCTHFPG (SEQ ID ID: 23). In some embodiments, L3 is a polypeptide containing the amino acid sequence QLDNLLL (SEQ ID NO: 8). In some embodiments, L4 is the amino acid glycine (“G”). In some embodiments, L4 contains the amino acid sequence “GY”. In some embodiments, L5 is a polypeptide containing the amino acid sequence QDPD (SEQ ID NO: 9).

[0161] In some embodiments, one or more (e.g., all) of L1, L2, L3, L4, and / or L5 contain a linker that differs from the wild-type linker sequence. For example, in some embodiments, each of L1, L2, L3, L4, and / or L5 contains a GS linker. The linker can be readily chosen and can be of any suitable length, such as 1 amino acid (e.g., glycine), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids. Examples of linkers available in this disclosure include, but are not limited to, glycine polymers (G)n, where n is an integer between 1 and 50. Examples of linkers available in this disclosure include, but are not limited to, glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers, also referred to herein as “GS linkers.” The structures of glycine and glycine-serine polymers are relatively unstructured, and therefore can serve as flexible links between peptide domains or subunits. In one embodiment, the GS connector is a polymer selected from the following molecular formulas: (GmSo)n (SEQ ID NO: 136), (GS)n (SEQ ID NO: 137), (GSGGS)n (SEQ ID NO: 56), (GGGS)n (SEQ ID NO: 57), (GGGGS)n (SEQ ID NO: 58), (GmSoGm)n (SEQ ID NO: 59), (GmSoGmSoGm)n (SEQ ID NO: 60), (GSGGSm)n (SEQ ID NO: 61), (GSGSmG)n (SEQ ID NO: 62), and (GGGSm)n (SEQ ID NO: 63), and (GGGGS)n (SEQ ID NO: 64). NO:64) and its combinations, where m, n and o are each independently selected from integers between 1 and 20, for example, 1-18, 2-16, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Examples of GS connectors include, but are not limited to, GGSG (SEQ ID NO:65), GGSGG (SEQ ID NO:66), GGSG (SEQ ID NO:67), GGSGG (SEQ ID NO:68), GGGSG (SEQ ID NO:69), and GSSSG (SEQ ID NO:70), GGGS (SEQ ID NO:71), GGGGS (SEQ ID NO:72), GGGGSGGGGS (SEQ ID NO:73), GGGGSGGGGSGGGGS (SEQ ID NO:74), GGSG (SEQ ID NO:75), GGSGG (SEQ ID NO:76), GGSG (SEQ ID NO:77), GGSGG (SEQ ID NO:78), GGGSG (SEQ ID NO:79), GGGGSGGGGS (SEQ ID NO:80), GGGGSGGGGSGSSSG (SEQ ID NO:81), and their polymers.

[0162] In some embodiments, the reverse monomeric polypeptide comprises or consists of an amino acid sequence derived from human IL10. In some embodiments, the reverse monomeric polypeptide comprises or consists of SEQ ID NO:10, or comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:10: KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCEN (SEQ ID NO: 10).

[0163] In some embodiments, the reverse monomeric polypeptide comprises or consists of SEQ ID NO:10, or comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:10.

[0164] In some embodiments, the reverse monomeric polypeptide comprises or consists of an amino acid sequence derived from human IL10. In some embodiments, the reverse monomeric polypeptide comprises or consists of SEQ ID NO:10, or comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:11: KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNTSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCEN (SEQ ID NO: 11).

[0165] In some embodiments, the reverse monomeric polypeptide comprises or consists of SEQ ID NO:10, or comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:11.

[0166] In some embodiments, this disclosure provides a polypeptide comprising, in the following order from amino to carboxyl groups: (a) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acids numbered according to mature human IL-10 (SEQ ID NO:20): (i) Amino acid residues 117-158 of human IL-10 (KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKI; SEQ ID NO: 12); (ii) Amino acid residues 117-159 of human IL-10 (KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIR; SEQ ID NO: 13); and (iii) Amino acid residues 117-160 of human IL-10 (KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN; SEQ ID NO:14). (b) Connector x , where x = 0 (connector does not exist) or 1 (connector exists). and (c) A second polypeptide comprising an amino acid sequence selected from the group consisting of: (i) Amino acid residues 1-116 of human IL-10 (SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ ID NO:15); (ii) Amino acid residues 2-116 of human IL-10 (PGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ ID NO:16); (iii) Amino acid residues 3-116 of human IL-10 (GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ ID NO:17); (iv) Amino acid residues 4-116 of human IL-10 (QGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLR LRRCHRFLPCEN; SEQ ID NO:18); (v) Amino acid residues 5-116 of human IL-10 (GTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ ID NO:19), numbered according to mature human IL-10 (SEQ ID NO:20).

[0167] (vi) Amino acid residues 6-116 of human IL-10 (TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQID NO: 29); (vii) Amino acid residues 7-116 of human IL-10 (QSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ ID NO:30); (viii) Amino acid residues 8-116 of human IL-10 (SENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ ID NO:31); (ix) Amino acid residues 9-116 of human IL-10 (ENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ IDNO: 32); (x) Amino acid residues 10-116 of human IL-10 (NSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ IDNO: 33); (xi) Amino acid residues 11-116 of human IL-10 (SCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ ID NO: 34); and (xii) Amino acid residues 12-116 of human IL-10 (CTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLLRLRLRRCHRFLPCEN; SEQ IDNO: 35).

[0168] In some embodiments, the fusion polypeptide comprises, in the following order from amino to carboxyl groups: (i) a first polypeptide comprising amino acid residues 117-158 of human IL-10 (SEQ ID NO:12) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12; and (ii) A second polypeptide comprising amino acid residues 5-116 of human IL-10 (SEQ ID NO:19) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:19.

[0169] In some embodiments, the fusion polypeptide comprises, in the following order from amino to carboxyl groups: (i) a first polypeptide comprising amino acid residues 117-158 of human IL-10 (SEQ ID NO:12), or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and (i) A second polypeptide comprising amino acid residues 5-116 of human IL-10 (SEQ ID NO:19), or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:19.

[0170] In some embodiments, the first polypeptide contains one, two, or three amino acid substitutions relative to SEQ ID NO:12. In some embodiments, the second polypeptide contains one, two, or three amino acid substitutions relative to SEQ ID NO:19.

[0171] In some embodiments, the first polypeptide and the second polypeptide are covalently linked by peptide bonds (i.e., no additional adapter sequences are required). In some embodiments, the C-terminus of the first polypeptide is covalently linked to the N-terminus of the second polypeptide by a peptide bond (i.e., no additional adapter sequences are required). In some embodiments, the first polypeptide and the second polypeptide are covalently linked by adapters as described herein.

[0172] In some embodiments, the reverse monomer comprises an amino acid sequence from Table 6 or Table 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence from Table 6, Table 8, Table 10, or Table 16.

[0173] In some embodiments, the reverse monomer comprises an amino acid sequence from Table 6, Table 10, or Table 16, or an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with an amino acid sequence from Table 6, Table 8, Table 10, or Table 16.

[0174] In some embodiments, the reverse monomer comprises an amino acid sequence having at least 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the hIL10 reverse monomer selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.

[0175] In some embodiments, the reverse monomer comprises an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity with the hIL10 reverse monomer selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98.

[0176]

[0177] To facilitate the evaluation of the activity of the reverse monomers of this disclosure in a mouse model of human disease, this disclosure also provides a mouse reverse IL10 monomer as a substitute for the human reverse IL10 monomer of this disclosure. The preparation of the mouse substitute reverse monomer can be achieved by substituting the corresponding human peptide into the mouse peptide, the substitution being based on the following alignment of mouse and human sequences (see...). Figure 11 ).

[0178] exist Figure 11 In the sequence DR1060_aa / 1-171, the amino acid sequence is as follows: KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRENGLRLRRCHRFLPCGSHHHHHHHH (SEQ ID NO: 27) WC161_aa / 1-172 contains the following amino acid sequence: NKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINAIEAYMMIKMKSSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENGGSHHHHHHHH (SEQ ID NO: 28).

[0179] reverse monomer dimer

[0180] In some embodiments, this disclosure provides polypeptides of formula (1) linked in a dimer form. In some embodiments, the polypeptide of formula (1) has the structure of formula (2): [Single Unit 1] - Connector x -[Single 2] (2) In this embodiment, monomer 1 and monomer 2 are polypeptides of formula 1. In some embodiments, monomer 1 and monomer 2 are identical (i.e., contain the same amino acid sequence). In some embodiments, monomer 1 and monomer 2 are identical or different (i.e., contain different amino acid sequences). In some embodiments, x = 0 (linker not present) or 1 (linker present).

[0181] In some embodiments, the reverse monomer dimer of formula (2) comprises, in the following order from amino to carboxyl groups: (a) Monomer 1, which comprises: (i) A first polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 of human IL-10 (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector x Where x = 0 (connector does not exist) or 1 (connector exists); and (iii) A second polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-160 of human IL-10 (SEQ ID NO:20); amino acid residues 2-160 of human IL-10 (SEQ ID NO:36); amino acid residues 3-160 of human IL-10 (SEQ ID NO:37); amino acid residues 4-160 of human IL-10 (SEQ ID NO:38); amino acid residues 5-160 of human IL-10 (SEQ ID NO:39); amino acid residues 6-160 of human IL-10 (SEQ ID NO:40); amino acid residues 7-160 of human IL-10 (SEQ ID NO:41); amino acid residues 8 ...7-160 of human IL (SEQ ID NO:42); amino acid residues 9-160 of human IL-10 (SEQ ID NO:43); amino acid residues 10-160 of human IL-10 (SEQ ID NO:44); and amino acid residues 11-160 of human IL-10 (SEQ ID NO:45). (b) Second connector y Where y = 0 (joint does not exist) or 1 (joint exists); and (c) Monomer 2, comprising a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-116 (SEQ ID NO:15), amino acid residues 2-116 (SEQ ID NO:16), amino acid residues 3-116 (SEQ ID NO:17), amino acid residues 4-116 (SEQ ID NO:18), amino acid residues 5-116 (SEQ ID NO:19), amino acid residues 6-116 (SEQ ID NO:29), amino acid residues 7-116 (SEQ ID NO:30), amino acid residues 8-116 (SEQ ID NO:20), amino acid residues 9 ... SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID NO:32), amino acid residues 10-116 of human IL-10 (SEQ ID NO:33), and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34).

[0182] In some embodiments, the reverse monomer dimer of formula (2) comprises, in the following order from amino to carboxyl groups: (b) Monomer 1, which comprises: (j) A first polypeptide comprising an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 of human IL-10 (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector x Where x = 0 (connector does not exist) or 1 (connector exists); and (iii) A second polypeptide comprising an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-160 of human IL-10 (SEQ ID NO: 20); amino acid residues 2-160 of human IL-10 (SEQ ID NO: 36); amino acid residues 3-160 of human IL-10 (SEQ ID NO: 37); amino acid residues 4-160 of human IL-10 (SEQ ID NO: 38); amino acid residues 5-160 of human IL-10 (SEQ ID NO: 39); amino acid residues 6-160 of human IL-10 (SEQ ID NO: 40); amino acid residues 7 ...7-160 of human IL-10 (SEQ ID NO: (SEQ ID NO:41); amino acid residues 8-160 of human IL-10 (SEQ ID NO:42); amino acid residues 9-160 of human IL-10 (SEQ ID NO:43); amino acid residues 10-160 of human IL-10 (SEQ ID NO:44); and amino acid residues 11-160 of human IL-10 (SEQ ID NO:45). (b) Second connector y Where y = 0 (joint does not exist) or 1 (joint exists); and (c) Monomer 2, comprising a polypeptide having at least 990%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-116 (SEQ ID NO:15), amino acid residues 2-116 (SEQ ID NO:16), amino acid residues 3-116 (SEQ ID NO:17), amino acid residues 4-116 (SEQ ID NO:18), amino acid residues 5-116 (SEQ ID NO:19), amino acid residues 6-116 (SEQ ID NO:29), amino acid residues 7-116 (SEQ ID NO:19), amino acid residues 6-116 (SEQ ID NO:29), amino acid residues 7-116 (SEQ ID NO:19), amino acid residues 8-116 (SEQ ID NO:19), amino acid residues 8-116 (SEQ ID NO:29), amino acid residues 9-116 (SEQ ID NO:19), amino acid residues 8-116 (SEQ ID NO:1 ... (SEQ ID NO:30), amino acid residues 8-116 of human IL-10 (SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID NO:32), amino acid residues 10-116 of human IL-10 (SEQ ID NO:33) and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34).

[0183] In some embodiments of the reverse monomer of formula (2), the first connector x With the second connector y They are independent and distinct. In some embodiments, in the reverse monomer of equation (2), x = 0 (no connector) or 1 (connector exists). In some embodiments, y = 0 (no connector) or 1 (connector exists). In some embodiments, if a first connector exists... x Second connector y Therefore, they can be the same or different. In some embodiments, there is no linker between the first and second polypeptides of monomer 1 in formula (2) and / or between monomer 1 and monomer 2. In some embodiments, there is a second linker (b) between monomer 1 and monomer 2 in formula (2) (e.g., linker y ), and / or there is a first linker (ii) between the first polypeptide and the second polypeptide of monomer 1 (e.g., linker x In one embodiment, the first and second connectors comprise an amino acid sequence of 1, 2, 3, 4, or 5 amino acids.

[0184] Table 13 below provides exemplary embodiments of the polypeptide of formula (2).

[0185]

[0186] In some embodiments, the reverse hIL10 monomer comprises having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reverse hIL10 monomer selected from the group consisting of: SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51.

[0187] In some embodiments, the reverse hIL10 monomer comprises an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity with the reverse hIL10 monomer selected from the group consisting of: SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51.

[0188] In some embodiments, the reverse monomer dimer of formula (2) comprises, in the following order from amino to carboxyl groups: (a) Monomer 1, which comprises: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 of mature human IL-10 (SEQ ID NO:20) (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector x ; (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 of human IL-10 (SEQ ID NO:15). (b) Second connector y ; (c) Monomer 2, which comprises: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 of mature human IL-10 (SEQ ID NO:20) (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) Third connector z ;and (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 of human IL-10 (SEQ ID NO:15).

[0189] In some implementations, the first, second, and third connectors are independent and distinct from each other. In some implementations, x, y, and / or z = 0 (connector not present) or 1 (connector present). In some implementations, if the first, second, and / or third connectors are present (e.g., connectors...),... x connector y and connector z If the first, second, and / or third linkers are identical (e.g., containing the same amino acid sequence), then they can be independently identical or different. For example, in some embodiments, the first, second, and / or third linkers are identical (e.g., containing the same amino acid sequence). In some embodiments, the first, second, and / or third linkers are different (e.g., each linker contains a different amino acid sequence, or one linker contains an amino acid sequence different from the other two linkers). In some embodiments, there are no linkers between the first and second peptides of monomer 1, between monomer 1 and monomer 2, or between the first and second peptides of monomer 2 in formula (2). In one embodiment, there is a first linker between the first and second peptides of monomer 1 (e.g., linker...). x In some embodiments, a second joint (e.g., a joint) exists between monomer 1 and monomer 2 of formula (2). y In some embodiments, a third linker (e.g., a linker) exists between the first and second polypeptides of monomer 2. z In one embodiment, the first, second, and / or third connectors comprise an amino acid sequence of 1, 2, 3, 4, or 5 amino acids.

[0190] In any embodiment of this disclosure, the reverse monomer or reverse monomer dimer polypeptide may also selectively include a polypeptide sequence located at the carboxyl terminus (-COOH), which facilitates polypeptide purification. In some embodiments, the sequence located at the carboxyl terminus (-COOH) is GGS-8xHis (GGSHHHHHHHHH (SEQ ID NO:52)).

[0191] In some embodiments, the reverse monomer dimer comprises an amino acid sequence from Table 8, Table 12, Table 13 or Table 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence from Table 8, Table 12, Table 13 or Table 16.

[0192] In some embodiments, the reverse monomer dimer comprises an amino acid sequence from Table 8, Table 12, Table 13, or Table 16, or an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with an amino acid sequence from Table 8, Table 12, Table 13, or Table 16.

[0193] reverse monomer variant

[0194] In addition to the reference sequences of domains [A], [B], [C], [D], [E], and [F] (containing amino acid sequences derived from the wild-type hIL10 sequence), this disclosure also provides polypeptides having one or two amino acid substitutions relative to the reference sequences of one or more (e.g., 1, 2, 3, 4, 5, or all six) of domains [A], [B], [C], [D], [E], and [F]. The reference sequences of domains [A], [B], [C], [D], [E], and [F] consist primarily of α-helical domains. As used herein, the term "helix" is as described in the RCSB Protein Database entry 2ILK (version 1.2) (see www.rcsb.org / sequence / 2ILK). Therefore, in some embodiments of this disclosure, amino acid substitutions included in one or more of the domains [A], [B], [C], [D], [E], and [F] of the polypeptide of Formula 1 are conducive to α-helix formation (and / or do not lead to α-helix instability). In some embodiments, the substitution of an amino acid at a given position in one or more of the domains [A], [B], [C], [D], [E], and [F] of the polypeptide of Formula 1 relative to the amino acid at the same position in the reference sequence produces a minimum free energy difference (Δ(ΔG)). In one embodiment, one or more amino acids in one or more of the domains [A], [B], [C], [D], [E], and [F] to be substituted are selected from methionine (M), alanine (A), leucine (L), glutamic acid (E), and lysine (K). In some embodiments, the polypeptide of Formula 1 contains one or two amino acid substitutions relative to the reference sequence of one or more of the domains [A], [B], [C], [D], [E], and [F], provided that the substituted amino acid is not one or more amino acids selected from glycine (G) and proline (P).

[0195] In some embodiments, the structural domain [C] includes a D to K substitution at position 8 of SEQ ID NO:3. In some embodiments, the structural domain [F] includes a T to L substitution at position 14 of SEQ ID NO:6.

[0196] In some embodiments, this disclosure provides a variant reverse monomer or a dimer thereof comprising one or more amino acid substitutions at positions corresponding to mature human or mouse IL10 residues. In some embodiments, the reverse monomer or its dimer comprises one or more amino acid substitutions at positions corresponding to D25 or T100 residues of the mature human or mouse IL10 sequence. In some embodiments, the variant reverse monomer comprises the amino acid substitution D25K according to the mature human or mouse IL10 sequence number. In some embodiments, the reverse monomer comprises the following amino acid sequence or a sequence having at least 95% sequence identity with SEQ ID NO:53: KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCEN (SEQ ID NO: 53).

[0197] In some embodiments, the amino acid corresponding to the residue of T100 is replaced with T100L.

[0198] In some embodiments, the variant reverse monomer comprises an amino acid substituted T100L according to the sequence number of mature human or mouse IL10. In some embodiments, the reverse monomer comprises the following amino acid sequence or a sequence having at least 95% sequence identity with SEQ ID NO:91: KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKLLLRLRLRRCHRFLPCEN (SEQ ID NO: 91).

[0199] In one embodiment, one reverse monomer of the dimerizing reverse monomer contains an amino acid substitution at position D25 or T100 (according to the mature human or mouse IL10 sequence number) or both. In some embodiments, each reverse monomer in the dimerizing reverse monomer contains an amino acid substitution at position D25 or T100 (according to the mature human or mouse IL10 sequence number) or both. In some embodiments, one reverse monomer of the dimerizing reverse monomer contains an amino acid substitution D25K (according to the mature human or mouse IL10 sequence number). In some embodiments, one reverse monomer of the dimerizing reverse monomer contains an amino acid substitution T100L (according to the mature human or mouse IL10 sequence number). In some embodiments, each reverse monomer in the dimerizing reverse monomer contains an amino acid substitution at position D25 or T100 (according to the mature human or mouse IL10 sequence number) or both. In some embodiments, the variant reverse monomer contains an amino acid substitution D25K according to the mature human or mouse IL10 sequence number. In some embodiments, the reverse monomer dimer contains the following amino acid sequence or a sequence having at least 95% sequence identity with SEQ ID NO:54: KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKS KAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCEN (SEQ ID NO:54).

[0200] In some embodiments, the amino acid corresponding to the T100 residue in the reverse monomer dimer is substituted with T100L. In some embodiments, the reverse monomer dimer comprises the following amino acid sequence or a sequence having at least 95% sequence identity with SEQ ID NO:55: KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKLLLRLRRCHRFLPCENKS KAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKLLLRLRRCHRFLPCEN (SEQ ID NO:55).

[0201] Biased activity

[0202] The inflammatory response is a series of biological events initiated in mammals following infection and / or traumatic stimuli, aimed at mitigating the risk of systemic infection. Typically, the inflammatory response in mammals is mediated by myeloid cells, particularly macrophages, which are activated by exogenous stimuli such as bacterial cell wall components, like lipopolysaccharide (“LPS”) from Gram-negative bacteria. Activated myeloid cells act as a precursor to infection and / or injury by secreting a variety of pro-inflammatory signaling molecules, including but not limited to interleukin-6 (IL-6), interleukin-1 (IL-1, especially IL-1β), and tumor necrosis factor-α (TNF-α).

[0203] While inflammatory responses are crucial for protecting mammals from infection, excessive and / or chronic activation of immune cells, particularly myeloid cells, is associated with tissue damage, organ dysfunction, and autoimmune diseases. Numerous human diseases are associated with excessive and / or chronic inflammation, including but not limited to inflammatory bowel disease (IBD), rheumatoid arthritis (RA), Alzheimer's disease, asthma, type 1 and type 2 diabetes, and cancer. Subjects produce certain molecules, such as IL-10, which, among other activities, can suppress inflammatory responses, thereby preventing the harmful effects of excessive inflammation. IL-10 gene deletion in mice and humans is associated with severe inflammatory bowel disease (IBD). IL-10 expression and secretion are associated with the suppression of inflammatory responses by immune cells, including but not limited to the suppression of the expression and / or secretion of pro-inflammatory cytokines and antigen presentation by activated myeloid cells. Although IL-10 plays a central role in suppressing inflammatory responses, it is also associated with pro-inflammatory activity, particularly in activated CD8+ T cells. Activated CD8+ T cells were observed to enhance the secretion of the pro-inflammatory cytokine interferon-γ (IFN-γ) and release cytolytic factors, such as granzyme A and granzyme B, upon contact with IL-10. These contradictory pro-inflammatory and anti-inflammatory effects pose a challenge to the therapeutic application of IL-10 in the treatment of inflammatory diseases in mammalian subjects.

[0204] In some embodiments, the reverse IL10 monomer is a biased IL10 partial agonist comprising a polypeptide of formula (1) or (2) that exhibits cell-type biased activity relative to the wild-type IL10 species from which it is derived. As used herein, the term “biased” in the context of the reverse IL10 monomer (and / or its dimer) refers to a higher proportion of wild-type IL10 activity levels exhibited by the biased IL10 reverse monomer (or its dimer) in a first cell type than in a second cell type, relative to the wild-type IL10 species from which it is derived. In one embodiment, the first cell type is a myeloid-derived cell, including myeloid cells. In one embodiment, myeloid cells are selected from myeloid cells, granulocytes (e.g., neutrophils, eosinophils, or basophils), mast cells, or monocytes. In some embodiments, the monocytes are macrophages or dendritic cells. In some embodiments, the cells are Kupffer cells. In one embodiment, the first cell type is activated myeloid cells. In one embodiment, the first cell type is LPS-activated human myeloid cells. In some embodiments, the second cell type is T cells.

[0205] The reversed IL10 monomer and / or its dimer disclosed herein can inhibit pro-inflammatory responses and / or STAT3-mediated signaling in a cell type-dependent manner, thereby suppressing inflammatory macrophage activation without significantly promoting T cell production of inflammatory cytokines such as interferon-γ. In some embodiments, the reversed monomer and / or its dimer of this disclosure retain the immunosuppressive function of wild-type hIL10, such as inhibiting the production of inflammatory cytokines, while reducing the immunostimulatory function of wild-type hIL10, such as CD8+. + T cells produce IFN-γ. For example, in some embodiments, the reverse monomer and / or its dimer of this disclosure retains activity comparable to wild-type hIL10, inhibiting myeloid cell activation (e.g., assessed by increased STAT3-mediated signaling in myeloid cells), but significantly reducing activation in PBMCs, T cells, B cells, and NK cells (e.g., assessed by reduced IFN-γ production).

[0206] In some embodiments, the reverse monomer and / or its dimer of this disclosure are hIL10 partial agonists.

[0207] Pro-inflammatory and anti-inflammatory activities

[0208] In some embodiments, the reverse IL10 monomer comprising a peptide of formula (1) or (2) is a biased IL10 partial agonist that: (a) exhibits a significant level of at least one anti-inflammatory property of wild-type IL10; and (b) exhibits a significantly reduced level of at least one pro-inflammatory property of wild-type IL10. In some embodiments, “a significant level of at least one anti-inflammatory property” refers to the Ei of the biased IL10 reverse monomer against such anti-inflammatory property. max Compared to the anti-inflammatory properties exhibited by wild-type IL10 as measured in the testing system, E max The levels are greater than 10%, or greater than 20%, or greater than 30%, or greater than 40%, or greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%. Examples of anti-inflammatory properties measurable in the testing system include, but are not limited to: (a) inhibition of activated human myeloid cells' expression and / or secretion of human interleukin β (hILβ); (b) inhibition of activated human myeloid cells' expression and / or secretion of human interleukin 6 (hIL6); and (c) inhibition of activated human myeloid cells' expression and / or secretion of human tumor necrosis factor α (hTNFα). In some embodiments, activated human myeloid cells can be obtained by isolating human monocytes from the erythrocyte sedimentation rate (ESR) layer of centrifuged anticoagulated human blood samples and activating the isolated monocytes by contacting them with lipopolysaccharide (LPS) according to methods known in the art. The levels of hILβ, hIL6, and hTNFα expressed and / or secreted by activated monocytes can be determined by immunoassay or flow cytometry according to methods known in the art. The embodiments described herein provide a protocol for evaluating the inhibition of hILβ, hIL6, and hTNF expression and / or secretion by LPS-activated human monocytes.

[0209] In some implementations, "a significant reduction in the level of at least one pro-inflammatory property" refers to the E2O2 of the biased IL10 reverse monomer against such pro-inflammatory properties. max Compared to the pro-inflammatory properties of wild-type IL10 measured in the testing system, E maxLess than 90%, or less than 80%, or less than 70%, or less than 60%, or less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10%. Examples of pro-inflammatory properties include, but are not limited to: (a) inhibition of IFNγ expression and / or secretion by activated human CD8+ T cells; (b) inhibition of granzyme A expression and / or secretion by activated human CD8+ T cells; and (c) inhibition of granzyme A expression and / or secretion by activated human CD8+ T cells. In one embodiment, activated human T cells are obtained by isolating CD8+ T cells from human whole blood and contacting the isolated CD8+ cells with anti-CD3 and anti-CD28 antibodies according to methods known in the art. The levels of IFNγ, granzyme A, and granzyme B expressed and / or secreted by the isolated CD8+ T cells can be determined by immunoassay or flow cytometry according to methods known in the art. The following examples provide a protocol for evaluating the expression and / or secretion of IFNγ, granzyme A, and granzyme B by CD3 / CD28 activated CD8+ T cells.

[0210] In some embodiments, the reverse IL10 monomer comprises a polypeptide of formula (1) or formula (2), which is a biased hIL10 partial agonist exhibiting a significant level of at least one anti-inflammatory property of wild-type hIL10 and a significantly reduced level of at least one pro-inflammatory property of wild-type IL10, wherein: (a) the significant level of at least one anti-inflammatory property of wild-type hIL10 is E of at least one anti-inflammatory property. max E greater than that of wild-type hIL10 exhibiting such anti-inflammatory properties max 30% of the total, wherein at least one anti-inflammatory property is selected from the group consisting of: (i) inhibition of expression and / or secretion of hILβ in LPS-activated human monocytes; (ii) inhibition of expression and / or secretion of hIL6 in LPS-activated human monocytes; or (iii) inhibition of expression and / or secretion of hTNFα in LPS-activated human monocytes; and (b) a significant reduction in at least one pro-inflammatory property of wild-type hIL10 is an E-value of at least one anti-inflammatory property. max E is smaller than that of wild-type hIL10 in exhibiting these anti-inflammatory properties. max 30% of which, at least one of the pro-inflammatory properties is selected from the group consisting of: (i) inhibition of expression and / or secretion of IFNγ by activated human CD8+ T cells, (ii) inhibition of expression and / or secretion of granzyme A by activated human CD8+ T cells, and (iii) inhibition of expression and / or secretion of granzyme A by activated human CD8+ T cells.

[0211] In some embodiments, the reverse IL10 monomer of this disclosure comprises a polypeptide of formula (1) or formula (2), and is a biased hIL10 partial agonist exhibiting a significant level of at least one anti-inflammatory property of wild-type hIL10 and a significantly reduced level of at least one pro-inflammatory property of wild-type IL10, wherein: the significant level of at least one anti-inflammatory property of wild-type hIL10 is E of at least one anti-inflammatory property. max E greater than that of wild-type hIL10 exhibiting such anti-inflammatory properties max 30% of the total, wherein at least one anti-inflammatory property is selected from the group consisting of: (i) inhibition of expression and / or secretion of hILb in LPS-activated human monocytes; (ii) inhibition of expression and / or secretion of hIL6 in LPS-activated human monocytes; or (iii) inhibition of expression and / or secretion of hTNFa in LPS-activated human monocytes; and (b) a significant reduction in at least one pro-inflammatory property of wild-type hIL10 is an E-value of at least one anti-inflammatory property. max E is smaller than that of wild-type hIL10 in exhibiting these anti-inflammatory properties. max 30% of which, at least one of the pro-inflammatory properties is selected from the group consisting of: (i) inhibition of expression and / or secretion of IFNγ by activated human CD8+ T cells, (ii) inhibition of expression and / or secretion of granzyme A by activated human CD8+ T cells, and (iii) inhibition of expression and / or secretion of granzyme A by activated human CD8+ T cells.

[0212] In some embodiments, the reverse IL10 monomer of this disclosure comprises a polypeptide of formula (1) or formula (2), and is a biased reverse hIL10 monomer, wherein: (a) the reverse hIL10 monomer exhibits E in an anti-inflammatory activity assay selected from the group consisting of... max E greater than wild-type hIL10 max 30% of: (i) inhibition of hILβ expression and / or secretion in LPS-activated human monocytes, (ii) inhibition of hIL6 expression and / or secretion in LPS-activated human monocytes, or (iii) inhibition of hTNFα expression and / or secretion in LPS-activated human monocytes; (b) E in the reverse hIL10 monomer in the pro-inflammatory activity assay selected from the following groups. max E smaller than wild-type hIL10 max 10% of: (i) inhibiting the expression and / or secretion of IFNγ by activated human CD8+ T cells; (ii) inhibiting the expression and / or secretion of granzyme A by activated human CD8+ T cells; and (iii) inhibiting the expression and / or secretion of granzyme A by activated human CD8+ T cells.

[0213] In some embodiments, the reverse IL10 monomer of this disclosure comprises a polypeptide of formula (1) or formula (2), and is a biased reverse hIL10 monomer, wherein: (a) the reverse hIL10 monomer is selected from the group consisting of E in an anti-inflammatory activity assay. max E greater than wild-type hIL10 max 50% of: (i) inhibition of hILβ expression and / or secretion in LPS-activated human monocytes, (ii) inhibition of hIL6 expression and / or secretion in LPS-activated human monocytes, or (iii) inhibition of hTNFα expression and / or secretion in LPS-activated human monocytes; (b) E in the reverse hIL10 monomer in the pro-inflammatory activity assay selected from the group below. max E smaller than wild-type hIL10 max 20% of: (i) inhibiting the expression and / or secretion of IFNγ by activated human CD8+ T cells; (ii) inhibiting the expression and / or secretion of granzyme A by activated human CD8+ T cells; and (iii) inhibiting the expression and / or secretion of granzyme A by activated human CD8+ T cells.

[0214] In some embodiments, the reverse IL10 monomer of this disclosure comprises a polypeptide of formula (1) or formula (2), and is a biased reverse hIL10 monomer, wherein: (a) the reverse hIL10 monomer is selected from the group consisting of E in an anti-inflammatory activity assay. max E greater than wild-type hIL10 max 50% of: (i) inhibition of hILβ expression and / or secretion in LPS-activated human monocytes, (ii) inhibition of hIL6 expression and / or secretion in LPS-activated human monocytes, or (iii) inhibition of hTNFα expression and / or secretion in LPS-activated human monocytes; (b) E in the reverse hIL10 monomer in the pro-inflammatory activity assay selected from the group below. max E smaller than wild-type hIL10 max 10% of: (i) inhibiting the expression and / or secretion of IFNγ by activated human CD8+ T cells; (ii) inhibiting the expression and / or secretion of granzyme A by activated human CD8+ T cells; and (iii) inhibiting the expression and / or secretion of granzyme A by activated human CD8+ T cells.

[0215] STAT3

[0216] As previously described, the interaction between IL-10 and its receptor leads to intracellular signaling, characterized by increased intracellular production of phosphorylated STAT3. Therefore, IL-10 activity can be assessed by detecting the intracellular production of phosphorylated STAT3 using cells expressing the IL-10 receptor (composed of IL-10Ra and IL-10Rb).

[0217] In one embodiment, the polypeptide of formula (1) or (2) is a biased hIL-10 partial agonist, the first cell type being activated human myeloid cells and the second cell type being activated human T cells, wherein the IL-10 activity level is determined by intracellular production of phosphorylated STAT3. In one embodiment, the polypeptide of formula (1) or (2) is a biased hIL-10 partial agonist that retains a higher proportion of hIL-10 activity on activated human monocytes than on activated human CD8+ T cells, wherein the IL-10 activity level is determined by intracellular production of phosphorylated STAT3. In some embodiments, the IL-10 activity levels in the first and second cell types are determined by phosphorylated STAT3 produced by the first cell type after contact between the first and second cell types with a reverse IL-10 monomer containing the polypeptide of formula (1) or (2).

[0218] In some embodiments, the relative activation of STAT3 signaling by the reverse monomer and / or its dimer described herein in the first and second cell types differs from the relative activation of STAT3 signaling by wild-type human or mouse IL-10 in the first and second cell types. In some embodiments, the level of intracellular phosphorylated STAT3 induced by contact with an effective amount of human IL-10 reverse monomer (or its dimer) in human myeloid cells is at least 10-fold, at least 100-fold, or at least 1000-fold higher than the level induced by contact with the same amount of human IL-10 reverse monomer (or its dimer) in human lymphocytes. In one embodiment, the ratio of STAT3 signaling induced by contact with human IL-10 reverse monomer in myeloid cells to that induced by contact with human IL-10 reverse monomer in lymphocytes is different from (greater than or less than) the ratio of STAT3 signaling induced by contact with wild-type hIL-10 in myeloid cells to that induced by contact with wild-type hIL-10 in lymphocytes. In some embodiments, the activity of human IL-10 reverse monomer (and / or its dimer) in human myeloid cells (as measured by intracellular phosphorylated STAT3 levels) relative to its activity in human lymphocytes is greater than the activity of wild-type human IL-10 in human myeloid cells relative to its activity in human lymphocytes. In some embodiments, myeloid cells are neutrophils, eosinophils, mast cells, basophils, or monocytes. In some embodiments, monocytes are macrophages or dendritic cells. In some embodiments, cells are Kupffer cells. In some embodiments, lymphocytes are CD8+ T cells, CD4+ T cells, B cells, or NK cells.

[0219] In some embodiments, the pSTAT3 E of the reverse monomer and / or its dimer of the present disclosure max pSTAT3 E greater than wild-type hIL10 in myeloid cells max 20%, 30%, 40%, 50%, 60%, or 70% (for example, see...) Figure 2A In some embodiments, compared to wild-type hIL10, the reverse monomers and / or their dimers of this disclosure exhibit reduced STAT3-mediated signaling in lymphocytes (e.g., T cells, B cells, or NK cells) (see, for example, see...). Figure 2B In one embodiment, the reverse monomer and / or its dimer of this disclosure are used in lymphocytes for pSTAT3 E. max pSTAT3 E in lymphocytes was lower than that in wild-type hIL10. max 70%, 60%, 50%, 40%, or 30%. In some embodiments, the reverse monomer and / or its dimer in lymphocytes pSTAT3 E max pSTAT3 E, a peptide smaller than wild-type or parental IL10, in lymphocytes max The percentage of lymphocytes is 70% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, or less than 30%) but greater than 20%. In some embodiments, the lymphocytes are selected from CD8+ T cells, CD4+ T cells, B cells, or NK cells.

[0220] connector

[0221] In some embodiments, this disclosure provides peptide linkers that can be used to covalently link functional subunits of peptides composed of multiple functional domains or subunits. The length of the linker is generally sufficient to allow some movement between the functional domains or subunits of the peptide to which it is linked. In some embodiments, the linker is present between one or more different helices or domains [A], [B], [C], [D], [E], and [F] of formula (1). In some embodiments, the linker is present between two monomers of formula (2).

[0222] The linker can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., glycine), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids. Examples of linkers available in this disclosure include, but are not limited to, glycine polymers (G)n, where n is an integer between 1 and 50 (SEQ ID NO: 138). Examples of linkers available in this disclosure include, but are not limited to, glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers, also referred to herein as “GS linkers.” The structures of glycine and glycine-serine polymers are relatively unstructured, and therefore can serve as flexible links between peptide domains or subunits. In one embodiment, the GS connector is a polymer selected from the following molecular formulas: (GmSo)n (SEQ ID NO: 136), (GS)n (SEQ ID NO: 137), (GSGGS)n (SEQ ID NO: 56), (GGGS)n (SEQ ID NO: 57), (GGGGS)n (SEQ ID NO: 58), (GmSoGm)n (SEQ ID NO: 59), (GmSoGmSoGm)n (SEQ ID NO: 60), (GSGGSm)n (SEQ ID NO: 61), (GSGSmG)n (SEQ ID NO: 62), and (GGGSm)n (SEQ ID NO: 63), and (GGGGS)n (SEQ ID NO: 64). NO:64) and its combinations, where m, n and o are each independently selected from integers between 1 and 20, for example, 1-18, 2-16, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Examples of GS connectors include, but are not limited to, GGSG (SEQ ID NO:65), GGSGG (SEQ ID NO:66), GGSG (SEQ ID NO:67), GGSGG (SEQ ID NO:68), GGGSG (SEQ ID NO:69), and GSSSG (SEQ ID NO:70), GGGS (SEQ ID NO:71), GGGGS (SEQ ID NO:72), GGGGSGGGGS (SEQ ID NO:73), GGGGSGGGGSGGGGS (SEQ ID NO:74), GGSG (SEQ ID NO:75), GGSGG (SEQ ID NO:76), GGSG (SEQ ID NO:77), GGSGG (SEQ ID NO:78), GGGSG (SEQ ID NO:79), GGGGSGGGGS (SEQ ID NO:80), GGGGSGGGGSGSSSG (SEQ ID NO:81), and their polymers.

[0223] Provides additional functionalities

[0224] In some embodiments, the reverse monomer and / or its dimer may include the functional domain of a chimeric polypeptide. The reverse monomer fusion protein of this disclosure can be readily prepared using recombinant DNA methods known in the art, which involve constructing a recombinant vector comprising a nucleic acid sequence including a nucleic acid sequence encoding the reverse monomer, which is located within a frame along with a nucleic acid sequence encoding a fusion partner at the N-terminus or C-terminus of the reverse monomer, optionally further comprising a nucleic acid sequence located within the frame encoding an adapter or spacer polypeptide.

[0225] In other embodiments, the reverse monomer and / or its dimer may be modified to include an additional polypeptide sequence, such as the FLAG sequence, as an antigen tag. As described herein, the FLAG sequence can be recognized by biotinylated, highly specific anti-FLAG antibodies (see, for example, Blanar et al. (1992) Science 256:1014 and LeClair et al. (1992) PNAS-USA 89:8145). In some embodiments, the binding molecule also includes a C-terminal c-myc epitope tag.

[0226] In some embodiments, the reverse monomer and / or its dimer are coupled to a molecule (“targeting domain”) to facilitate selective binding to a specific cell type or tissue expressing a cell surface molecule that specifically binds to such a targeting domain. Optionally, a linker molecule of 1-40 (or 2-20, or 5-20, or 10-20) amino acids is introduced between the reverse monomer sequence and the fusion protein targeting domain sequence.

[0227] In other embodiments, chimeric polypeptides comprising a reverse monomer and its antibody or antigen-binding portion can be generated. The antibody or antigen-binding component of the chimeric protein can serve as a targeting portion. For example, it can be used to localize the chimeric protein to a specific cell subpopulation or target molecule. In some embodiments, the targeting domain is an antibody. As used herein, the term “antibody” means any form of antibody (also known as an immunoglobulin (Ig)) that has the desired biological activity of binding to an antigenic epitope, as described herein. The term “antibody” specifically covers, but is not limited to, polyclonal antibodies, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), multispecific antibodies (e.g., bispecific antibodies that bind to two or more antigens or antigenic epitopes of a single antigen), fully human antibodies (huAb), humanized antibodies (hzAb), chimeric antibodies, single-chain variable fragment antibodies (scFv), single-domain antibodies (sdAb), variable heavy chain (VH) domain antibodies, bispecific antibodies (dAb), and antigen-binding fragments (VHH) of heavy chain-only antibodies containing the amino acid sequence of the variable region, as described herein. As used herein, the term “antibody” refers to: (a) glycosylated and non-glycosylated immunoglobulins (including, but not limited to, mammalian immunoglobulins IgG1, IgG2, IgG3, and IgG4) that specifically bind to target molecules (e.g., antigens); and (b) immunoglobulin derivatives, including, but not limited to, IgG(1-4)δC that compete with immunoglobulins of their origin for binding to target molecules. H 2, F(ab')2, Fab, ScFv, V H V LAntibodies include tetraclonal antibodies, triclonal antibodies, biclonal antibodies, dsFv, F(ab')3, scFv-Fc, and (scFv)2. The term "antibody" is not limited to immunoglobulins derived from any particular mammalian species, but includes antibodies from mice, humans, horses, camels, and humans. The term "antibody" includes "heavy chain antibodies," "VHHs," and "nanobodies," which are typically obtained by immunizing camelids (including camels, alpacas, and llamas) (e.g., as described in Hamers-Casterman et al. 1993. Nature. 363:446-448, see the definition of "VHH" below). The term "antibody" includes antibodies that can be isolated from animals of natural origin or after immunization with an antigen, as well as engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted, veneered, or deimmunized (e.g., B and / or T cell epitopes removed) antibodies. In some embodiments, the targeting domain specifically binds to cell surface markers of pro-inflammatory cells (e.g., activated immune cells). In some embodiments, the targeting domain is an antibody that selectively binds to cell surface markers, including but not limited to the IL1R1 receptor, IL-1 receptor accessory protein, IL6 receptor subunit (IL6R), HLA-DR, HLA-DR α chain, HLA-DR β chain, TNFR1, TNFR2, CD4, CD8, F4 / 80, CCR2, CD169, CX3CR1, CD206, CD163, or Lyve1. Methods for generating cytokine-antibody chimeric peptides are described, for example, in U.S. Patent No. 6,617,135.

[0228] Binding to carrier molecules to increase the duration of action

[0229] The reverse monomers and / or their dimers described herein can be modified to prolong their half-life in vivo and / or prolong their duration of action in a target body. In some embodiments, the reverse monomers and / or their dimers are coupled to a carrier molecule to provide desired pharmacological properties, such as prolonged half-life. In some embodiments, the reverse monomers and / or their dimers are covalently linked to the Fc domain of IgG, albumin, a water-soluble polymer, or other molecules to prolong their half-life, for example, by methods known in the art such as glycosylation, acylation, etc. In some embodiments, the reverse monomers and / or their dimers are modified to provide an extended duration of action in mammalian subjects, such that their half-life in mammalian subjects is greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 7 hours, greater than 8 hours, greater than 9 hours, greater than 10 hours, greater than 12 hours, greater than 18 hours, greater than 24 hours, greater than 2 days, greater than 3 days, greater than 4 days, greater than 5 days, greater than 6 days, greater than 7 days, greater than 10 days, greater than 14 days, greater than 21 days, or greater than 30 days.

[0230] Modifications to reverse monomers and / or their dimers to prolong their duration of action in mammalian subjects include (but are not limited to): • Couple the reverse monomer to one or more protein transport molecules. • The reverse monomer is coupled to a protein transport molecule, optionally fused with other polypeptide sequences in the form of a fusion protein (e.g., a reverse monomer-Fc fusion protein) and • Coupling with polymers (e.g., water-soluble polymers to provide PEGylated IL10 reverse monomeric peptides). It should be noted that for a given reverse monomer and / or its dimer, various modifications can be employed to prolong its duration of action in mammalian subjects. For example, the hIL10 reverse monomer and / or its dimer of this disclosure may include amino acid substitutions to prolong the duration of action, as well as coupling with a carrier molecule (e.g., a polyethylene glycol (PEG) molecule).

[0231] Protein transport molecules ：

[0232] Examples of protein carrier molecules that can be covalently linked to reverse monomers and / or their dimers to prolong their duration of action in vivo include, but are not limited to, albumins, antibodies, and antibody fragments, such as the Fc domain of IgG molecules.

[0233] Fc Fusion: In some embodiments, the reverse monomer of this disclosure is coupled to the Fc domain. Fc fusion conjugates have been shown to increase the systemic half-life of biologics, thus reducing the need for frequent dosing. Fc binds to nascent Fc receptors (FcRn) lining the endothelial cells of blood vessels. Upon binding, the Fc fusion molecule is protected from degradation and re-release into circulation, allowing the molecule to remain in circulation for a longer period. This Fc binding is considered a mechanism by which endogenous IgG maintains its long plasma half-life. Recent Fc fusion technologies link a single copy of a biologic to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biologic compared to conventional Fc fusion conjugates. The "Fc region" used to prepare the Fc fusion can be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by papain digestion of IgG. The molecular weight of the IgG Fc is approximately 50 kDa. The binding molecules described herein can be coupled to the entire Fc region or retain a smaller portion of its ability to extend the circulating half-life as a part of a chimeric polypeptide. Furthermore, the full-length or fragmented Fc region can be a variant of the wild-type molecule. In a typical presentation, each monomer of the dimer Fc can carry a heteropeptide, which may be the same or different.

[0234] As previously described, the connection between the reverse monomer and the Fc subunit can be achieved by incorporating a linker molecule between the reverse monomer and the Fc subunit. In some embodiments, the reverse monomer is expressed as a fusion protein whose Fc domain contains the amino acid sequence of the IgG antibody hinge region. The Fc domain engineered according to the aforementioned method can be derived from mammalian IgG species such as IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain can be derived from human IgG1, IgG2, IgG3, and IgG4 IgG species. In some embodiments, the hinge region is the hinge region of IgG1. In one specific embodiment, the reverse monomer is connected to the Fc domain via a human IgG1 hinge domain.

[0235] In some embodiments, the linker is a chemical linker. Examples of chemical linkers include arylaceyne, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. In some embodiments, the linker is a peptide linker. A peptide linker may contain 1 to 50 amino acids (e.g., between 2 and 50, 5 and 50, 10 and 50, 15 and 50, 20 and 50, 25 and 50, 30 and 50, 30 and 50, 35 and 50, 40 and 50, 45 and 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, or 2 to 5 amino acids). Glycine and glycine-serine polymers are relatively unstructured and can therefore be used as neutral tethers between components. Examples of glycine polymers include (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n (SEQ ID NO: 136), (GSGGS)n (SEQ ID NO: 56)), (GmSoGm)n (SEQ ID NO: 59), (GmSoGmSoGm)n (SEQ ID NO: 60), (GSGGSm)n (SEQ ID NO: 61), (GSGSmG)n (SEQ ID NO: 62) and (GGGSm)n (SEQ ID NO: 63) and combinations thereof, wherein m, n and o are each independently selected from at least 1 to 20 integers, such as 1-18, 216, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) and other flexible connectors.

[0236] In some embodiments, the amino acid sequence of the Fc domain is modified to reduce effector function. In some embodiments, the Fc domain may be modified to significantly reduce binding to Fc receptors (FcγR and FcR), which reduces or eliminates antibody-directed cytotoxicity (ADCC) effector function. Modifying the Fc domain to reduce effector function is well known in the art. See, for example, Wang et al. (2018) IgG Fc engineering to modulate antibody effector function modulate antibody effector functions) , Protein Cell 9(1):63-73. For example, the mutation of the lysine residue at position 235 (EU number) from leucine (L) to glutamate (E) is known to reduce effector function by decreasing FcgR and C1q binding. Alegre, et al. (1992) J. Immunology 148:3461-3468. In addition, the substitution of two leucine (L) residues at positions 234 and 235 (EU number) of the IgG1 hinge region with alanine (A) (i.e., L234A and L235A) results in a decrease in complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC). Hezereh et al., (2001) J. Virol75(24):12161-68. Furthermore, the mutation at position 329 (EU designation) of proline to alanine (P329A) or glycine (P329G) mitigates the effect and can be combined with L234A and L235A substitutions. In some embodiments, the Fc domain may contain amino acid substitutions referred to as "LALAPA" substitutions L234A / L235A / P329A (EU designation) or "LALAPG" substitutions L234A / L235A / P329G (EU designation). In some embodiments, the Fc domain may contain amino acid substitutions E233P / L234V / L235A / ΔG237 (referred to in the scientific literature as the PVAdelG mutation).

[0237] In some implementations, the Fc domain is derived from hIgG4. Studies have shown that glycosylation at position 297 (EU number) contributes to the effector function of IgG4. Edelman et al. (1969) PNAS (USA) 63:78-85. Examples of modifying N297 to eliminate glycosylation sites and effector function in the Fc domain of hIgG4 include amino acid substitutions selected from N297Q and N297G (EU numbers).

[0238] In some embodiments, the amino acid sequence of the Fc domain is modified to introduce amino acid substitutions to prolong the molecule's duration of action and prevent its clearance. In some embodiments, such modifications to the Fc domain include amino acid substitutions M428L and N434S (EU designations), referred to as "LS" modifications. LS modifications can be selectively used in conjunction with amino acid substitutions to reduce effector function.

[0239] In some embodiments, the amino acid sequence of the Fc domain may be further modified to eliminate N-linked or O-linked glycosylation sites. The non-glycosylated variants of the Fc domain, particularly those of the IgG1 subclass, are known to be undesirable mediators of effector function (Jefferies et al., 1998, Immol. Rev., Vol. 163, 50-76).

[0240] In some embodiments, the reverse monomer or its dimer of this disclosure may be further modified to prolong its duration of action in vivo. In some embodiments, coupling of the PEG moiety may be accomplished via a thiol (-SH) group on a cysteine ​​residue. In some embodiments, PEGylation of the reverse monomer / Fc fusion peptide is performed at the native cysteine ​​residue at position 220 (C220, EU number) of the hinge region on the Fc domain.

[0241] Albumin transport molecules

[0242] In some embodiments, the reverse monomer and / or its dimer are coupled to an albumin molecule (e.g., human serum albumin), which is known in the art to promote prolonged exposure time in vivo. In some embodiments, the reverse monomer and / or its dimer are coupled to albumin via chemical bonds or expressed as a protein fused to an albumin molecule (referred to herein as a "reverse monomer-albumin fusion protein"). In the context of reverse monomer-albumin fusion protein, the term "albumin" includes albumins such as human serum albumin (HSA), cynomolgus monkey serum albumin, and bovine serum albumin (BSA). In some embodiments, the HSA contains a C34S or K573P amino acid substitution relative to the wild-type HSA sequence. According to this disclosure, albumin can be coupled to the reverse monomer at the carboxyl terminus, amino terminus, carboxyl terminus and amino terminus, and internally (e.g., see US 5,876,969 and US 7,056,701). In the reverse IL10 monomer-HSA fusion protein contemplated in this disclosure, various forms of albumin can be used, such as albumin secretion precursor sequences and variants thereof, fragments and variants thereof, and HSA variants. This form typically possesses one or more desired albumin activities. In other embodiments, this disclosure relates to fusion proteins comprising a reverse monomer fused directly or indirectly to albumin, albumin fragments, and albumin variants, wherein the fusion protein exhibits greater plasma stability compared to the unfused drug molecule, and / or the fusion protein retains the therapeutic activity of the unfused drug molecule. As an alternative to the chemical link between the reverse monomer and the albumin molecule, the reverse monomer-albumin complex can be provided as a fusion protein comprising an albumin polypeptide sequence and a reverse monomer, recombinantly expressed in host cells as a single polypeptide chain, optionally comprising a linker molecule connecting the albumin and the reverse monomer. Such fusion proteins can be readily prepared using recombinant techniques by those skilled in the art. Nucleic acid sequences encoding such fusion proteins can be ordered from any of a variety of commercial sources. The nucleic acid sequence encoding the fusion protein is incorporated into an expression vector operatively linked to one or more expression control elements, the vector is introduced into a suitable host cell, and the fusion protein is isolated from the host cell culture using techniques well known in the art.

[0243] Polymer transporter

[0244] In some embodiments, the in vivo action time of the reverse monomer and / or its dimer can be prolonged by coupling with one or more polymer carrier molecules (e.g., XTEN polymers or water-soluble polymers).

[0245] XTEN Coupler

[0246] The reverse monomer and / or its dimer may also contain an XTEN polymer. XTEN polymers coupled to the reverse monomer and / or its dimer (chemically or as a fusion protein) can provide extended action time similar to PEGylation and can be produced as recombinant fusion proteins in E. coli. XTEN polymers suitable for use in conjunction with the reverse monomer and / or its dimer of this disclosure are provided in Podust et al. (2016), “Extension of in vivo half-life of biologically active molecules by XTEN protein polymers,” J Controlled Release 240:52-66, and Haeckel et al. (2016), “XTENs as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic,” PLOS ONE | DOI:10.1371 / journal.pone.0157193, June 13, 2016. XTEN polymer fusion proteins may contain a protease-sensitive cleavage site, such as an MMP-2 cleavage site, between the XTEN peptide and the reverse monomer and / or its dimer.

[0247] Water-soluble polymers

[0248] In some embodiments, the reverse monomer and / or its dimer may be coupled to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of this disclosure include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (copolymers of polyvinylpyrrolidone, ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefinicalcohol), polysaccharides, poly-α-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0249] In some embodiments, the reverse monomer and / or its dimer may be coupled to one or more polyethylene glycol molecules, or "PEGylated". Although the methods or sites of PEG attachment to the binding molecules may differ, in some embodiments, PEGylation does not change or only minimally alters the activity of the binding molecules.

[0250] PEGs suitable for coupling with polypeptide sequences are typically water-soluble at room temperature and have the general formula...

[0251] R(O-CH2-CH2) n OR Where R is hydrogen or a protecting group, such as alkyl or alkylol, and n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons. PEG can be linear or branched. This invention considers branched PEG derivatives, “star-PEG”, and multi-arm PEG.

[0252] In some implementations, selective PEGylation can be used to PEGylate the reverse monomer and / or its dimer, for example, by introducing non-natural amino acids with side chains to facilitate selective PEG coupling. Specific PEGylation sites can be selected such that PEGylation of the binding molecule does not affect its binding to the target receptor.

[0253] In some embodiments, the increase in half-life outweighs any decrease in biological activity. PEGs suitable for coupling to polypeptide sequences are generally water-soluble at room temperature and have the general formula R(O-CH2-CH2)nO-R, where R is a hydrogen or protecting group, such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons. PEGs coupled to polypeptide sequences can be linear or branched. The present invention considers branched PEG derivatives, “star-PEG”, and multi-arm PEG.

[0254] The molecular weight of PEG used in this invention is not limited to any particular range. The PEG component of the bound molecule can have a molecular weight greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. The molecular weight of linear or branched PEG molecules is about 2,000 to about 80,000 Daltons, or about 2,000 to about 70,000 Daltons, or about 5,000 to about 50,000 Daltons, or about 10,000 to about 50,000 Daltons, or about 20,000 to about 50,000 Daltons, or about 30,000 to about 50,000 Daltons, or about 20,000 to about 40,000 Daltons, or about 30,000 to about 40,000 Daltons. In one embodiment of this disclosure, the PEG is a 40 kD branched PEG comprising two 20 kD arms.

[0255] In some embodiments, this disclosure provides a “mono-PEGylated” reverse monomer dimer (i.e., only one reverse monomer in the reverse monomer dimer is PEGylated) and a “dual-PEGylated” reverse monomer dimer (i.e., both reverse monomers in the reverse monomer dimer are PEGylated).

[0256] This disclosure also considers compositions of conjugates in which PEG has different n values, thus allowing for a variety of different PEGs at specific ratios. For example, some compositions comprise mixtures of conjugates where n = 1, 2, 3, and 4. In some compositions, the percentage of conjugate with n = 1 is 18-25%, the percentage of conjugate with n = 2 is 50-66%, the percentage of conjugate with n = 3 is 12-16%, and the percentage of conjugate with n = 4 is up to 5%. Such compositions can be produced using reaction conditions and purification methods known in the art. Chromatography can be used to distinguish the fractions of the conjugates, and then identify the fractions containing, for example, conjugates with the desired amount of PEG attached, purified from unmodified protein sequences and conjugates with other amounts of PEG attached.

[0257] PEGs suitable for coupling to peptide sequences are typically water-soluble at room temperature and have the general formula R(O-CH2-CH2). n OR, where R is a hydrogen or protecting group, such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons.

[0258] Two widely used first-generation active monomethoxy PEGs (mPEGs) are succinimide carbonate PEGs (SC-PEG; see, for example, Zalipsky, et al. (1992)). Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, for example, Dolence et al., U.S. Patent No. 5,650,234), which preferentially react with lysine residues to form carbamate bonds, but are also known to react with histidine and tyrosine residues. PEG-aldehyde linkers are used to target a single N-terminal site of the polypeptide via reductive amination.

[0259] PEGylation most frequently occurs at the α-amino group at the N-terminus of peptides, the ε-amino group on the side chain of lysine residues, and the imidazole group on the side chain of histidine residues. Since most recombinant peptides possess a single α-group and several ε- and imidazole groups, numerous positional isomers can be generated depending on the chemical nature of the linker. Conventional PEGylation strategies known in the art are applicable herein.

[0260] PEG can bind to the binding molecules of the present invention via terminal reactive groups (“spacers”) that mediate binding between a free amino or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEGs having spacers capable of binding free amino groups include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating a succinate of polyethylene glycol with N-hydroxysuccinimide.

[0261] In some embodiments, the PEGylation of the binding molecule is facilitated by incorporating a non-natural amino acid with a unique side chain to promote site-specific PEGylation. Incorporating non-natural amino acids into peptides to provide a functional moiety for site-specific PEGylation of such peptides is known in the art. See, for example, Ptacin et al., PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018, and published on February 7, 2019, with International Publication No. WO 2019 / 028419A1.

[0262] The PEG coupled to the polypeptide sequence can be linear or branched. This invention considers branched PEG derivatives, "star-PEG", and multi-arm PEG. The specific embodiments of PEG useful in practice according to this disclosure include 10kDa linear PEG-aldehydes (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10kDa linear PEG-NHS esters (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20kDa linear PEG-aldehydes (e.g., Sunbright® ME-200AL, NOF, 20kDa linear PEG-NHS esters (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), and 20kDa 2-arm branched PEG-aldehydes. PEG-aldehyde comprising two 10kDa straight-chain PEG molecules (e.g., Sunbright® GL2-200AL3, NOF), a 20kDa 2-arm branched PEG-NHS ester, the 20kDa PEG-NHS ester comprising two 10kDa straight-chain PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), a 40kDa 2-arm branched PEG-aldehyde, the 40kDa PEG-aldehyde comprising two 20kDa straight-chain PEG molecules (e.g., Sunbright® GL2-400AL3), a 40kDa 2-arm branched PEG-NHS ester, the 40kDa PEG-NHS ester comprising two 20kDa straight-chain PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), and a straight-chain 30kDa PEG-aldehyde (e.g., Sunbright® ME-300AL) and linear 30kDa PEG-NHS ester.

[0263] In some embodiments, a linker can be used to connect the reverse monomer and the PEG molecule. Suitable linkers include “flexible linkers” whose length is generally sufficient to allow some movement between the modified polypeptide sequence and the linked component and molecule. Linker molecules are typically about 6-50 atoms long. For example, linker molecules can also be arylaceyne, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily chosen and can be of any suitable length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids. Examples of flexible linkers are illustrated in Section IV. Furthermore, polymers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) can be linked together to provide a flexible linker that can be used to couple two molecules. Alternatively, a polypeptide linker may be used, which can be a chemical linker, such as a PEG-aldehyde linker. In some embodiments, the binding molecule is acetylated at the N-terminus by an enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, in addition to N-terminal acetylation, the binding molecule may be acetylated at one or more lysine residues, for example, by an enzymatic reaction with a lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325 (5942):834-840.

[0264] In some embodiments, this disclosure provides a PEGylated reverse monomer, wherein the PEG is coupled to the reverse monomer and the PEG is a straight-chain or branched PEG molecule with a molecular weight of about 2,000 to about 80,000 Daltons, about 2,000 to about 70,000 Daltons, about 5,000 to about 50,000 Daltons, about 10,000 to about 50,000 Daltons, about 20,000 to about 50,000 Daltons, about 30,000 to about 50,000 Daltons, about 20,000 to about 40,000 Daltons, or about 30,000 to about 40,000 Daltons. In one embodiment of this disclosure, the PEG is a 40kD branched PEG comprising two 20 kD arms.

[0265] In some embodiments, this disclosure provides a PEGylated reverse monomer, wherein the PEGylated reverse monomer is a molecule of formula 2 or formula 3: PEG-L m -[Reverse monomer] [2] [Reverse Monomer]-L m - PEG [3] in: (a) PEG is a straight-chain or branched PEG molecule with a molecular weight of approximately 10 kD to 80 kD; (v) “Reverse monomer” is the reverse monomer of Formula 1; (c) L is a polypeptide or chemical linker; (d) and m = 0 (does not exist) or 1 (exists); In some embodiments, the PEGylated reverse monomer of Formula 2 or 3 comprises a 40 kDa linear or branched PEG. In some embodiments, the 40 kDa PEG is a branched 40 kDa PEG comprising two 20 kDa arms. In some embodiments, the PEGylated reverse monomer of Formula 2 or 3 comprises a 40 kDa branched PEG having the following structure:

[0266] In some embodiments, the reverse monomer of this disclosure is a PEGylated reverse monomer of Formula 2, wherein: (a) the reverse monomer is selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:11; (b) m=1; and the PEG is a 40 kDa branched PEG having the following structure: . In some embodiments, the reverse monomer of this disclosure is a PEGylated reverse monomer of Formula 3, wherein: (a) the reverse monomer is selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:11; (b) m=1; and the PEG is a 40 kDa branched PEG having the following structure: .

[0267] fatty acid transporters

[0268] In some embodiments, as described in Resh (2016) Progress in Lipid Research 63:120–131, reverse monomers and / or their dimers, which have prolonged duration of action in mammalian subjects and are suitable for use in this disclosure, can be obtained by covalently linking the reverse monomer and / or its dimer to a fatty acid molecule. Examples of fatty acids that can be coupled include myristic acid, palmitic acid, and palmitoleic acid. Myristic acid is typically linked to an N-terminal glycine, but lysine can also be myristicated. Palmitoylation is typically achieved by enzymatic modification of a free cysteine ​​-SH group, such as S-palmitoylation catalyzed by DHHC proteins. Palmitoylation of serine and threonine residues is typically achieved by enzymatic catalysis using a PORCN enzyme. In one embodiment, the reverse monomer and / or its dimer are acetylated at the N-terminus by an enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, in addition to N-terminal acetylation, the reverse monomer and / or its dimer may also be acetylated at one or more lysine residues, for example, by enzymatic reaction with lysine acetyltransferases. See, for example, Choudhary et al. (2009) Science 325 (5942):834L2 ortho840.

[0269] Reorganization of production

[0270] In some embodiments, the reverse monomers and / or dimers of this disclosure are produced via recombinant DNA technology. In typical practice of recombinant peptide production, a nucleic acid sequence encoding the desired peptide is incorporated into a host cell expression vector suitable for expression in which the peptide is fully expressed. The nucleic acid sequence is operatively linked to one or more expression control sequences encoded by the vector and functioning in the target host cell. If a secretory leader sequence (signal peptide) is incorporated into the peptide, the recombinant protein can be recovered by disrupting the host cell or from the cellular medium.

[0271] In some embodiments, the reverse monomer and / or its dimer of this disclosure comprises amino acid substitutions that provide enhanced recombinant expression compared to wild-type hIL10 or mutants without such substitutions; pharmaceutical compositions comprising the reverse monomer and / or its dimer; recombinant nucleic acid molecules comprising nucleic acid sequences encoding the reverse monomer and / or its dimer; recombinant cells engineered to express the reverse monomer and / or its dimer; and kits comprising the reverse monomer and / or its dimer, nucleic acids encoding the reverse monomer and / or its dimer, or recombinant cells expressing the reverse monomer and / or its dimer.

[0272] Nucleic acid sequences encoding reverse monomers and / or their dimers

[0273] In some embodiments, the reverse monomer and / or its dimer are produced via recombination methods using nucleic acid sequences encoding the reverse monomer and / or its dimer (or fusion proteins containing the reverse monomer and / or its dimer). The nucleic acid sequences encoding the desired reverse monomer and / or its dimer can be synthesized chemically using an oligonucleotide synthesizer.

[0274] In some embodiments, the reverse monomer and / or its dimer are produced via recombination methods using nucleic acid sequences encoding the reverse monomer and / or its dimer (or fusion proteins containing the reverse monomer and / or its dimer). The nucleic acid sequences encoding the desired reverse monomer and / or its dimer can be synthesized chemically using an oligonucleotide synthesizer.

[0275] Nucleic acid sequences are not limited to sequences encoding polypeptides; they may also include partial or complete non-coding sequences located upstream or downstream of the coding sequence. Those skilled in molecular biology are familiar with the routine procedures used to isolate nucleic acid molecules. For example, they can be produced by treating genomic DNA with restriction endonucleases or by performing a polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0276] Nucleic acid molecules encoding inverse monomers and / or their dimers (and fusions) may contain naturally occurring sequences or sequences different from naturally occurring sequences, but which encode the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules may be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis) or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, nucleic acid molecules may be double-stranded or single-stranded (i.e., sense or antisense strands).

[0277] The nucleic acid sequences encoding the reverse monomer and / or its dimer are available from various commercial sources that provide custom nucleic acid sequences. Amino acid sequence variants of the reverse monomer and / or its dimer disclosed herein can be prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code known in the art. Such variants represent insertions, substitutions, and / or specific deletions of the indicated residues. Any combination of insertions, substitutions, and / or specific deletions can yield the final construct, provided that the final construct possesses the desired biological activity as defined herein.

[0278] The methods disclosed herein for constructing DNA sequences encoding inverse monomers and / or their dimers and expressing these sequences in appropriately transformed hosts include, but are not limited to, the use of PCR-assisted mutagenesis. Mutations consisting of deletions or additions of amino acid residues can also be made to inverse monomers and / or their dimers using standard recombination techniques. In the case of deletions or additions, the nucleic acid molecule encoding the inverse monomer and / or its dimer is optionally digested with a suitable restriction endonuclease. The resulting fragment can be expressed directly or further manipulated, for example, by ligation to a second fragment. Ligation can be facilitated if the two ends of the nucleic acid molecule contain mutually overlapping complementary nucleotides, but blunt-ended fragments can also be ligated. PCR-generated nucleic acids can also be used to generate a variety of mutant sequences.

[0279] The reverse monomer and / or its dimer disclosed herein can be produced not only directly via recombination but also fused with heterologous polypeptides (e.g., signal sequences or other polypeptides having specific cleavage sites at the N- or C-terminus of the reverse monomer) to form fusion polypeptides. In some embodiments, the nucleic acid molecule also contains a nucleic acid sequence encoding a signal peptide. Typically, the signal sequence can be a component of the vector or a portion of the coding sequence inserted into the vector. The selected heterologous signal sequence is preferably a sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). Whether a signal sequence is included depends on whether the reverse monomer and / or its dimer needs to be secreted from the recombinant cell that produces it. If the selected cell is a prokaryotic cell, it is generally preferred that the DNA sequence does not encode the signal sequence. When the recombinant host cell is a yeast cell (e.g., Saccharomyces cerevisiae), an α-mating factor secretion signal sequence can be used to achieve extracellular secretion of the reverse monomer and / or its dimer into the culture medium, as described in U.S. Patent No. 7,198,919 B1 issued by Singh on April 3, 2007. In some embodiments, the signal peptide comprises an endogenous or wild-type IL10 signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of a human IL10 polypeptide: MHSSALLCCLVLLTGVRA (SEQ ID NO:86). In some embodiments, the signal peptide comprises the amino acid sequence of a mouse IL10 polypeptide: MPGSALLCCLLLLTGMRI (SEQ ID NO:87).

[0280] If the reverse monomer and / or its dimer will be expressed in a chimeric form (e.g., a fusion protein comprising the reverse monomer and / or its dimer and a heterologous polypeptide sequence), then the chimeric protein may be encoded by a hybrid nucleic acid molecule comprising a first sequence encoding all or part of the reverse monomer and / or its dimer and a second sequence encoding all or part of the heterologous polypeptide. For example, the reverse monomer and / or its dimer described herein may be fused with a hexahistidine / octahistidine tag (“HHHHHH” and “HHHHHHHHH”, disclosed as SEQ ID NO: 139-140, respectively) to facilitate the purification of proteins expressed in bacteria; or fused with a hemagglutinin tag to facilitate the purification of proteins expressed in eukaryotic cells. The terms “first” and “second” should not be construed as restrictions on the orientation of the elements of the fusion protein; the heterologous polypeptide may be attached to the N-terminus and / or C-terminus of the reverse monomer and / or its dimer. For example, the N-terminus may be attached to a targeting domain, while the C-terminus may be attached to a purification handle of a hexahistidine (His6) tag (SEQ ID NO: 139).

[0281] The complete amino acid sequence of the polypeptide (or fusion / chimera) to be expressed can be used to construct a back-translated gene. DNA oligonucleotides containing nucleotide sequences encoding inverse monomers and / or their dimers can be synthesized. For example, small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0282] Codon optimization:

[0283] In some implementations, nucleic acid sequences encoding inverse monomers and / or their dimers can be “codon-optimized” to facilitate their expression in specific host cell types. Techniques for codon optimization in various expression systems, including mammalian, yeast, and bacterial host cells, are well-known in the art, and online tools are available to provide codon-optimized sequences for expression in multiple host cell types. See, for example, Hawash, et al. (2017) 9:46-53 and Mauro and Chappell in... Recombinant Protein Expression in Mammalian Cells: Methods and Protocols Expression in Mammalian Cells: Methods and Protocols), Edited by David Hacker (Human Press, New York). In addition, several web-based online software programs are available free of charge to assist in the preparation of codon-optimized nucleic acid sequences.

[0284] Vehicle for expression:

[0285] Once the nucleic acid sequence encoding the reverse monomer and / or its dimer is assembled (through synthesis, site-directed mutagenesis, or other methods), it can be inserted into an expression vector. A variety of expression vectors can be used for various host cells, typically based on the host cell chosen for expression. Expression vectors generally include, but are not limited to, one or more of the following: origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Vectors include viral vectors, plasmid vectors, integration vectors, etc. Plasmids are examples of non-viral vectors.

[0286] In some embodiments, such as when the first and second reverse monomers of the dimer are different (e.g., in the case of heterodimeric reverse monomers), the vector comprises a first nucleic acid sequence encoding the first reverse monomer and a second nucleic acid sequence encoding the second reverse monomer, wherein the first and second nucleic acid sequences are operatively linked to expression control elements (e.g., promoters), and the first and second nucleic acid sequences are separated by a sequence that promotes co-expression (e.g., an IRES or T2A sequence). Alternatively, the vector comprises a first nucleic acid sequence encoding the first reverse monomer and a second nucleic acid sequence encoding the second reverse monomer, wherein the first and second nucleic acid sequences are each operatively linked to expression control sequences, which may be the same or different.

[0287] To promote efficient expression of recombinant peptides, the nucleic acid sequence encoding the peptide to be expressed can be operatively linked to transcriptional and translational regulatory sequences that are functional in the selected expression host.

[0288] Optional markers:

[0289] Expression vectors typically contain a select gene, also known as a selectable marker. This gene encodes a protein essential for the survival or growth of transformed host cells in selective media. Host cells not transformed with a vector containing a select gene cannot survive in the medium. A typical select gene encodes a protein that will (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for auxotrophic deficiencies; or (c) provide a key nutrient that is not available from a complex culture medium.

[0290] Regulatory sequence:

[0291] The expression vectors of the reverse monomers and / or their dimers disclosed herein contain regulatory sequences that are recognizable by a host organism and are operatively linked to nucleic acid sequences encoding the reverse monomers and / or their dimers. The terms “regulatory sequence,” “regulatory sequence,” or “expression control sequence” are used interchangeably herein to refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in *Gene Expression Technology: Methods in Enzymology*, 185 (Academic Press, San Diego, California, USA). Regulatory sequences include nucleotide sequences that guide constitutive expression in many types of host cells and nucleotide sequences that guide expression only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of expression vectors may depend on factors such as the choice of host cells to be transformed, the desired protein expression level, etc. In the selection of expression control sequences, those skilled in the art will understand that a variety of factors will be considered. These factors include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the target inverse monomer and / or its dimer, particularly regarding potential secondary structures.

[0292] Promoter:

[0293] In some implementations, the regulatory sequence is a promoter, the selection of which is based, for example, on the cell type in which expression is sought. A promoter is a non-translated sequence (typically within about 100 to 1000 bp) located upstream (5') of the start codon of a structural gene that controls the transcription and translation of a specific nucleic acid sequence operatively linked to it. Such promoters are generally classified into two categories: inducible and constitutive. Inducible promoters are promoters that respond to certain changes in culture conditions (such as the presence or absence of nutrients or changes in temperature), increasing the level of transcription from the DNA they control. A large number of promoters recognized by a variety of potential host cells are well known.

[0294] In bacteria, the T7 promoter can be used; in insect cells, the polyhedrosis protein promoter can be used; and in mammalian cells, the cytomegalovirus or metallothionein promoter can be used. Additionally, tissue-specific and cell-specific promoters are widely used in the case of higher eukaryotic cells. These promoters are named for their ability to direct the expression of nucleic acid molecules in a given in vivo tissue or cell type. Technologists are familiar with many promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

[0295] Transcription of vectors in mammalian host cells can be controlled by promoters, for example, derived from viral genomes such as polyomavirus, vaccinia virus, adenovirus (e.g., human adenovirus serotype 5), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses (e.g., mouse stem cell virus), hepatitis B virus, and most ideally simian virus 40 (SV40); promoters from heterologous mammals such as actin promoters, PGK (phosphoglycerate kinase) or immunoglobulin promoters; and heat shock promoters, provided these promoters are compatible with the host cell system. Early and late promoters of SV40 virus are readily available as SV40 restriction fragments, which also contain the SV40 virus's origin of replication.

[0296] Enhancer:

[0297] Transcription in higher eukaryotes is typically amplified by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically 10 to 300 bp in length, that act on the promoter to amplify its transcription. The orientation and location of enhancers are relatively independent, found at the 5' and 3' of transcription units, within introns, and within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic viruses are commonly used. Examples include the SV40 enhancer post-OMI, the cytomegalovirus early promoter enhancer, the polyoma enhancer post-OMI, and the adenovirus enhancer. Enhancers can be spliced ​​into the 5' or 3' position of the coding sequence in the expression vector, but are preferably located 5' from the promoter. Expression vectors for eukaryotic host cells will also contain sequences necessary for terminating transcription and for stabilizing mRNA. These sequences are typically obtained from the 5', and occasionally 3', untranslated regions of eukaryotic or viral DNA or cDNA. The construction of suitable carriers containing one or more of the above components employed standard techniques.

[0298] In addition to sequences that promote transcription of the inserted nucleic acid molecule, the vector may contain genes encoding origins of replication and other optional markers. For example, the neomycin resistance (neoR) gene confers resistance to G418 on its expressing cells, thus allowing phenotypic selection of transfected cells. Other examples of markers or reporter genes include β-lactamases, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-β-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine-guanine phosphoribosyltransferase (XGPRT). Those skilled in the art can readily determine whether a given regulatory element or optional marker is suitable or unsuitable for a particular expression environment.

[0299] The correct assembly of expression vectors can be confirmed by nucleic acid sequencing, restriction mapping, and expression of bioactive peptides in a suitable host.

[0300] Host cell:

[0301] This disclosure further provides prokaryotic or eukaryotic cells containing and expressing one or more nucleic acid molecules encoding inverse monomers and / or their dimers. The cells disclosed herein are transfected cells, i.e., cells in which nucleic acid molecules (e.g., nucleic acid molecules encoding inverse monomers and / or their dimers) have been introduced via recombinant DNA technology.

[0302] In some embodiments, such as when the first and second reverse monomers of the dimer are different, for example in the case of a heterodimer reverse monomer, the recombinant-modified cell comprises a vector comprising a first nucleic acid sequence encoding a first reverse monomer and a second nucleic acid sequence encoding a second reverse monomer, wherein the first and second nucleic acid sequences are operatively linked to a single expression control sequence, and the first and second nucleic acid sequences are separated by a sequence promoting co-expression. In other embodiments, the recombinant-modified cell comprises a vector comprising a first nucleic acid sequence encoding a first reverse monomer and a second nucleic acid sequence encoding a second reverse monomer, wherein each of the first and second nucleic acid sequences is operatively linked to an expression control sequence. In other embodiments, when the first and second reverse monomers of the dimer are different, for example in the case of a heterodimer reverse monomer, the recombinant-modified cell may comprise two vectors, wherein a first vector comprises a first nucleic acid sequence encoding a first reverse monomer operatively linked to an expression control sequence, and a second vector comprises a nucleic acid sequence encoding a second reverse monomer. In some embodiments, the recombinant-modified cell is a prokaryotic cell, such as a bacterial cell. In some embodiments, the recombinant-modified cell is a eukaryotic cell, such as a mammalian cell.

[0303] Host cells are typically selected based on their compatibility with the chosen expression vector, the toxicity of the product encoded by the DNA sequence of this invention, their secretion characteristics, their ability to correctly fold polypeptides, their fermentation or culture requirements, and the ease of purification of the product encoded by the DNA sequence. Suitable host cells for cloning or expressing the DNA in the vectors described herein are the aforementioned prokaryotic cells, yeast, or higher eukaryotic cells.

[0304] In some implementations, the recombinant reverse monomer can also be produced in eukaryotes, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors that can be used to express proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors used for expression in Saccharomyces cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123), pYES2 (Invitrogen... Corporation, San Diego, California) and pPicZ (Ingenieur Corporation, San Diego, California)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al., (1987) EMBO J. 6:187:195)).

[0305] Examples of useful mammalian host cell lines include: mouse L cells (LM[TK-], ATCC CRL-2648); monkey kidney CV1 cell line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (HEK293 or HEK293 cell subclones, grown in suspension culture); young hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); and human lung cells (W138, ATCC CCL 1651). 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). In mammalian cells, the control function of expression vectors is usually provided by viral regulatory elements. For example, commonly used promoters are derived from polyomaviruses, adenovirus type 2, cytomegaloviruses, and simian virus 40.

[0306] Reverse monomers and / or their dimers can be generated in prokaryotic hosts (e.g., *E. coli*) or eukaryotic hosts (e.g., insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells)). These cells can be obtained from many sources, including the U.S. Type Culture Collection (Massanas, Virginia). When selecting an expression system, compatibility between the components is of paramount importance. Those skilled in the art are capable of making this decision. Furthermore, if guidance is needed in selecting an expression system, those skilled in the art can consult Ausubel et al. (*Current Protocols in Molecular Biology*, John Wiley and Sons, NY, 1993) and Pouwels et al. (*Cloning Vectors: A Laboratory Manual*, 1985, Supplement 1987).

[0307] In some implementations, whether the resulting reverse monomer is glycosylated depends on the host organism used to produce the peptide. If bacteria are chosen as the host, the resulting reverse monomer will not be glycosylated. On the other hand, eukaryotic cells typically lead to glycosylation of the reverse monomer.

[0308] In one embodiment, the amino acid sequence (particularly its CDR sequence) of the sdAb to be integrated into the reverse monomer and / or its dimer may contain a glycosylation motif, particularly an N-linked glycosylation motif with the sequence Asn-X-Ser (NXS) or Asn-X-Thr (NXT), where X is any amino acid other than proline. In this case, it is desirable to eliminate such an N-linked glycosylation motif by modifying the sequence of the N-linked glycosylation motif to prevent glycosylation. In some embodiments, the N-linked glycosylation motif is disrupted by the inclusion of a conserved amino acid substitution of the Asn(N) residue of the N-linked glycosylation motif.

[0309] For other expression systems for prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY). See also Goeddel (1990), *Gene Expression Technology: Methods in Enzymology* (Academic Press, San Diego, CA).

[0310] Transfection:

[0311] The expression constructs disclosed herein can be introduced into host cells to produce inverse monomers and / or their dimers. Expression vectors containing nucleic acid sequences encoding these inverse monomers and / or their dimers can be introduced into prokaryotic or eukaryotic host cells using conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY), and other standard molecular biology laboratory manuals. To facilitate transfection of target cells, target cells can be directly exposed to non-viral vectors under conditions favorable to their uptake. Examples of conditions that promote the uptake of exogenous nucleic acids by mammalian cells are well known in the art, including but not limited to chemical methods (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt, and magnetic fields (electroporation).

[0312] Cell culture:

[0313] Cells can be cultured in conventional nutrient media modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. Mammalian host cells can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI 1640 (Sigma), and Darwin's modified Ehrlich medium (DMEM, Sigma) are suitable for culturing host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or equivalent energy sources. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used for selecting expression host cells and are obvious to those skilled in the art.

[0314] Recombinant protein recovery:

[0315] If a secretory leader sequence is used, the recombinant-produced reverse monomer and / or its dimer can be recovered from the culture medium via secretory peptides. Alternatively, the reverse monomer and / or its dimer can also be recovered from host cell lysates. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), can be used in the recovery phase from cell lysates to inhibit protease degradation during purification and may include antibiotics to prevent the growth of exogenous contaminants.

[0316] Various purification steps are known and available in the art, such as affinity chromatography. Affinity chromatography utilizes highly specific binding sites commonly found in biological macromolecules to separate them based on their ability to bind to specific ligands. Covalent bonds attach ligands to an insoluble porous support medium, making the ligands readily visible on the protein sample, thus utilizing the natural specific binding of one type of molecule to separate and purify a second type from the mixture. Antibodies are commonly used in affinity chromatography. Size selection steps can also be used, for example, gel filtration chromatography (also known as size exclusion or molecular sieve chromatography) for separating proteins based on their size. In gel filtration, a protein solution is passed through a column filled with a semi-permeable porous resin. The semi-permeable resin has a range of pore sizes that determines the size of proteins that can be separated using the column.

[0317] The recombinant reverse monomer and / or its dimer expressed by the transformed host can be purified using any suitable method. The recombinant reverse monomer and / or its dimer can be isolated from inclusion bodies produced by *E. coli*, or from conditioned media of mammalian or yeast cultures producing specific peptides, using methods including cation exchange, gel filtration, and / or reversed-phase liquid chromatography. The substantially purified recombinant reverse monomer and / or its dimer can be purified from the expression system using conventional biochemical methods and can be used as therapeutic agents, for example, as described herein.

[0318] In some embodiments, as described above, the purification handle can be used to separate the reverse monomer and / or its dimer from cell lysate or cell culture medium when the reverse monomer and / or its dimer are expressed together with the purification tag. In the case that the purification tag is a chelate peptide, methods for separating such molecules using immobilized metal affinity chromatography are well known in the art. See, for example, U.S. Patent 4,569,794 to Smith et al.

[0319] The bioactivity of the recovered reverse monomer and / or its dimer can be determined by any suitable method known in the art, and when expressed using a secretory leader sequence, it can be evaluated either as a basically purified form or as part of a cell lysate or cell culture medium.

[0320] pharmaceutical preparations

[0321] In some embodiments, the reverse monomer and / or its dimer (and / or nucleic acid encoding the reverse monomer and / or its dimer, or recombinant cells containing a nucleic acid sequence and modified to express the reverse monomer and / or its dimer) can be incorporated into compositions including pharmaceutical compositions, etc. Such compositions typically comprise peptide or nucleic acid molecules and pharmaceutically acceptable carriers. The pharmaceutical composition is formulated to be compatible with its intended route of administration and with the therapeutic use of the reverse monomer and / or its dimer intended for treating a subject requiring treatment.

[0322] carrier ：

[0323] The carrier includes sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable flowability can be maintained, for example by using coating materials such as lecithin, maintaining the desired particle size in the case of dispersions, and by using surfactants such as sodium dodecyl sulfate. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsipney, NJ), or phosphate-buffered saline (PBS).

[0324] Buffer solution:

[0325] The term "buffer" includes buffers such as acetate, citrate, or phosphate, as well as reagents such as sodium chloride or glucose to adjust osmotic pressure. The pH value can be adjusted with acids or bases, such as sodium dihydrogen phosphate and / or disodium hydrogen phosphate, hydrochloric acid, or sodium hydroxide (e.g., adjusted to a pH value of approximately 7.2-7.8, such as 7.5).

[0326] dispersion ：

[0327] Typically, the active ingredient is incorporated into a sterile carrier containing an alkaline dispersion medium and the other desired components mentioned above to prepare a dispersion. In preparing sterile powders for sterile injections, the preferred preparation method is vacuum drying and freeze-drying, yielding a powder of the active component and any other desired components from a previously sterile filtered solution.

[0328] preservative ：

[0329] Pharmaceutical preparations intended for parenteral administration should be sterile and fluid to facilitate injectability. They should be stable under manufacturing and storage conditions and resistant to contamination during storage. Microbial protection can be achieved through various antimicrobial and antifungal agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; and chelating agents, such as ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. A sterile solution can be prepared by incorporating the desired amount of the active compound and one or more of the above components, or combinations thereof, into a suitable solvent, followed by filtration and sterilization.

[0330] tension agent ：

[0331] In many cases, the composition may preferably contain isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride.

[0332] Provide a means

[0333] In some embodiments, the treatment methods of this disclosure involve administering to a subject in need of treatment a pharmaceutical formulation comprising a reverse monomer and / or its dimer (and / or a nucleic acid encoding the reverse monomer and / or its dimer, or a recombinant-modified host cell expressing the reverse monomer and / or its dimer). The pharmaceutical formulations of this disclosure comprising the reverse monomer and / or its dimer can be administered to a subject in need of treatment or prevention via a variety of routes of administration, including parenteral, oral, topical, or inhalation routes.

[0334] Parenteral administration:

[0335] In some embodiments, the methods of this disclosure involve administering a pharmaceutical formulation comprising a reverse monomer and / or its dimer (and / or a nucleic acid encoding the reverse monomer and / or its dimer, or a recombinant-modified host cell expressing the reverse monomer and / or its dimer) to a subject requiring treatment via a parenteral route. Examples of parenteral routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), mucosal, and rectal administration. Parenteral formulations include solutions or suspensions for parenteral application and may include carriers and buffers. Pharmaceutical formulations suitable for parenteral administration include sterile aqueous solutions (when water-soluble) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In one embodiment, the formulation is provided in a pre-filled syringe.

[0336] Oral administration:

[0337] In some embodiments, the methods of this disclosure involve orally administering to a subject requiring treatment a pharmaceutical formulation comprising a reverse monomer and / or its dimer (and / or a nucleic acid encoding the reverse monomer and / or its dimer, or a recombinant-modified host cell expressing the reverse monomer and / or its dimer). If an oral composition is used, it typically includes an inert diluent or an edible carrier. For oral therapeutic administration purposes, the active compound may be incorporated with excipients and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions may also be prepared using liquid carriers for use as mouthwashes. Pharmaceutically compatible binder and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginate, carboxymethyl starch (Primogel). TMOr corn starch; lubricants such as magnesium stearate or fully hydrogenated vegetable oils (Sterotes) TM ); flow aids such as silica gel; sweeteners such as sucrose or saccharin; or flavorings such as mint, methyl salicylate, or orange flavoring.

[0338] Inhaled formulations ：

[0339] In some embodiments, the methods of this disclosure involve administering a pharmaceutical formulation comprising a reverse monomer and / or its dimer (and / or a nucleic acid encoding the reverse monomer and / or its dimer, or a recombinant modified host cell expressing the reverse monomer and / or its dimer) to a subject requiring treatment via inhalation. In the case of inhalation administration, the present reverse monomer and / or its dimer, or the nucleic acid encoding them, is delivered as an aerosol spray from a pressurized vessel, dispenser, or nebulizer containing a suitable propellant, such as a gas like carbon dioxide. Such methods include those described in U.S. Patent No. 6,468,798.

[0340] Mucosal and transdermal preparations:

[0341] In some embodiments, the methods of this disclosure involve administering, via mucosa or transdermally, a pharmaceutical formulation comprising a reverse monomer and / or its dimer (and / or a nucleic acid encoding the reverse monomer and / or its dimer, or a recombinant-modified host cell expressing the reverse monomer and / or its dimer) to a subject requiring treatment. For mucosal or transdermal administration, a permeabilizer suitable for the permeation barrier is employed in the formulation. Such permeabilizers are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for mucosal administration. Mucosal administration may be performed using nasal sprays or suppositories (e.g., using conventional suppository bases such as cocoa butter or other glycerides) or retention enemas for rectal delivery. For transdermal administration, the active compound is formulated as an ointment, ointment, gel, or cream, as is generally known in the art, and may incorporate a penetration enhancer such as ethanol or lanolin.

[0342] Extended release and storage formulations:

[0343] In some embodiments of the methods disclosed herein, the reverse monomer and / or its dimer are administered to the subject in need of treatment in the form of a formulation providing sustained release of the reverse monomer and / or its dimer. Examples of extended-release formulations of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition. In one embodiment, the current reverse monomer and / or its dimer or nucleic acid is prepared together with a carrier that protects the reverse monomer and / or its dimer from rapid clearance from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Such formulations can be prepared using standard techniques. The materials are also available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes of cells infected with viral antigens targeted by monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0344] Give nucleic acids encoding reverse monomers and / or their dimers

[0345] In some embodiments of the methods disclosed herein, delivery of the reverse monomer and / or its dimer to a subject requiring treatment is achieved by administering a nucleic acid encoding the reverse monomer and / or its dimer. The method of administering the nucleic acid encoding the reverse monomer and / or its dimer to the subject is achieved by transfection or infection using methods known in the art, including but not limited to those described in McCaffrey et al. (Nature (2002) 418:6893), Xia et al. (Nature Biotechnol. (2002) 20:1006-1010), or Putnam (Am. J. Health Syst. Pharm. (1996) 53: 151-160). In one embodiment, the reverse monomer and / or its dimer are administered to a subject via a pharmaceutically acceptable recombinant expression vector formulation comprising a nucleic acid sequence encoding the reverse monomer and / or its dimer, the nucleic acid sequence being operatively linked to one or more expression control sequences operable in mammalian subjects. In some embodiments, the expression control sequence operable in a limited range of cell types (or a single cell type) may be selected to facilitate selective expression of the reverse monomer and / or its dimer in a specific target cell type. In some embodiments, the recombinant expression vector is a viral vector. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or a recombinant adenovirus (rAd), particularly a replication-deficient adenovirus derived from human adenovirus serotypes 3 and / or 5. In some embodiments, the replication-deficient adenovirus has one or more modifications to the E1 region, which interfere with the virus's ability to initiate cell circulation and / or apoptosis pathways in human cells. The replication-deficient adenoviral vector may optionally include a deletion in the E3 domain. In some embodiments, the adenovirus is a replication-capable adenovirus. In some implementations, the adenovirus is a replicative recombinant virus that has been engineered to selectively replicate in target cell types.

[0346] In some embodiments, particularly for treating intestinal diseases or bacterial infections in subjects, nucleic acids encoding inverse monomers and / or their dimers can be delivered to the subject by administering a recombinant-modified phage vector encoding inverse monomers and / or their dimers. As used herein, the terms “prokaryotic virus,” “phage,” and “bacteriophage” are used interchangeably to describe any of a variety of bacterial viruses that infect and replicate within bacteria. Phages selectively infect prokaryotic cells, restricting the expression of inverse monomers and / or their dimers to the target prokaryotic cells while avoiding expression in mammalian cells. A variety of phages capable of selecting a variety of bacterial cells have been identified and characterized in the scientific literature. In some embodiments, phages are modified to remove adjacent motifs (PAMs). Removing the Cas9 sequence from the phage genome reduces the ability of the Cas9 endonuclease in the target prokaryotic cells to neutralize invading phages encoding inverse monomers and / or their dimers.

[0347] Recombinant modified cells expressing reverse monomers or dimers were given

[0348] In some embodiments of the methods disclosed herein, delivery of the reverse monomer and / or its dimer to a subject requiring treatment is achieved by administering recombinant host cells modified to express the reverse monomer and / or its dimer. The recombinant host cells can be used for the therapeutic and preventative applications described herein. In some embodiments, the recombinant host cells are mammalian cells, such as human cells. In some embodiments, the recombinant host cells are prokaryotic cells, such as bacterial cells associated with the gut microbiota, such as *Escherichia coli* or *Lactobacillus*.

[0349] In some embodiments, the nucleic acid sequence encoding the reverse monomer and / or its dimer (or a vector containing the sequence) may be stored in an extrachromosomal form in a recombinant-modified host cell for administration. In other embodiments, the nucleic acid sequence encoding the reverse monomer and / or its dimer may be incorporated into the genome of the host cell to be administered using at least one endonuclease to facilitate the insertion of the nucleic acid sequence into the cell's genomic sequence. As used herein, the term "endonuclease" refers to a wild-type or variant enzyme capable of catalyzing the cleavage of bonds between nucleic acids within a DNA or RNA molecule (preferably a DNA molecule). When such an endonuclease has a polynucleotide recognition site longer than about 12 base pairs (bp) (more preferably 14-55 bp), it is referred to as a "rare-cut" endonuclease. Rare-cut endonucleases may be used to inactivate genes at loci or to integrate transgenes via homologous recombination (HR), i.e., by inducing DNA double-strand breaks (DSBs) at the locus and by inserting foreign DNA at the locus through gene repair mechanisms. Examples of rare cleaving endonucleases include homing endonucleases (Grizot et al. (2009) Nucleic Acids Research 37(16):5405-5419), chimeric zinc finger nucleases (ZFNs) resulting from the fusion of engineered zinc finger domains (Porteus M and Carroll D., using zinc finger nucleases to target genes ( Gene targeting using zinc finger nucleases ) (2005) Nature Biotechnology 23(3):967-973), TALEN-nuclease, Cas9 nuclease from the CRISPR system, or modified restriction endonuclease with extended sequence specificity (Eisenschmidt, et al. 2005; 33(22): 7039–7047).

[0350] In some embodiments, particularly for the delivery of the reverse monomer and / or its dimer to the gut, the reverse monomer and / or its dimer may be delivered to the subject via recombinant-modified prokaryotic cells (e.g., Lactobacillus). The use of engineered prokaryotic cells to deliver recombinant proteins to the gut is known in the art. See, for example, Lin, et al. (2017) Microb Cell Fact 16:148. In some embodiments, engineered bacterial cells expressing the reverse monomer and / or its dimer may be administered orally (typically as an aqueous suspension) or rectally (e.g., via enema).

[0351] How to use

[0352] This disclosure also provides a method of treating a subject suffering from a disease or condition by administering a therapeutically effective amount of the following substances: (a) a reverse IL10 monomer and / or its dimer; (b) a pharmaceutically acceptable formulation containing a reverse IL10 monomer and / or its dimer as an active ingredient; (c) a recombinant nonviral or eukaryotic viral or bacteriophage vector comprising a nucleic acid sequence encoding a reverse monomer and / or its dimer, the nucleic acid sequence being operatively linked to one or more expression control sequences; (d) a genome-modified recombinant prokaryotic or mammalian cell comprising a nucleic acid sequence encoding a reverse monomer and / or its dimer, the nucleic acid sequence being operatively linked to one or more expression control sequences; or (e) a recombinant prokaryotic or mammalian cell comprising a nucleic acid encoding a reverse monomer and / or its dimer, the nucleic acid being operatively linked to one or more expression control sequences.

[0353] Dosage / Administration

[0354] Compared to wild-type IL10, the biased IL10 reverse monomer (and its dimer) of this disclosure has a wider therapeutic window. "Therapeutic window" refers to the range of administration of the biased IL10 reverse monomer (and / or its dimer) that provides in vivo concentrations of the biased IL10 reverse monomer (and its dimer) sufficient to produce significant IL10 activity (e.g., STAT3 signaling) in monocytes, but below concentrations sufficient to induce significant IL10 activity signaling in T cells. Therefore, in some embodiments, the biased IL-10 reverse monomer is administered to the subject at a level that produces concentrations sufficient to provide significant STAT3 signaling in monocytes, but below concentrations sufficient to induce significant STAT3 signaling in T cells. In some embodiments, this disclosure provides a method for treating a subject suffering from an inflammatory or autoimmune disease, disorder, or condition, characterized by administering a therapeutically effective amount of a reverse IL-10 monomer and / or its dimer (or a pharmaceutically acceptable formulation containing a reverse IL-10 monomer and / or its dimer as an active ingredient), wherein a therapeutically effective amount refers to an amount in the subject that produces a concentration sufficient to provide significant STAT3 signaling in monocytes, but less than a dose in the subject that reaches a concentration sufficient to induce significant STAT3 signaling in T cells.

[0355] In one embodiment, this disclosure provides a method for treating subjects suffering from inflammatory or autoimmune diseases, disorders, or conditions by administering a therapeutically effective amount of a reverse IL10 monomer and / or its dimer (or a pharmaceutically acceptable formulation containing a reverse IL10 monomer and / or its dimer as an active ingredient), wherein the dose level of the monomer maximizes the activity of IL10 on monocytes. max With the activity of IL10 on T cells Emax The ratio. In one embodiment, this disclosure provides a method of treating a subject suffering from a disease, disorder, or condition by administering a therapeutically effective amount of reverse IL10 monomer and / or its dimer (or a pharmaceutically acceptable formulation containing reverse IL10 monomer and / or its dimer as an active ingredient), wherein a therapeutically effective amount is a dose providing a concentration greater than that of activated human monocytes STAT3 EC. 20 Or larger than STAT3 EC in activated human monocytes 30 Or larger than STAT3 EC in activated human monocytes 40 Or larger than STAT3 EC in activated human monocytes 50 Or larger than STAT3 EC in activated human monocytes 60 Or larger than STAT3 EC in activated human monocytes 70 Or larger than STAT3 EC in activated human monocytes 80 Or larger than STAT3 EC in activated human monocytes 90 Or greater than the E of activated human monocytes max (EC) 100 (b) smaller than STAT3EC in activated human T cells; 40 Or smaller than STAT3 EC in activated human T cells 30 Or smaller than STAT3 EC in activated human T cells 20 Or smaller than STAT3 EC in activated human T cells 10 Or less than STAT3 EC5 of activated human T cells. In some embodiments, this disclosure provides a method of treating a subject suffering from a disease or condition by administering a therapeutically effective amount of reverse IL-10 monomer and / or its dimer (or a pharmaceutically acceptable formulation containing reverse IL-10 monomer and / or its dimer as an active ingredient), wherein the therapeutically effective amount is a dose providing a concentration greater than STAT3 EC5 of activated human monocytes. 20 However, it is smaller than STAT3 EC in activated human T cells. 30 Or, larger than STAT3 EC in activated human monocytes. 50 However, it is smaller than STAT3 EC in activated human T cells. 20 Or, larger than STAT3 EC in activated human monocytes. 70 However, it is smaller than the STAT3 EC in activated human T cells. 20 Or, larger than STAT3 EC in activated human monocytes. 80 However, it is smaller than the STAT3 EC in activated human T cells. 20Or, greater than STAT3 E in activated human monocytes. max However, it is smaller than the STAT3ECs in activated human T cells. 30 Or, larger than STAT3 EC in activated human monocytes. 50 However, it is smaller than the STAT3 EC in activated human T cells. 10 .

[0356] Inflammation and autoimmune diseases

[0357] Diseases treatable with the reverse monomers and / or their dimers (including pharmaceutically acceptable formulations comprising the reverse monomers and / or their dimers and / or nucleic acid molecules encoding them, including recombinant viruses encoding such reverse monomers and / or their dimers) of this disclosure include inflammatory or autoimmune diseases, including but not limited to organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases (including Alzheimer's disease, systemic lupus erythematosus (SLE)), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes (including type 1 or type 2 diabetes), inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, chondritis, and arthritis. Rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (serone-negative tenoarthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (serone-negative tenoarthropathy syndrome).

[0358] Examples of other proliferative and / or differentiation disorders for which the reverse monomers and / or their dimers (including pharmaceutically acceptable formulations comprising the reverse monomers and / or their dimers and / or nucleic acid molecules encoding them, including recombinant viruses encoding such reverse monomers and / or their dimers) of this disclosure can be used for treatment include, but are not limited to, skin diseases. Such skin diseases may involve abnormal activity of one or more cells or layers in the dermis, epidermis, or subcutaneous layer, or abnormalities at the dermal-epidermal junction. For example, skin diseases may involve abnormal activity of keratinocytes (such as overproliferating basal and superbasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cells, and other cells found in one or more epidermal layers (such as the basal layer (germinal layer), spinous layer, granular layer, stratum lucidum, or stratum corneum). In other embodiments, the disorder may involve abnormal activity of dermal cells, such as dermal endothelial cells, fibroblasts, and immune cells (such as mast cells or macrophages) found in the dermis, such as the papillary layer or reticular layer.

[0359] Examples of inflammatory or autoimmune skin diseases include psoriasis, psoriatic arthritis, dermatitis (eczema), such as exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosacea, psoriasis-like diseases, lichenoid pityriasis, lichen planus, lichen luster, ichthyosis-like skin diseases, keratosis, skin diseases, alopecia areata, pyoderma gangrenosa, vitiligo, pemphigoid (e.g., ocular cicatricial pemphigoid or bullous pemphigoid), urticaria, keratosis, and rheumatoid arthritis. This includes conditions involving excessive proliferation and inflammation of the epithelial-associated cells lining the joint capsule; dermatitis such as seborrheic dermatitis and photodermatitis; keratosis such as seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis, and follicular keratosis; acne vulgaris; keloids and prevention of keloid formation; moles; skin tags, including warts, condyloma acuminata, or genital warts, and human papillomavirus (HPV) infection, such as genital warts; leukoplakia; lichen planus, and keratitis. Skin diseases can be dermatitis, such as atopic dermatitis or allergic dermatitis, such as psoriasis.

[0360] The compositions disclosed herein (including pharmaceutically acceptable formulations comprising inverse monomers and / or their dimers, and / or nucleic acid molecules encoding such inverse monomers and / or their dimers, including recombinant viruses encoding such inverse monomers and / or their dimers) may also be used to treat patients who have (or may have) psoriasis or psoriasis-related diseases. The term "psoriasis" is intended to have its medical meaning as a disease that primarily affects the skin and produces raised, thickened, scaly, non-scarring lesions. Lesions are typically well-defined erythematous papules covered with overlapping, shiny scales. The scales are usually silvery or slightly milky white. Nails are frequently affected, resulting in pitting, onycholysis, thickening, and discoloration. Psoriasis is sometimes associated with arthritis and can have serious consequences. Excessive keratinocyte proliferation is a key feature of epidermal hyperplasia in psoriatic arthritis, accompanied by epidermal inflammation and reduced keratinocyte differentiation. Several mechanisms have been cited to explain the keratinocyte proliferation characteristic of psoriasis. Disordered cellular immunity is also thought to be involved in the pathogenesis of psoriasis. Examples of psoriasis include chronic quiescent psoriasis, plaque psoriasis, moderate to severe plaque psoriasis, common psoriasis, eruptive psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or focal pustular psoriasis.

[0361] Ulcerative colitis

[0362] In some embodiments, this disclosure provides a method for treating a mammalian subject suffering from ulcerative colitis, the method comprising the step of administering to the subject a hIL10 reverse monomer or hIL10 reverse monomer dimer (or a pharmaceutical preparation comprising hIL10 reverse monomer or hIL10 reverse monomer dimer), the administration of which improves one or more symptoms of ulcerative colitis. In some embodiments, the method comprises administering to the subject a reverse monomer of formula 1 or 2 or a pharmaceutical preparation comprising a reverse monomer of formula 1 or 2, the administration of which improves one or more symptoms of ulcerative colitis. In one embodiment, this disclosure provides a method for treating a mammalian subject suffering from ulcerative colitis, the method comprising the step of administering to the subject an hIL10 reverse monomer or hIL10 reverse monomer dimer having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a polypeptide selected from the group consisting of: SEQ ID NO: 10, 11, 27, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 53, 108, 109, 110, 111, 112, 113, 114, 46, 47, 48, 49, 50, 51, 52, 53, 54, 121, 122, 123, 124, 125, and 126, wherein the administration improves one or more symptoms of ulcerative colitis.

[0363] IFNγ-induced anemia

[0364] In some embodiments, this disclosure provides a method for treating and / or preventing IFNγ-induced anemia in a subject. The method includes the step of administering a therapeutically effective amount of the disclosed fusion IL-10 dimer to the subject, wherein the administration improves symptoms of one or more IFNγ-induced anemias in the subject and / or reduces serum IFNγ levels. In some embodiments, the method includes administering a therapeutically effective amount of the reverse monomer of Formula 1 to the subject, wherein the administration improves symptoms of one or more IFNγ-induced anemias in the subject and / or reduces serum IFNγ levels. IFNγ-induced anemia is a common complication in patients with chronic inflammatory diseases. Chronic inflammation can be caused by a variety of diseases, such as infectious diseases, HIV / AIDS, malignancies, and / or autoimmune diseases, such as inflammatory bowel disease and rheumatoid arthritis. Activated T cells secrete IFNγ and interleukin-2 (IL-2). IFNγ has been observed to enhance the cytotoxic effects of TNFα. The compositions of this disclosure have been evaluated in a mouse model of IFNγ-induced anemia (Example 32). In one embodiment, this disclosure provides a method for treating and / or preventing IFNγ-induced anemia in a subject, the method comprising the step of administering to the subject an hIL10 reverse monomer or hIL10 reverse monomer dimer having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a polypeptide selected from the group consisting of: SEQ ID NO: 10, 11, 27, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 53, 108, 109, 110, 111, 112, 113, 114, 46, 47, 48, 49, 50, 51, 52, 53, 54, 121, 122, 123, 124, 125, and 126, wherein the administration improves one or more symptoms of IFNγ-induced anemia.

[0365] Macrophage activation syndrome

[0366] In some embodiments, this disclosure provides a method for treating and / or preventing macrophage activation syndrome (MAS) in a subject. The method includes administering to the subject a therapeutically effective amount of the reversed IL-10 monomer of Formula 1 or Formula 2 of this disclosure. Macrophage activation syndrome (MAS) is a potentially life-threatening complication of systemic inflammatory diseases, including but not limited to systemic juvenile idiopathic arthritis (sJIA), Kawasaki disease, systemic lupus erythematosus (SLE), and infections, particularly Epstein-Barr virus (EBV) infection, malignancies, and primary immunodeficiency. Elevated CD163 is associated with MAS. In some embodiments, this disclosure provides a method for treating and / or preventing macrophage activation syndrome (MAS) in a subject. The method includes administering to the subject a therapeutically effective amount of the fusion IL-10 dimer of this disclosure, wherein the administration improves one or more symptoms of macrophage activation syndrome. In one embodiment, this disclosure provides a method for treating and / or preventing macrophage activation syndrome in a subject, the method comprising the step of administering to the subject an hIL10 reverse monomer or hIL10 reverse monomer dimer having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a polypeptide selected from the group consisting of: SEQ ID NO: 10, 11, 27, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 53, 108, 109, 110, 111, 112, 113, 114, 46, 47, 48, 49, 50, 51, 52, 53, 54, 121, 122, 123, 124, 125, and 126, wherein the administration improves one or more symptoms of macrophage activation syndrome.

[0367] As previously mentioned, chronic inflammation is associated with the development of various cancers. See Coussens and Werb (2002). Nature 420: 860–867. Therefore, the reverse compositions of this disclosure can be used to treat and / or prevent cancers caused by chronic inflammation. In one embodiment, the cancer associated with chronic inflammation is selected from colon cancer, colorectal cancer, pancreatic cancer, and liver cancer. In one embodiment, this disclosure provides a method for preventing a human subject from developing cancers associated with chronic inflammation by administering a preventatively effective amount of the composition to a subject, the composition comprising a vector encoding the reverse hIL10 monomer (or a dimer thereof) of this disclosure or recombinant cells expressing the latter.

[0368] Methods for modulating IL10-mediated signal transduction

[0369] In another aspect, this disclosure provides a method for modulating IL10-mediated signaling in a subject. In some embodiments, the method includes administering an effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises the reverse monomer and / or its dimer as described herein, a nucleic acid molecule encoding the reverse monomer and / or its dimer, or a recombinant-modified cell comprising a nucleic acid molecule encoding the reverse monomer and / or its dimer. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0370] In some embodiments, methods for modulating IL10-mediated signaling in a subject include measuring STAT3-mediated signaling from one or more cells obtained from the subject. In some embodiments, STAT3-mediated signaling is measured by an assay selected from the group consisting of gene expression assays, phosphorylation signaling assays, and enzyme-linked immunosorbent assays (ELISA). In some embodiments, STAT3-mediated signaling in the subject is reduced by about 20% to about 100% compared to a reference level. In some embodiments, the administered composition results in a reduced ability in the subject to induce the expression of pro-inflammatory genes selected from the group consisting of IFN-γ, granzyme B, granzyme A, perforin, TNF-α, GM-CSF, and MIP-1α.

[0371] Reagent test kit

[0372] The present invention also provides a kit comprising the reverse monomers and / or their dimers disclosed herein. In some embodiments, the kit comprises one or more components for modulating IL-10-mediated signaling in a subject or treating a health condition in a subject of need, wherein said components are selected from the reverse monomers and / or their dimers described herein, nucleic acid molecules encoding the reverse monomers and / or their dimers described herein, recombinant modified cells comprising nucleic acid molecules encoding the reverse monomers and / or their dimers described herein, or pharmaceutical compositions comprising said one or more components. In some embodiments, the pharmaceutical composition of the kit comprises a pharmaceutically acceptable carrier.

[0373] Example

[0374] The following embodiments are provided to illustrate, but not to limit, the claimed invention.

[0375] Example 1: Method

[0376] This embodiment describes a method for preparing reverse monomers and dimers of the present invention and testing their biophysical properties.

[0377] Protein expression and purification

[0378] The protein-coding DNA sequence was inserted between the EcoRI and NotI restriction endonuclease sites of pEXSyn 2.0. The C-terminal His-tagged fusion peptide has a natural signal peptide for human molecules (DNA sequence: ATGCACAGCAGCGCCCTGCTGTGCTGCCTGGTGCTGCTGACCGGCGTGAGAGCC (SEQ ID NO:85); amino acid sequence: MHSSALLCCLVLLTGVRA (SEQ ID NO:86)) or a mouse Ig heavy chain signal peptide for mouse molecules (DNA sequence: ATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCCACCGGTGT GCACAGC (SEQ ID NO:88); amino acid sequence: MGWSCIILFLVATATGVHS (SEQ ID NO:89)). The nucleic acid and amino acid sequences of the human and mouse inverse monomers and dimers are shown in Table 5-12.

[0379] Proteins were expressed in expi293 cells via transient transfection. The C-terminal His-tagged peptide was purified from the collected cell culture supernatant using nickel affinity chromatography. The culture supernatant was supplemented with 5 mM imidazole, and purification was performed on a gravity column containing Ni-Excel (Cytiva) resin, followed by equilibration with PBS containing 5 mM imidazole. Washing and elution were performed in PBS supplemented with 5 mM and 250 mM imidazole, respectively. The protein was dialyzed in PBS buffer before storage and testing. Protein purity and quality were assessed using SD-PAGE, LCMS, and analytical SEC.

[0380] Protein PEGylation

[0381] The purified protein was concentrated to 2–3 mg / ml in 10k Amicon (Millipore) in 100 mM sodium phosphate buffer (pH 6.3, 100 mM NaCl). The N-terminus of the protein was PEGylated via aldehyde chemistry, using 20 kDa linear PEG (Sunbright® ME-200AL, NOF) for native IL10 and 40 kDa branched PEG (Sunbright® GL2-400AL3) for the reverse monomer. The PEGylation reaction was carried out at room temperature with end-to-end rotation for 24–48 hours, followed by the addition of a 5-fold molar excess of PEG relative to the protein and 10 mM sodium cyanoborohydride (NaCNBH3, Sigma). The PEGylation process was evaluated by analytical size exclusion chromatography (SEC). The PEGylated reaction mixture was diluted 10-fold and purified using a cation exchange column (SP HP, Topvan) with stepwise elution in 20 mM sodium phosphate buffer (pH 6.3, 0.7 M NaCl) to separate the protein from free polyethylene glycol (PEG) and sodium cyanoborohydride (NaCNBH3). The eluted protein contained a mixture of unPEGylated, mono-PEGylated, and poly-PEGylated proteins, which were then purified by size exclusion chromatography using a Superose 6 increase column (Topvan) equilibrated with phosphate-buffered saline (PBS). The elution peaks containing PEGylated proteins were collected, concentrated to 0.5–2 mg / ml, and sterilely filtered before use.

[0382] Biacore bonding

[0383] All experiments were performed on a Biacore T200 instrument equipped with an anti-human capture (AHC)-CM5 chip (Stenford) in 10 mM Hepes, 150 mM NaCl, 0.05% (v / v) polysorbate 20 (PS20) and 3 mM EDTA (HBS-EP+ buffer).

[0384] Human or mouse IL10RαFc (human: R&D Systems, Serial No. 9044-RI; mouse: Sino Biological, Serial No. 51298-M02H) were captured on the AHC surface. Since the IL10 analyte may be divalent, the ligand capture density was set to 12–25 RU to limit R... max <8.5 RU (determined in a separate experiment) was used to measure binding kinetics under non-avid conditions. IL10 analytes were injected at 0.74, 2.22, 6.66, 20, 60, and 180 nM in high-efficiency mode (binding for 90 seconds, dissociation for 360 seconds), followed by surface regeneration with 3M MgCl2 (40 seconds, 30 μL / min). Sensing plots with buffer subtraction were processed using Biacore T200 evaluation software, and a 1:1 Langmuir binding model was used for global fitting to extract kinetic and affinity constants (ka, kd, KD). max The range of 4 to 8.5 RU indicates that the surface density is sufficient to support real-world binding kinetic measurements in the absence of affinity.

[0385] Since the affinity of IL10 for IL10Rβ is expected to be weak (μM to mM) and cannot be detected under these conditions, binding to human or mouse IL10Rβ has not been reported.

[0386] Compared to IL10 WT, the affinity K of the reverse monomer for IL10Ra is... D It has been reduced by 3-6 times. See Tables 3 and 4 below.

[0387]

[0388] Thermal stability measurement

[0389] Proteins were prepared into PBS solutions with a concentration of approximately 1 mg / mL and filtered through a 0.1 μm filter to remove aggregates. Each protein was loaded into four capillary tubes, and dynamic light scattering (DLS) and turbidity were measured using a Prometheus Panta instrument at a rate of 1 °C / min from 25 °C to 95 °C. Due to the lack of tryptophan in the IL10 molecule, resulting in a weak signal, the unfolded data obtained by differential scanning fluorometry were not analyzed. The data indicate that the engineered molecules have equivalent thermal stability to IL10 WT, with an aggregation initiation temperature (DLS) of approximately 52 °C. Data are shown in... Figure 1 .

[0390] Example 2. STAT3 Activity Assessment

[0391] This embodiment describes the method and results for testing the peptide activity of this disclosure.

[0392] pSTAT3 flow cytometry assay: PBMCs were purified from blood collected in a leukocyte removal system (LRS) chamber using the Miltenyi MACSprep™ PBMC Isolation Kit. Purified PBMCs (500,000 cells / well) were stimulated for 20 min at 37°C with the IL-10 proteins h_DR1061_AA (SEQ ID NO:108) and h_SM0043_AA at concentrations ranging from 0.1 pM to 100 nM. h_SM0043_AA is the wild-type human IL-10 sequence expressed in *E. coli*, with the following amino acid sequence: MSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 163).

[0393] Cells were fixed at 37°C for 15 minutes with BD Phosflow™ Fix Buffer I (BD Biosciences, catalog number 557870). Cells were then washed and permeabilized overnight at -20°C with pre-chilled BD Phosflow™ Perm Buffer III (catalog number 558050). Cells were washed to remove the permeabilization buffer and blocked at room temperature for 5 minutes with human Trustain Fcx (Biolegend, catalog number 422301) and mouse serum. Cells were then treated with an antibody mixture (see Table 14 below) at room temperature for 1 hour.

[0394]

[0395] After antibody staining, cells were washed, fixed, and analyzed using a Cytek® Aurora Spectral flow cytometer. Data were analyzed using FlowJo software (www.flowjo.com). Different cell lineages were gated using lineage markers, and the geometric mean fluorescence intensity of pSTAT3 expression was calculated in FlowJo. Data are shown below. Figure 2A and 2B The results showed that, compared with wild-type hIL10 molecule (h_SM0043_AA), reverse monomeric IL10 molecule (h_DR1060_AA) exhibited reduced pSTAT3 signaling in monocytes and CD8 T cells.

[0396] Example 3. Monocyte IL10 activity assay: Human PBMCs were isolated from the LRS chamber of non-smoking donors using Ficoll® gradient purification. Human monocytes were purified by positive selection of PBMCs using human CD14 microbeads (Miltenyi Biotech, catalog number 130-050-201) according to the manufacturer's protocol. Monocytes were treated with 1 ng / ml lipopolysaccharide (LPS) (Millipore Sigma, catalog number L2630) at 37°C for 48 h in the presence of varying concentrations of IL-10 protein (0.05 pM to 200 nM). After treatment, cells were centrifuged at 400 g for 5 min, and the supernatant was collected. Pro-inflammatory cytokines in the supernatant were detected using the MSD kit (Meso Scale Discovery, catalog number K151A9H-4), largely according to the manufacturer's protocol. Data were plotted using Prism software (www.prismsoftware.com). Data are shown in... Figure 3 . Data showed that the reverse monomeric IL-10 molecule (h_DR1061_AA) inhibited LPS-induced IL-1β secretion in monocytes, although its potency was 150 times lower than that of the natural dimeric IL-10 molecule (h_SM0043_AA).

[0397] Example 4. CD8 T cell blasts IL10 activity assay: Human PBMCs were isolated from LRS chambers obtained from non-smoking donors using Ficoll gradient purification. Human CD8 T cells were purified from PBMCs using a CD8+ T cell isolation kit (Medrin Biotechnology, Catalog No. 130-096-495), largely following the manufacturer's protocol, via negative selection. The isolated CD8 T cells were activated for 72 hours at 37°C in the presence of 100 pM human IL-2 using a human T cell activation / expansion kit (Medrin Biotechnology, Catalog No. 130-091-441). The activated CD8 T cells were washed, and magnetic beads were removed by magnetic force. The cells were then treated with varying concentrations of IL-10 protein (0.05 pM to 200 nM) at 37°C for 72 hours. After treatment, the cells were centrifuged at 400 g for 5 minutes, and the supernatant was collected. Using the MSD kit (Meso Scale Discovery, catalog number K151AEL-4), the cytokines IFNγ, granzyme A, and granzyme B in the supernatant were detected, largely following the manufacturer's protocol. Data were plotted using Prism software and are shown below. Figures 4A-4C .

[0398] Data indicate that, compared to the natural hIL10 dimer (h-SM0043_AA), the reverse monomeric IL10 molecule (h_DR1061_AA) does not induce activated CD8 T cells to secrete IFN-γ, granzyme A, or granzyme B.

[0399] Example 5. Mouse pSTAT3 flow cytometry assay: Spleens were harvested from naïve C57BL / 6 mice. The spleens were placed in complete RPMI medium (Gibco RPMI 1640 + 10% FBS + 1% penicillin-streptomycin), and spleen cells were isolated by squeezing with the syringe plunger. The cells were then filtered through a 70 µM sieve. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 10 ml of ammonium chloride-potassium chloride (ACK) lysis buffer (ThermoFisher) and incubated at 37°C for 2 min. After washing the cells, they were resuspended in complete RPMI medium. The purified spleen cells (500,000 cells / well) were stimulated with IL-10 protein at concentrations ranging from 0.1 pM to 100 nM at 37°C for 20 min. The cells were then fixed with BD Phosflow™ Fix Buffer I (BD Biosciences, catalog number 557870) at 37°C for 15 min. Cells were then washed and permeabilized overnight at -20°C with pre-chilled BD Phosflow™ PermBuffer III (BD Biosciences, catalog number 558050). Cells were washed again to remove the permeabilization buffer and blocked for 5 minutes at room temperature with Mouse Trustain Fcx (Byrlazen, catalog number 101320). Cells were then treated with an antibody mixture (see Table 15 below) at room temperature for 1 hour.

[0400]

[0401] After antibody staining, cells were washed, fixed, and analyzed on a Cytek® Aurora Spectral flow cytometer. Data were analyzed using FlowJo software. Lineage markers were used to gate different cell lineages, and the geometric mean fluorescence intensity of pSTAT3 expression was calculated in FlowJo. Mouse pSTAT3 flow cytometry data are shown below. Figure 5A and 5B As shown.

[0402] Data showed that, compared with the natural dimer mIL-10 (m_DR768_AA), the mouse reverse monomeric IL-10 molecule (m_WC161_AA) had a greater effect on pSTAT3-induced ECGs in mouse myeloid cells and CD8 T cells. 50 Lower.

[0403] Example 6. Mouse spleen cell LPS test: Spleens were harvested from naïve C57BL / 6 mice. The spleens were placed in complete RPMI medium (Gibco RPMI 1640 medium + 10% FBS + 1% penicillin-streptomycin), and the spleen cells were isolated by squeezing with the plunger of a syringe and filtered through a 70 µM sieve. The cells were centrifuged at 400g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 10 ml ACK lysis buffer and incubated at 37°C for 2 minutes. After washing the cells, they were resuspended in complete RPMI medium. The purified spleen cells (500,000 cells / well) were treated with 1 ng / ml LPS (Millibos Sigma, catalog number L2630) at 37°C for 48 hours in the presence of different concentrations of IL-10 protein (0.1 pM to 100 nM). After treatment, the cells were centrifuged at 400g for 5 minutes, and the supernatant was collected. The pro-inflammatory cytokines IL-6 and TNF-α in the supernatant were detected using the MSD kit (Meso Scale Discovery, catalog number K152A0H-2), largely following the manufacturer's protocol. Prism software was used to plot the results as shown below. Figure 6A and 6B The data shown in the figure indicate that the reverse monomeric mouse IL-10 molecule (m_WC161_AA) inhibits the secretion of IL-6 and TNF-α by mouse spleen cells, but its efficacy is lower than that of the natural dimer IL-10 molecule (m_DR768_AA).

[0404] Example 7. Mouse CD8 T cell blast cell assay

[0405] Spleens were harvested from naïve C57BL / 6 mice. The spleens were placed in complete RPMI medium (Gibco RPMI 1640 + 10% FBS + 1% penicillin-streptomycin), and spleen cells were isolated by squeezing with the syringe plunger. The cells were then filtered through a 70 µM sieve. The cells were centrifuged at 400g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 10 ml ACK lysis buffer and incubated at 37°C for 2 minutes. After washing the cells, they were resuspended in complete RPMI medium. Mouse CD8 T cells were purified by negative selection of PBMCs using a kit (Medlani Biotechnology, Catalog No. 130-104-075) according to the manufacturer's protocol. The isolated CD8 T cells were activated at 37°C for 72 hours in the presence of 100 pM mouse IL-2 using a mouse T cell activation and expansion kit (Medlani Biotechnology, Catalog No. 130-093-627). Activated CD8 T cells were washed, magnetic beads were removed using a magnetic method, and then treated with different concentrations of IL-10 protein (0.1 pM to 100 nM) at 37°C for 72 hours in the presence of 100 pM mouse IL-2. After treatment, the cells were centrifuged at 400 g for 5 minutes, and the supernatant was collected. Cytokines (granzyme B) in the supernatant were detected using the MSD kit (Meso Scale Discovery, catalog number K151A9H-2), largely following the manufacturer's protocol. Data were plotted using Prism software. Figure 7 Data from a mouse CD8 T cell blast cell assay are shown.

[0406] Data showed that, compared with the natural dimer mIL-10 molecule (m_DR756_AA), the mouse monomeric IL10 molecule (m_WC161_AA) could not induce activated CD8+ T cells to secrete granzyme B.

[0407] Example 8. Mouse CD8 T cell blast cell survival assay

[0408] Spleens were harvested from naïve C57BL / 6 mice. The spleens were placed in complete RPMI medium (RPMI + 10% FBS + 1% penicillin-streptomycin), and the spleen cells were isolated by squeezing with the syringe plunger and filtered through a 70 µM sieve. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 10 ml ACK lysis buffer and incubated at 37°C for 2 min. After washing the cells, they were resuspended in complete RPMI medium. Mouse CD8 T cells were purified by negative selection of PBMCs using a kit (Mitengene Biotech, catalog number 130-104-075), essentially following the manufacturer's protocol. 200,000 CD8 T cells were added to each well of a 96-well plate pre-coated with 2.5 µg / ml anti-mouse CD3ε antibody (Byrlazen 100302). Cells were then treated with IL-10 protein at concentrations ranging from 0.1 pM to 100 nM in the presence of 5 µg / ml anti-mouse CD28 antibody (Byrlazen 102102) at 37°C for 72 hours. Cells were then centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Cells were stained with annexin V and propidium iodide using a cell death and apoptosis detection kit for flow cytometry (Ingenieur, catalog number V13241), basically following the manufacturer's instructions. Data were analyzed using FlowJo software. After staining, cells were analyzed on a Cytek Aurora Spectral flow cytometer. Data were analyzed using FlowJo software. Plots were generated using Prism software as shown below. Figure 8 The data chart shown.

[0409] Data showed that, compared with the natural dimer mIL-10 molecule (m_DR756_AA), the mouse reverse monomeric IL-10 molecule (m_WC161_AA) did not enhance the survival of activated mouse CD8+ T cells.

[0410] Example 9. Serum protein concentration:

[0411] IL-10 protein, prepared substantially according to the method described in Example 1, was subcutaneously injected at a specified dose into 10-week-old female C57BL / 6J mice (RRID:IMSR_JAX:000664). Blood was collected via the submandibular vein, and serum was prepared using serum microcentrifuge tubes (Sarstedt, product number 41.1378.005). Serum IL-10 concentrations were determined using an MSD kit (Meso Scale Discovery, K15069M). Concentrations were interpolated using a four-parameter (4PL) model in GraphPad Prism 9.3.1 software, and the results are shown below. Figure 9 As shown.

[0412] Data showed that within 72 hours after a single injection, the serum concentration of PEGylated mouse monomeric IL-10 molecules (m_WC161_AAPEG) was higher than that of natural dimer IL-10 molecules (m_DR756_AA_PEG).

[0413] Example 10. CD64 staining on the surface of monocytes : Human PBMCs were isolated from the LRS chamber of non-smoking donors using Ficoll gradient purification. Human monocytes were purified by positive selection of PBMCs using human CD14 microbeads (Medrin Biotechnology, No. 130-050-201) according to the manufacturer's protocol. Monocytes were treated with different concentrations of the fusion IL-10 peptide (0.1 pM - 100 nM) at 37 °C for 48 h. After treatment, cells were centrifuged at 400 g for 5 min. Cells were then washed and blocked at 4 °C for 15 min with human Trustain Fcx (Byrlazen, No. 422301) and mouse serum. Subsequently, cells were treated with CD64 antibody (Byrlazen, No. 305006) at 4 °C for 30 min. After antibody staining, cells were washed, fixed, and analyzed on a Cytek AuroraSpectral flow cytometer. Data were analyzed using FlowJo software. Plotting was performed using Prism software. Figure 10 The data chart shown.

[0414] Data showed that the reverse monomer (h_DR1061_AA) induced much lower CD64 expression on the surface of monocytes compared to the natural dimer IL-10 molecule (h_SM0043_AA).

[0415] Example 11: Evaluation of reverse monomers in pSTAT3 flow cytometry assay: Using the Miltenyi MACSprep PBMC Isolation Kit (Miltenyi Biotechnology, San Diego, CA), PBMCs were purified from healthy non-smoking donor blood collected in a leukocyte removal system (LRS), largely following the manufacturer's instructions. The purified PBMCs (500,000 cells / well) were stimulated with IL-10 protein at concentrations ranging from 0.1 pM to 100 nM at 37°C for 20 min. Cells were then fixed with BD fix buffer I (BD Biosciences, Catalog No. 557870) at 37°C for 15 min. Cells were then washed and permeabilized overnight with pre-chilled BD perm buffer III (BD Biosciences, Catalog No. 558050) at -20°C. Cells were washed to remove the permeabilization buffer and blocked for 5 min at room temperature with human Trustain Fcx (Bylazin, Catalog No. 422301) and mouse serum. Cells were then treated with the antibody mixture provided in Table 16 below at room temperature for 1 hour.

[0416]

[0417] After antibody staining, cells were washed, fixed, and analyzed on a Cytek Aurora Spectral flow cytometer (Cytek Biosciences). Data were analyzed using FlowJo software (BD Biosciences, www.flowjo.com). Different cell lineages were gated using lineage markers, and the geometric mean fluorescence intensity of pSTAT3 expression was calculated using FlowJo. The data from this experiment are shown in Figures 12A and 12B. As shown in Figures 12A and 12B, monomeric IL10 molecules (h_DR1060_AA and h_DR1061_AA) exhibited reduced pSTAT3 signaling in monocytes and CD8 T cells compared to the native dimer IL10 molecule (h_DR757_AA).

[0418] Example 12. In vivo evaluation of a DSS model of ulcerative colitis

[0419] Evaluation of the therapeutic effect of the IL10 variant disclosed herein in a mouse DSS model of ulcerative colitis. Mouse DSS-induced colitis models are widely used and share many similarities with human ulcerative colitis. Chassaing, B., et al. (2014) Current Protocols in Immunology 104:15.25.1-15.25.14.

[0420] The typical sequence of a mature (excluding the endogenous 18-amino acid signal peptide) wild-type mouse IL10 molecule (UniProt reference number P18893) is a 160-amino acid polypeptide with the following amino acid sequence: SRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMK (SEQ ID NO: 141).

[0421] Refer to SEQ ID NO: 141 when referring to amino acid substitutions or deletions in the mouse IL10 variant molecule described herein.

[0422] Table 17 below describes the PEGylated mouse IL10 (mIL10) assays used in the DSS study.

[0423]

[0424] Table 18 below lists the amino acid sequences of the parent peptides of the m_DR756_AA_PEG and m_WC161_AA_PEG test samples:

[0425] In summary, the DSS study was conducted as follows. Female C57Bl / 6 mice (Jackson Laboratories) had free access to drinking water containing 2.5% DSS from day 0 to day 6 of the study. Mice that drank plain water served as negative disease controls. Mice were treated with PBS, m_DR756_AA_PEG (3 or 10 μg subcutaneously every other day), or m_WC161_AA_PEG (10 or 30 μg subcutaneously every other day), according to Table 19 below.

[0426]

[0427] Throughout the study, the mice's weight was assessed. Mice were weighed three days before the start of DSS (day -3 of the study) and after treatment with DSS and the test drug. Weight data for each treatment group are attached. Figure 13The figure also shows mean body weight and mean body weight percentage + / - SEM. The body weight percentage was calculated as: (body weight on day X of the study – baseline body weight on day -3 of the study) / (baseline body weight on day -3 of the study). Final body weight of mice was assessed on day 15 of the study; the mean body weight percentage + / - SEM on day 15 is attached. Figure 14 One-way ANOVA and Holm- were used to analyze the variance. ídák Multiple comparison tests were used to determine significance. p<0.0001.

[0428] On day 15 of the study, terminal blood was collected via cardiac puncture and deposited into serum collection tubes. The blood clot was centrifuged at 10,000 RPM for 5 minutes to separate antenatal and terminal serum. The serum was collected after centrifugation, temporarily placed on ice, and then frozen for later analysis. Serum cytokines were assessed using the Meso Scale Discovery multiplex assay kit, largely following the manufacturer's instructions.

[0429] On day 15 of the experiment, the mice were euthanized by carbon dioxide asphyxiation. The colons were harvested, feces were removed, and their length was measured. Figure 15 The data shows the changes in colon length across different treatment groups. The figure displays the mean colon length plus / minus SEM. Figure 15 As shown, m_DR756_AA_PEG, m_DR2677_AA_PEG and m_DR2678_AA_PEG exhibit protective effects against DSS-related lesions and pathology.

[0430] Hematocrit levels were assessed on day 4 (D4) of the study. Blood samples collected via the submandibular vein on day 4 were placed in serum collection tubes and quantified using a Hemata Stat II hematocrit microcentrifuge (EKF Diagnostics, Inc.) following the manufacturer's instructions. Relevant data on hematocrit levels are attached. Figure 16 The figure shows the mean hematocrit + / - standard error (SEM). As shown in the figure, a putative decrease in HCT may be observed on day 4 using m_DR756_AA_PEG.

[0431] Example 13. Assessment of the time-dependent expression changes of peritoneal exudate cell clearance factors CD164 and CD64.

[0432] For the collection of peritoneal exudate cells, peritoneal lavage was performed on euthanized mice, and exudate (peritoneal exudate cells, PEC) was collected. The peritoneal lavage fluid was centrifuged (1500 rpm, 5 min), the supernatant was removed, and the cell pellet was resuspended in 1 ml PBS and placed on ice. Blood was collected in EDTA-containing tubes, transferred to 5 ml centrifuge tubes, and erythrocytes were lysed using ACK lysis buffer (ThernoFisher #A1059201), followed by centrifugation (1500 rpm, 5 min). The supernatant was removed, and the cell pellet was resuspended in 1 ml PBS. Peritoneal exudate cells were collected over time, and CD163 or CD64 were detected by flow cytometry. Single and mean median fluorescence intensity + / - SEM images are also shown. APC = antigen-presenting cells. Figure 17 As shown, treatment with PEGylated wild-type mouse IL-10 substitute molecule (m_DR756_AA_PEG) upregulated CD163 expression in macrophages. CD64 expression was also evaluated, and the results are as follows: Figure 18 As shown.

[0433] Example 14. Assessment of the time-dependent expression changes of peritoneal exudate cell clearance factors CD164 and CD64.

[0434] Serum was collected at different time points during the study, and serum cytokines (including IFNγ, IL-10, IL-1β, IL-6, TNF-α, IL-4, IL-5, IP10, IL-17A / F, and MIP1α) were measured. Serum cytokines were assessed using the MesoScale Discovery multiplex assay kit, largely following the manufacturer's instructions. Data for each treatment group are shown in the attached figure. Figure 19 and Figure 20 The figure shows the average analyte concentration (pg / mL) + / - SEM. For example... Figure 19 and Figure 20 As shown, treatment with the reverse monomer (m_WC161_AA_PEG) was generally associated with decreased systemic IFNγ and TNFα levels compared to wild-type molecule (m_DR756_AA_PEG). Treatment with m_DR756_AA_PEG was associated with increased serum IP-10 and MIP1a. Treatment with the reverse monomer (m_WC161_AA_PEG) was associated with increased serum IL-4 and IL-5 (Th2 cytokines).

[0435] After assessing the colon length, the dissected colon was placed in formalin solution and incubated at room temperature for 24 hours. It was then processed using a tissue processor according to standard procedures and embedded into FFPE blocks. The FFPE blocks were cut into 5 μm thick sections and placed on charged glass slides.

[0436] Multiplex immunofluorescence analysis was performed on the collected tissues to detect the presence of the following markers: RORγ, CD11b, and CD3e. The primary antibodies used for immunohistochemical analysis were: anti-mouse RORγ antibody (Abcam, catalog number ab207802), working concentration 1:750; anti-CD11b antibody (Abcam, catalog number ab216445), working concentration 1:3000; and anti-CD3e antibody (Thermo / Invitrogen, catalog number MA1-90582), working concentration 1:200. All antibodies were incubated at room temperature for 60 minutes.

[0437] Antibody staining was performed on the slides using the Leica Bond Rx (Leica Biosystems) fully automated staining system. The automated staining system program is set to perform the following steps: (a) In BOND-PRIME Epitope repair Liquid 2 (a) Heat-induced epitope repair (HIER) and dewaxing were performed in the presence of (ER2) (Leica Biosystems) at 97°C for 20 min; (b) peroxidase quenching was performed; (c) incubation with primary antibody for 60 min; (d) incubation with secondary antibody polymer for 30 min; and (e) detection with fluorescent dyes at room temperature for 10 min. Fluorescent dyes used to display the signal included Opal 520, Opal 620, and Opal 690 fluorophores (Akoya Biosciences, Marlborough, MA).

[0438] For labeling, the staining steps are performed sequentially, with a second antigen retrieval step inserted between the two staining steps to utilize BOND-PRIME. Epitope Repair Solution 1 (ER1) (Leica Biosystems) removes any unbound reagents from previous markers. Appropriate cross-reactivity controls are included for quality control.

[0439] After fluorescence staining, tissues were counterstained with Akoya Spectroscopy DAPI (Akoya Biosciences) and mounted with ProLong™ Gold anti-fading mounting medium (ThermoFisher Scientific / Invitrogen, catalog number: P36930) aqueous mounting medium. Whole-slide scanning and signal quantification were performed using the Akoya Vectra multispectral imaging system (Akoya Biosciences) and Akoya Vectra InForm analysis software (Akoya Biosciences). Specific staining signals were digitally quantified, and data from all images were processed and summarized using the Akoya Phenoptr Reports software suite (Akoya Biosciences).

[0440] Colonic epithelial damage was assessed in the tissues. Whole sections of mouse colonic tissue stained with hematoxylin and eosin (H&E) were evaluated using Visiopharm software (Visiopharm A / S, DK 2970-Hørsholm, Denmark) to determine the presence of an intact epithelial layer. Data for each treatment group are shown in the attached figure. Figure 21 The figure shows the average percentage of colonic epithelial damage + / - SEM.

[0441] Whole-section mouse colon tissue samples stained with multiplex immunofluorescence were evaluated using inForm image analysis software (Akoya Biosciences) to determine the presence of CD11b and CD4+ RorG T cells. Data for each treatment group are shown in the attached figure. Figure 22 The figure shows the average cell count / mm² + / - SEM.

[0442] In summary, the data obtained in the ulcerative colitis DSS model indicate that the reverse monomer (m_WC161_AA_PEG) exhibits: (a) a protective effect against DSS-related outcomes and pathology; (b) a protective effect against DSS-related epithelial damage; and (c) a reduction in myeloid and Th17 infiltration in the intestinal mucosa.

[0443] Example 15: In vivo assessment of pharmacokinetic parameters:

[0444] Pharmacokinetic parameters of the m_DR756_AA_PEG and m_WC161_AA_PEG analytes were evaluated in an in vivo study in C57Bl / 6 mice. Briefly, on study day 0 (D0), female C57Bl / 6 mice received a single subcutaneous dose of 10 μg of m_DR756_AA_PEG, or a single subcutaneous dose of 10, 30, or 90 μg of m_WC161_AA_PEG. Pre-mortem serum samples were collected via submandibular vein collection tubes at different time points. Terminal blood samples were harvested via cardiac puncture and collected in serum collection tubes. The blood clots were centrifuged at 10,000 RPM for 5 min to separate pre-mortem and terminal serum. The serum was collected after centrifugation, temporarily placed on ice, and then frozen for subsequent analysis. Quantitative analysis of the analytes in the serum was performed using the Mouse IL-10MSD U-plex Kit (Meso Scale Discovery), largely following the manufacturer's instructions. Each analyte was used as a detection standard.

[0445] Serum was collected over time, and the serum concentration of the test substance was quantified over time; see attached figure. Figure 23 The data provided reflects the average concentration (ng / mL) of the test substance + / - SEM. For example... Figure 23As shown, m_WC161_AA_PEG exhibited higher systemic exposure compared to m_DR756_AA_PEG. Furthermore, the reverse monomer (m_WC161_AA_PEG) maintained high sustained exposure even after multiple doses.

[0446] Example 16: Assessment of the time-dependent changes in pSTAT3 induction in myeloid cells

[0447] The ability of the test substance to induce pSTAT3 in myeloid cells was evaluated as follows. Pre-mortem or terminal whole blood from the experiments described in the above examples was collected in EDTA tubes for pSTAT or surface immunophenotyping flow cytometry assays. 100–200 μL of whole blood collected in the EDTA tubes was transferred to 96-well deep-well plates. BD Phosflow lysis / fixation buffer (catalog number 558049) was added to a total volume of 2 ml, and the plates were incubated at 37°C for 10 min. Cells were centrifuged at 600 g for 7 min, the supernatant was removed, and the cells were washed twice with 1 ml PBS / 2% FBS per well. The cell pellet was vortexed and resuspended, 1 ml of pre-chilled BDPerm Buffer III (catalog number 558050) was added, and the cells were stored at -80°C. After pre-mortem sample collection, the cells were thawed and washed three times to remove permeabilization buffer. Cells were blocked on ice for 10 min with 1:25 TruStain FcX (Byrlazen #101320) and 1:50 rat serum, and then incubated with the antibody mixture at 4°C for 30 min (see Table 12 below). Cells were washed twice and resuspended in PBS / 2% FBS, and then analyzed on a Cytek Aurora Spectral flow cytometer. Data were analyzed using Cell Engine software. Cells were gated using lineage markers, and the median fluorescence intensity of pSTAT1 and pSTAT3 was calculated.

[0448]

[0449] To assess the changes in pSTAT3 induction in myeloid cells over time, peripheral blood samples were collected over time, and pSTAT3 levels were detected by flow cytometry. The data are shown in the attached figure. Figure 24 Provided in [the text]. For example... Figure 24 As shown, the reverse monomer (m_WC161_AA_PEG) showed signs of pSTAT3 induction in PBMCs at 48 and 72 hours post-dose.

[0450] sequence

[0451] The nucleic acid sequences of the reverse monomers in the coding table 6 are shown in Table 5 below.

[0452]

[0453] Table 7. Human dimerized reverse monomer nucleic acid sequences

[0454] Table 8. Amino acid sequences of human dimerized reverse monomers

[0455] Table 9. Mouse reversed monomeric nucleic acid sequences

[0456] Table 10. Mouse reverse monomer amino acid sequence

[0457] Table 10B provides other mouse inverse dimers of this disclosure. All substitutions mentioned in the specification are numbered according to the following mature mIL10 typical reference sequence: SRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS (SEQ ID NO: 141).

[0458] The binding specificity of the mouse reverse monomers provided in Table 10A to the mIL10Ra receptor subunits coupled to each arm of the immobilized Fc was confirmed by surface plasmon resonance (SPR), and the results are substantially consistent with the teachings of the examples and the data in Table 10A. As discussed in the examples, in the absence of IL10, the affinity of IL10 for the IL10Rb subunit is very low and cannot be measured by SPR. Therefore, we used ELISA to assess the binding of the molecules to IL10Ra and IL10Rb. The results showed that some of these reverse monomers had a higher binding affinity to the IL10Ra receptor than the WC131 control molecule described below. Based on the sequence homology of the mouse and human molecules, the amino acid substitutions in the mouse reverse monomers described in Tables 10 and 10B can be incorporated into the human reverse monomer of Formula 1. In some embodiments, this disclosure provides a human reverse monomer in which the positions substituted in the homologous mouse reverse monomers in Tables 10 or 10B contain one or more amino acid substitutions.

[0459]

[0460] Table 11. Mouse dimerized reverse monomeric nucleic acid sequences

[0461] Table 12. Amino acid sequences of mouse dimerized reverse monomers

Claims

1. A polypeptide comprising the amino acid sequence of Formula 1: [A]-L1x-[B]-L2-[C]-L3-[D]-L4y-[E]-L5-[F] (1) in: x and y are independently selected from 0 (not present) or 1 (present); [A] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence SKAVEQVKNAFNKL (SEQ ID NO:1); x = 0 (no connector, L1 is missing); or x = 1, and L1 contains a polypeptide linker consisting of 1 to 5 amino acids; [B] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence "EKGIYKAMSEFDIFINYIEAYMTMKIR" (SEQ ID NO:2); L2 contains a linker consisting of 10 to 25 amino acids; [C] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence "NLPNMLRDLRDAFSRVKTFFQMKD" (SEQ ID NO:3); L3 contains a linker consisting of 4 to 11 amino acids; [D] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence "KESLLEDFKG" (SEQ ID NO:4); y = 0 (L4 is missing), or y = 1, and L4 contains a polypeptide linker consisting of 1 to 5 amino acids; [E] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence "LGCQALSEMIQFYLEEVMPQAEN" (SEQ ID NO:5); L5 contains a linker consisting of 1 to 7 amino acids; and [F] is a polypeptide with 0, 1 or 2 amino acid substitutions relative to the amino acid sequence "IKAHVNSLGENLKTLLRLRLRRC" (SEQ ID NO:6).

2. The polypeptide of claim 1, wherein L1, L2, L3, L4 and / or L5 comprise a GS linker.

3. The polypeptide of claim 1 or 2, wherein L1 is the amino acid glutamine (Q).

4. The polypeptide according to any one of claims 1 to 3, wherein L2 is a polypeptide having the amino acid sequence NSPGQGTQSENSCTHFPG (SEQ ID NO:7) or NTSPGQGTQSENSCTHFPG (SEQ ID NO:23).

5. The polypeptide according to any one of claims 1 to 4, wherein L3 is a polypeptide having the amino acid sequence QLDNLLL (SEQ ID NO: 8).

6. The polypeptide according to any one of claims 1 to 5, wherein L4 is the amino acid glycine ("G") or contains the amino acid sequence GY.

7. The polypeptide according to any one of claims 1 to 6, wherein L5 is a polypeptide having the amino acid sequence QDPD (SEQ ID NO: 9).

8. The polypeptide of any one of claims 1 to 7, wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:10 or SEQ ID NO:

11.

9. The polypeptide of claim 8, wherein the polypeptide comprises an amino acid sequence. KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCEN (SEQ ID NO: 10).

10. The polypeptide of claim 8, wherein the polypeptide comprises an amino acid sequence KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNTSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCEN (SEQ ID NO: 11).

11. The polypeptide of any one of claims 1 to 8, wherein the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with the hIL10 reverse monomer selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO:

98.

12. A polypeptide comprising formula (2): [Single Unit 1] - Connector x -[Single 2] (2) in, Monomer 1 and monomer 2 each independently comprise a polypeptide selected from the polypeptide of claim 1, and x = 0 (linker not present) or 1 (linker present).

13. The polypeptide of claim 12, wherein monomer 1 and monomer 2 each independently comprise a polypeptide, said polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:

98.

14. The polypeptide of claim 13, wherein monomer 1 and monomer 2 each comprise a polypeptide, the polypeptide comprising the amino acid sequence of SEQ ID NO:

10.

15. The polypeptide of claim 13, wherein monomer 1 and monomer 2 each comprise a polypeptide, said polypeptide comprising the amino acid sequence of SEQ ID NO:

11.

16. The polypeptide of any one of claims 12 to 15, wherein x is 1 (a linker is present), and the linker includes a GS linker.

17. The polypeptide of any one of claims 12 to 15, wherein x is 0 (the linker does not exist).

18. The polypeptide of claim 12, wherein the polypeptide of formula (2) comprises, in the following order from amino to carboxyl groups: (a) Monomer 1, which comprises: (i) A first polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 of human IL-10 (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector x Where x = 0 (connector does not exist) or 1 (connector exists); and (iii) A second polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-160 of human IL-10 (SEQ ID NO:20); amino acid residues 2-160 of human IL-10 (SEQ ID NO:36); amino acid residues 3-160 of human IL-10 (SEQ ID NO:37); amino acid residues 4-160 of human IL-10 (SEQ ID NO:38); amino acid residues 5-160 of human IL-10 (SEQ ID NO:39); amino acid residues 6-160 of human IL-10 (SEQ ID NO:40); amino acid residues 7-160 of human IL-10 (SEQ ID NO:41); amino acid residues 8-160 of human IL-10 (SEQ ID NO:41); amino acid residues 8-160 of human IL-10 (SEQ ID NO:39); amino acid residues 6-160 of human IL-10 (SEQ ID NO:40); amino acid residues 7 ... (SEQ ID NO:42); amino acid residues 9-160 of human IL-10 (SEQ ID NO:43); amino acid residues 10-160 of human IL-10 (SEQ ID NO:44); and amino acid residues 11-160 of human IL-10 (SEQ ID NO:45). (b) Second connector y Where y = 0 (joint does not exist) or 1 (joint exists); and (c) Monomer 2, comprising a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 1-116 (SEQ ID NO:15), 2-116 (SEQ ID NO:16), 3-116 (SEQ ID NO:17), 4-116 (SEQ ID NO:18), 5-116 (SEQ ID NO:19), 6-116 (SEQ ID NO:29), 7-116 (SEQ ID NO:30), 8-116 (SEQ ID NO:20), and 99% or 100% sequence identity with a polypeptide sequence numbered according to mature human IL-10 (SEQ ID NO:20): amino acid residues 1-116 (SEQ ID NO:15), 2-116 (SEQ ID NO:16), 3-116 (SEQ ID NO:17), 4-116 (SEQ ID NO:18), 5-116 (SEQ ID NO:19), 6-116 (SEQ ID NO:29), 7-116 (SEQ ID NO:30), and 8-116 (SEQ ID NO:20). (SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID NO:32), amino acid residues 10-116 of human IL-10 (SEQ ID NO:33), and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34).

19. The polypeptide of claim 12, wherein the polypeptide of formula (2) comprises, in the following order from amino to carboxyl groups: (a) Monomer 1, which comprises: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 of human IL-10 (SEQ ID NO:20) (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) First connector x , where x = 0 (connector does not exist) or 1 (connector exists); (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 of human IL-10 (SEQ ID NO:15). (b) Second connector y , where y = 0 (connector does not exist) or 1 (connector exists); (c) Monomer 2, which comprises: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 of human IL-10 (SEQ ID NO:20) (SEQ ID NO:12); amino acid residues 117-159 of human IL-10 (SEQ ID NO:13); and amino acid residues 117-160 of human IL-10 (SEQ ID NO:14). (ii) Third connector z Where z = 0 (connector does not exist) or 1 (connector exists); and (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 of human IL-10 (SEQ ID NO: 15).

20. The polypeptide of claim 12, wherein the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an hIL10 reverse monomer selected from the group consisting of SEQ ID NO: 46-51.

21. The polypeptide of any one of claims 1 to 20, wherein the polypeptide is PEGylated.

22. The polypeptide of claim 21, wherein the PEG molecule is linear or branched and has a molecular weight of about 10 kD to about 80 kD.

23. The polypeptide of claim 22, wherein the PEG molecule is a 40kD branched PEG molecule comprising two 20kD arms.

24. The polypeptide of any one of claims 21 to 23, wherein the PEG molecule is covalently linked to the N-terminus of the polypeptide.

25. The polypeptide of any one of claims 1 to 24, wherein the polypeptide exhibits cell type biased activity relative to the wild-type IL10 substance derived from the reverse IL10 monomer.

26. The polypeptide of claim 25, wherein (a) it exhibits a significant level of at least one anti-inflammatory property of wild-type IL10; and (b) it exhibits a significantly reduced level of at least one pro-inflammatory property of wild-type IL10.

27. The polypeptide of claim 26, wherein the at least one anti-inflammatory property is selected from the group consisting of: inhibiting the expression or secretion of IL1β, TNFα or IL6 in myeloid cells.

28. The polypeptide of claim 27, wherein the at least one pro-inflammatory property is selected from the group consisting of: inhibiting the expression or secretion of IFNγ, granzyme A, or granzyme B in T cells.

29. The polypeptide of claim 27, wherein the at least one anti-inflammatory property is selected from the group consisting of: inhibiting the expression or secretion of IL1b, TNFα or IL6 in myeloid cells; and the at least one pro-inflammatory property is selected from the group consisting of: inhibiting the expression or secretion of IFNγ, granzyme A or granzyme B in T cells.

30. The polypeptide of claim 25, wherein cell type-biased activity is the production of phosphorylated STAT3.

31. The polypeptide of claim 30, wherein, pSTAT3 E of the polypeptide max pSTAT3 E greater than wild-type hIL10 in myeloid cells max 20%, 30%, 40%, 50%, 60% or 70%.

32. The polypeptide of claim 31, wherein pSTAT3 E in lymphocytes max pSTAT3 E is smaller than wild-type hIL10 in lymphocytes max 70%, 60%, 50%, 40% or 30%.

33. The polypeptide of claim 30, wherein (a) the polypeptide has pSTAT3 E max (a) pSTAT3 Emax greater than 20%, 30%, 40%, 50%, 60%, or 70% of wild-type hIL10 in myeloid cells; and (b) pSTAT3 Emax in lymphocytes max pSTAT3 E is smaller than wild-type hIL10 in lymphocytes max 70%, 60%, 50%, 40% or 30%.

34. A nucleic acid sequence encoding the polypeptide of any one of claims 1-33.

35. A recombinant vector comprising the nucleic acid sequence of claim 34 operably linked with one or more expression control sequences.

36. A recombinant cell transformed using the recombinant vector of claim 35.

37. A method for preparing the polypeptide according to any one of claims 1 to 33, the method comprising the following steps: (a) Culturing the host cells of claim 36 under conditions suitable for expressing the polypeptide; (b) Recover the polypeptide from the host cell culture.

38. The method of claim 37, wherein the host cell is a mammalian host cell.

39. The method of claim 37, wherein the host cell is a bacterial cell.

40. A composition comprising a polypeptide of any one of claims 1 to 33, a nucleic acid sequence of claim 34 or a recombinant vector of claim 35, and one or more pharmaceutically acceptable salts, excipients and / or diluents.

41. A method of treating a mammalian subject suffering from a disease, disorder, or symptom, the method comprising administering to the subject a therapeutically effective amount of any one of claims 1 to 33 or the composition of claim 40.

42. The method of claim 41, wherein the disease, disorder, or symptom is an autoimmune disease, disorder, or symptom.

43. The method of claim 42, wherein the autoimmune disease, disorder, or condition is selected from the group consisting of: ulcerative colitis, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, adjacent autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarthritis. Juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (serone-negative tenoarthropathy), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic flare-up rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (serone-negative tenoarthropathy).

44. The method of claim 41, wherein the disease, disorder, or symptom is cancer associated with chronic inflammation.

45. The method according to any one of claims 41 to 44, wherein, The treatment prevents the progression of the disease, disorder, or symptom.

46. ​​The method according to any one of claims 41 to 44, wherein, The treatment improves one or more symptoms of the disease, disorder, or condition.

47. A method for preventing a mammalian subject at risk of developing a disease, disorder, or condition from contracting said disease, disorder, or condition, the method comprising administering to the subject a preventatively effective amount of the composition of claim 40 prior to the onset of symptoms of said disease, disorder, or condition.

48. The method of claim 47, wherein the disease, disorder, or symptom is cancer associated with chronic inflammation.

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