Uses of IL-40 and methods for detecting IL-40 activity

Abstract

The present technology relates in part to methods for detecting the activity of IL-40 and its modified forms. The present technology also relates in part to the use of IL-40 for promoting cell differentiation.

Description

[0001] Related patent applications

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 803,037, entitled USES OF IL-40 AND METHODS FOR DETECTING IL-40 ACTIVITY, filed on February 8, 2019, with Marcela Hernandez Ruiz et al. as inventors, and designated by Attorney Docket No. BLD-2001-PV. The entire contents of the foregoing application are incorporated herein by reference, including all text, tables, and drawings. Technical Field

[0003] The present technology relates in part to methods for detecting the activity of IL-40 and its modified forms. The present technology also relates in part to the use of IL-40 for promoting cell differentiation. Background Art

[0004] Cytokines are small secreted proteins involved in immune responses, host defense, inflammation, and immune system development. Cytokines typically exert their effects by binding to specific receptors on the target cell membrane. Examples of cytokines include members of the interleukin, chemokine, interferon, and tumor necrosis factor superfamily. Certain cytokines are involved in autoimmune diseases, cancer, endocrine disorders, and other ailments; and can be used in immunotherapy.

[0005] IL-40 is generally considered to be a cytokine associated with B cells and can be used for certain research applications (e.g., studying the pathogenesis of certain diseases; studying immune cell activation and differentiation), diagnosis and / or certain types of immunotherapy. Provided herein are methods for detecting the activity of IL-40 (e.g., recombinant IL-40 and its modified forms). IL-40 may be involved in promoting the differentiation of certain immune cells. Provided herein are methods for inducing immune cell differentiation using IL-40. Summary of the invention

[0006] In some aspects, provided herein is a method for evaluating the activity of an IL-40 polypeptide, the method comprising: a) contacting a cell with a first composition comprising an IL-40 polypeptide; b) measuring the production of one or more cytokines, chemokines and / or growth factors selected from CCL2, CCL3, CCL4, CCL5, CCL11, CXCL8, CXCL10, IL-1RA, erythropoietin, PDGF-AA and VEGF by the cell, thereby measuring cytokine, chemokine and / or growth factor production; and c) detecting the activity of the IL-40 polypeptide in the first composition based on the cytokine, chemokine and / or growth factor production measured in (b).

[0007] In some aspects, the present invention also provides a method for evaluating the activity of an IL-40 polypeptide, the method comprising: a) contacting a cell with a first composition comprising an IL-40 polypeptide and a second composition comprising a co-stimulatory agent; b) measuring the production of one or more cytokines, chemokines and / or growth factors selected from CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, IL-1RA, IL-6, erythropoietin, PDGF-AA and VEGF by the cell, thereby measuring the cytokine, chemokine and / or growth factor production; and c) detecting the activity of the IL-40 polypeptide in the first composition according to the cytokine, chemokine and / or growth factor production measured in (b).

[0008] In some aspects, the present invention also provides a method for evaluating the activity of an IL-40 polypeptide, the method comprising: a) contacting a population of monocytes with a first composition comprising an IL-40 polypeptide; b) detecting differentiation of monocytes into macrophages and / or monocyte activation in the population; and c) evaluating the activity of the IL-40 polypeptide in the first composition based on the differentiation of monocytes into macrophages and / or monocyte activation detected in b).

[0009] In some aspects, provided herein is a method for inducing differentiation of monocytes into macrophages, the method comprising contacting the monocytes with a first composition comprising an IL-40 polypeptide.

[0010] In some aspects, the present invention also provides a kit comprising: a) a first composition comprising one or more polypeptides selected from IFN-γ, GM-CSF, IFN-α, IL-1β, IL-4, IL-10, IL-13, M-CSF, TGF-β and TNF-α; b) one or more components for measuring the production of cytokines, chemokines and / or growth factors, wherein the cytokines, chemokines and / or growth factors are selected from one or more of CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, IL-1RA, IL-6, erythropoietin, PDGF-AA and VEGF; and c) instructions for use.

[0011] In some aspects, the present invention also provides a method for detecting the presence of an IL-40 receptor on a cell, the method comprising: a) contacting the cell with a first composition comprising a stimulatory agent and a second composition comprising a recombinant human IL-40 polypeptide; b) incubating the cell with a labeled anti-IL-40 antibody; and c) measuring the amount of the labeled anti-IL-40 antibody bound to the cell, thereby detecting the presence of the IL-40 receptor.

[0012] Certain embodiments are further described in the following detailed description, examples, claims, and figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings illustrate certain embodiments of the present technology and are not limiting. For clarity and ease of illustration, the accompanying drawings are not drawn to scale, and in some cases, various aspects may be shown exaggerated or enlarged to facilitate understanding of specific embodiments.

[0014] Figure 1 The effect of rhIL-40 on CXCL8 production is shown and its activity is aggravated by IFN-γ. The results are shown as median ± SEM, and the stimuli are as follows: Fc part (shaded column), Fc part plus IFN-γ (black column), rhIL-40 (white column), and rhIL-40 plus IFN-γ (dotted column).

[0015] Figure 2 The effect of rhIL-40 plus IFN-γ on CXCL9 production is shown. Results are shown as mean ± SEM, and the stimuli are indicated as follows: Fc portion (shaded bars), Fc portion plus IFN-γ (black bars), rhIL-40 (white bars), and rhIL-40 plus IFN-γ (dotted bars).

[0016] Figure 3 The effect of rhIL-40 on CCL2 production is shown and its activity is aggravated by IFN-γ. The results are shown as median ± SEM, and the stimuli are indicated as follows: Fc part (shaded column), Fc part plus IFN-γ (black column), rhIL-40 (white column), and rhIL-40 plus IFN-γ (dotted column).

[0017] Figure 4 The effect of rhIL-40 on CCL3 production is shown and its activity is aggravated by IFN-γ. The results are shown as median ± SEM, and the stimuli are indicated as follows: Fc part (shaded column), Fc part plus IFN-γ (black column), rhIL-40 (white column), and rhIL-40 plus IFN-γ (dotted column).

[0018] Figure 5The effect of rhIL-40 on CCL4 production is shown and its activity is aggravated by IFN-γ. The results are shown as median ± SEM, and the stimuli are indicated as follows: Fc part (shaded column), Fc part plus IFN-γ (black column), rhIL-40 (white column), and rhIL-40 plus IFN-γ (dotted column).

[0019] Figure 6 The effect of rhIL-40 on CCL5 production is shown and its activity is aggravated by IFN-γ. The results are shown as median ± SEM, and the stimuli are indicated as follows: Fc part (shaded column), Fc part plus IFN-γ (black column), rhIL-40 (white column), and rhIL-40 plus IFN-γ (dotted column).

[0020] Figure 7 The effect of rhIL-40 plus IFN-γ on CXCL10 production is shown. Results are shown as mean ± SEM, and the stimuli are indicated as follows: Fc portion (shaded bars), Fc portion plus IFN-γ (black bars), rhIL-40 (white bars), and rhIL-40 plus IFN-γ (dotted bars).

[0021] Figure 8 The effect of rhIL-40 plus IFN-γ on IL1RA production is shown. Results are shown as mean ± SEM, and stimuli are indicated as follows: Fc portion (shaded bars), Fc portion plus IFN-γ (black bars), rhIL-40 (white bars), and rhIL-40 plus IFN-γ (dotted bars).

[0022] Fig. 9 The effect of rhIL-40 plus IFN-γ on IL-6 production is shown. Results are shown as median ± SEM, and the stimuli are indicated as follows: Fc portion (shaded bars), Fc portion plus IFN-γ (black bars), rhIL-40 (white bars), and rhIL-40 plus IFN-γ (dotted bars).

[0023] Fig.10 The effect of rhIL40 on erythropoietin production is shown and its activity is aggravated by TGF-β, M-CSF, IL-10, GM-CSF, IFN-γ or IL-1 β. The results are shown as median ± SEM, and the stimuli are represented as follows: control or cytokine (white column), Fc part or Fc part + cytokine (black column), rhIL40 or rhIL40 + cytokine (dotted column).

[0024] Fig.11The effect of rhIL40 on PDGF-AA production is shown and its activity is aggravated by TGF-β, IL-10 or IL-1 β. The results are shown as median ± SEM, and the stimuli are represented as follows: control or cytokine (white bars), Fc part or Fc part + cytokine (black bars), rhIL40 or rhIL40 + cytokine (dotted bars).

[0025] Fig.12 The effect of rhIL40 on VEGF production and its activity is shown to be aggravated by IL-4, IL-13, M-CSF, GM-CSF, TNF-α or IL-1 β. The results are shown as median ± SEM, and the stimuli are represented as follows: control or cytokine (white bars), Fc part or Fc part + cytokine (black bars), rhIL40 or rhIL40 + cytokine (dotted bars).

[0026] Fig.13 Figure AM in shows the influence of rhIL-40 on chemokine production in PBMC.In PBMC, observe the statistically significant difference of generation of CXCL8 (Figure A), CXCL10 (Figure B), CCL11 (Figure C), CCL17 (Figure D), CCL2 (Figure E), CCL3 (Figure G), CXCL5 (Figure I), CCL20 (Figure J), ​​CXCL1 (Figure K), CXCL11 (Figure L) and CCL4 (Figure M).Result is shown with median ± SEM.

[0027] Fig.14 Figures AF in FIG. 5 show the morphological changes of THP-1 induced by rhIL-40 in the presence or absence of IFN-γ.

[0028] Fig.15 Figures A to C in show the effect of rhIL-40 plus IFN-γ on the expression of HLA-A, B, C (Figure A), CD40 (Figure B) and CD11b (Figure C) on the surface of THP-1 monocytes. The results are shown in bar graphs, and the stimuli are represented as follows: unstimulated cells (gray dotted line), Fc part (gray dashed line), rhIL-40 (gray solid line), IFN-γ (black dotted line), Fc part plus IFN-γ (black dashed line), and rhIL-40 plus IFN-γ (black solid line).

[0029] Fig.16The effect of rhIL40 on the expression of IL40 receptor on THP-1 cells is shown. The results are shown in a bar graph and are represented as follows: unstained cells (grey dotted line), rhFc part + isotype-APC (grey dashed line), rhFc part + anti-Fc-APC (grey solid line), rhIL40 + isotype-APC (black dashed line), rhIL40 + anti-Fc-APC (black solid line). DETAILED DESCRIPTION

[0030] IL-40 can be used for certain research applications, diagnosis and / or immunotherapy. Therefore, bioassays for evaluating the activity of IL-40, recombinant IL-40, IL-40 variants, IL-40 fragments and other modified forms of IL-40 are useful for developing practical applications of IL-40. Methods for evaluating the activity of IL-40 are provided herein. Methods for inducing cell differentiation using IL-40 are also provided herein.

[0031] IL-40

[0032] Provided herein are methods for evaluating the activity of interleukin-40 (IL-40) polypeptides. Also provided herein are methods for inducing cell differentiation using interleukin-40 (IL-40). Interleukin-40 (which may be referred to as IL-40, IL40, C17orf99 (chromosome 17 open reading frame 99), GLPG464 or UNQ464) is a cytokine involved in humoral immune regulation and is generally associated with B cells. IL-40 is expressed in bone marrow, fetal liver, and certain human B cell lymphomas, and IL-40 expression can be induced in peripheral B cells after activation. IL-40 is present in the mammalian genome, and the IL-40 gene (C17orf99) encodes a small (~27-kDa) secreted protein that is unrelated to other cytokine families, indicating a function in mammalian immune responses. Naive B cells can express IL-40 after activation, and production increases after culture with various cytokines such as IL-4 and TGF-β1.

[0033] IL-40 is generally present in mammals, including humans, mice, rats and chimpanzees. Examples of human IL-40 nucleic acid sequences are provided herein as SEQ ID NO: 2 (GENBANK accession number NM_001163075.1), and examples of human IL-40 amino acid sequences are provided herein as SEQ ID NO: 1 (GENBANK accession number NP_001156547.1). Examples of mouse IL-40 nucleic acid sequences are provided herein as SEQ ID NO: 4 (GENBANK accession number NM_029964.1), and examples of mouse IL-40 amino acid sequences are provided herein as SEQ ID NO: 3 (GENBANK accession number NP_084240.1).

[0034] The IL-40 polypeptide may refer to a precursor IL-40 polypeptide (including a signal peptide) or a mature IL-40 polypeptide (excluding a signal peptide). In some embodiments, the IL-40 polypeptide is a precursor IL-40 polypeptide (e.g., a precursor human IL-40 polypeptide comprising amino acids 1-265 of SEQ ID NO: 1; a precursor mouse IL-40 polypeptide comprising amino acids 1-252 of SEQ ID NO: 3). In some embodiments, the IL-40 polypeptide is a mature IL-40 polypeptide (e.g., a mature human IL-40 polypeptide comprising amino acids 21-265 of SEQ ID NO: 1; a mature mouse IL-40 polypeptide comprising amino acids 19-252 of SEQ ID NO: 3).

[0035] In some embodiments, the IL-40 polypeptide is a recombinant IL-40 polypeptide. A recombinant IL-40 polypeptide is typically an IL-40 polypeptide encoded by a DNA (i.e., an IL-40 nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, the IL-40 polypeptide is a recombinant human IL-40 polypeptide (rhIL-40). In some embodiments, the IL-40 polypeptide is a recombinant mouse IL-40 polypeptide (rmIL-40).

[0036] The IL-40 polypeptide herein may refer to an unmodified IL-40 polypeptide. An unmodified polypeptide generally refers to a natural or wild-type full-length (precursor or mature) polypeptide, which has no amino acid substitution, no insertion, no deletion, no chemical modification, no amino acid side chain modification, no tag, no detectable marker, no fusion, etc.

[0037] The IL-40 polypeptide herein may refer to a modified IL-40 polypeptide. A modified polypeptide generally refers to a polypeptide comprising one or more amino acid substitutions, one or more insertions, one or more deletions, one or more chemical modifications, one or more amino acid side chain modifications, one or more tags, one or more detectable labels, one or more fusions, etc., and combinations thereof. Modifications may include, for example, the addition of one or more fluorophores, glycosylation, isoprenylation, pegylation, attachment to a solid surface, biotinylation, antibody conjugation, conjugation with a therapeutic agent, chemical modification at cysteine ​​(e.g., aminoethylation, iodoacetamide, maleimide, Dha formation, disulfide formation, reaction of Dha with thiols, and desulfurization of disulfides), introduction of one or more unnatural amino acids, etc., and combinations thereof.

[0038] In some embodiments, the IL-40 polypeptide refers to an IL-40 variant or mutant. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the IL-40 polypeptide. The IL-40 variant may include any combination of deletions, insertions, and substitutions. In some embodiments, the IL-40 polypeptide comprises one or more amino acid substitutions. These variants have at least one amino acid residue removed from the IL-40 polypeptide and a different residue inserted in its place. For example, the IL-40 variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions. The IL-40 variant may include conservative substitutions and / or non-conservative substitutions, and the variants may be screened using one or more bioassays described herein for assessing IL-40 activity. Examples of substitutions are listed below:

[0039] Examples of amino acid residue substitutions

[0040] Ala(A) val;leu;ile;val

[0041] Arg(R)lys;gln;asn;lys

[0042] Asn(N) gln;his;asp,lys;gln;arg

[0043] Asp(D) glu;asn

[0044] Cys(C) ser;ala

[0045] Gln(Q) asn;glu

[0046] Glu(E) asp;gln

[0047] Gly(G)ala

[0048] His(H) asn;gln;lys;arg

[0049] Ile(I) leu; val; met; ala; leu; phe; norleucine

[0050] Leu(L) norleucine;ile;val;ile;met;ala;phe

[0051] Lys(K) arg;gln;asn

[0052] Met(M) leu;phe;ile

[0053] Phe (F) leu; val; ile; ala; tyr

[0054] Pro(P)ala

[0055] Ser(S) thr

[0056] Thr(T) ser

[0057] Trp(W)tyr;phe

[0058] Tyr(Y) trp;phe;thr;ser

[0059] Val(V) ile; leu; met; phe; ala; norleucine

[0060] Substantial modification of the biological properties of the IL-40 polypeptide can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and / or (c) the bulk of the side chain. Naturally occurring residues are divided into the following groups based on common side chain properties:

[0061] (1) Hydrophobicity: norleucine, met, ala, val, leu, ile;

[0062] (2) Neutral hydrophilicity: Cys, Ser, Thr;

[0063] (3) Acidic: asp, glu;

[0064] (4) Basic: asn, gln, his, lys, arg;

[0065] (5) Residues that affect chain orientation: gly, pro; and

[0066] (6) Aromatic: trp, tyr, phe.

[0067] Non-conservative substitutions will generally entail exchanging a member of one of these classes for another class.

[0068] In some embodiments, the IL-40 polypeptide comprises one or more insertions. In some embodiments, the IL-40 polypeptide comprises one or more insertions, wherein each insertion comprises one or more amino acids. For example, each insertion may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more inserted amino acids. In some embodiments, the IL-40 polypeptide comprises one or more deletions. In some embodiments, the IL-40 polypeptide comprises one or more deletions, wherein each deletion removes one or more amino acids from the full-length amino acid sequence. For example, each deletion may remove 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0069] In some embodiments, the IL-40 polypeptide comprises a fusion polypeptide. A fusion polypeptide may be referred to as a fusion protein or a chimeric protein. A fusion polypeptide is generally produced by connecting two or more genes encoding independent proteins. Translation of the fusion construct may result in a single or multiple polypeptides having functional properties derived from each original protein. Recombinant fusion proteins may be artificially produced by recombinant DNA techniques for biological research or therapy. Examples of fusion polypeptides include IL-40 fused to a fluorescent protein tag (e.g., green fluorescent protein (GFP)), a therapeutic protein (e.g., an antibody), or any protein tag described herein. In some embodiments, the fusion polypeptide comprises a linker (e.g., a flexible linker, a rigid linker, a cleavable linker).

[0070] In some embodiments, the IL-40 polypeptide comprises one or more tags (e.g., one or more amino acid or peptide tags; one or more affinity tags). Tags can facilitate detection, separation and / or purification of IL-40 polypeptides. Tags sometimes specifically bind to molecules or moieties such as solid phase or detectable labels, thereby having the utility of separating, purifying and / or detecting IL-40 polypeptides. In some embodiments, the tag comprises one or more of the following elements: Fc (derived from an immunoglobulin Fc domain), FLAG (e.g., DYKDDDDKG), V5 (e.g., GKPIPNPLLGLDST), c-MYC (e.g., EQKLISEEDL), HSV (e.g., QPELAPEDPED), influenza virus hemagglutinin HA (e.g., YPYDVPDYA), VSV-G (e.g., YTDIEMNRLGK), bacterial glutathione-S-transferase, maltose binding protein, streptavidin- or avidin-binding tags (e.g., pcDNA3-binding proteins), IgG4-binding proteins, IgG5-binding proteins, IgG6-binding proteins, IgG7-binding proteins, IgG8-binding proteins, IgG9-binding proteins, IgG1-binding proteins, IgG2-binding proteins, IgG1 ... TM 6 BioEase TM Biotinylation System (Invitrogen)), thioredoxin, β-galactosidase, VSV-glycoprotein, fluorescent protein (e.g., green fluorescent protein or one of its multiple color variants (e.g., yellow, red, blue)), polylysine or polyarginine sequences, polyhistidine sequences (e.g., His6) or other sequences that chelate metals (e.g., cobalt, zinc, copper), and / or cysteine-rich sequences that bind to arsenic-containing molecules. In certain embodiments, the cysteine-rich tag comprises the amino acid sequence CC-Xn-CC, where X is any amino acid and n is 1 to 3, and the cysteine-rich sequence is sometimes CCPGCC. In certain embodiments, the tag includes a cysteine-rich element and a polyhistidine element (e.g., CCPGCC and His6).

[0071] Tags can bind to binding partners. For example, some tags bind to antibodies (e.g., FLAG), and sometimes specifically bind to small molecules. For example, polyhistidine tags specifically chelate divalent metals such as copper, zinc, and cobalt; polylysine or polyarginine tags specifically bind to zinc fingers; glutathione S-transferase tags bind to glutathione; and cysteine-rich tags specifically bind to arsenic-containing molecules. Arsenic-containing molecules include LUMIO TM Reagents (Invitrogen, California), such as FlAsH TM (EDT2 [4',5'-bis(1,3,2-dithioarsenic-2-yl)fluorescein-(1,2-ethanedithiol)2]) and ReAsH reagents. Such antibodies and small molecules are sometimes linked to a solid phase for the isolation of target proteins or peptides.

[0072] In some embodiments, the IL-40 polypeptide comprises one or more detectable markers or labels. In some embodiments, the IL-40 polypeptide is conjugated to a detectable marker or label. For example, for research and diagnostic applications, the modified IL-40 polypeptide can be labeled with a detectable moiety. Many labels are available, which generally include radioactive isotopes (e.g. 35 S. 14 C. 125 I. 3 H and 131 I), fluorescent labels (e.g., rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, Texas Red and Brilliant Violet TM), and enzyme substrate labels (e.g., described in U.S. Pat. No. 4,275,149, which is incorporated herein by reference, luciferases (e.g., firefly luciferase and bacterial luciferin; U.S. Pat. No. 4,737,456, which is incorporated herein by reference), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc.).

[0073] In some cases, the label is indirectly conjugated to the IL-40 polypeptide. For example, the IL-40 polypeptide can be conjugated to biotin, and any suitable label described above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, and the label can be conjugated to the IL-40 polypeptide in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the IL-40 polypeptide, the IL-40 polypeptide is conjugated to a small hapten (e.g., digoxigenin), and one of the above label types is conjugated to an anti-hapten antibody (e.g., anti-digoxigenin antibody).

[0074] In some embodiments, the IL-40 polypeptide refers to a fragment of the IL-40 polypeptide. Typically, the IL-40 fragment contains fewer amino acids than the full-length mature IL-40. For example, the IL-40 fragment may include a portion of the mature human IL-40 polypeptide (i.e., a portion of amino acids 21-265 of SEQ ID NO: 1), or a portion of the mature mouse IL-40 polypeptide (i.e., a portion of amino acids 19-252 of SEQ ID NO: 3). The length of the full-length mature human IL-40 is 245 amino acids. Therefore, the length of the fragment of human IL-40 may be 244 amino acids or shorter. The length of the full-length mature mouse IL-40 is 234 amino acids. Therefore, the length of the fragment of mouse IL-40 may be 233 amino acids or shorter.

[0075] In some embodiments, the IL-40 polypeptide refers to a functional fragment of the IL-40 polypeptide. Provided herein are methods for evaluating the activity of IL-40 polypeptides and functional fragments of IL-40. In some embodiments, the functional fragment of IL-40 is a fragment that exhibits at least 50% of the activity of full-length mature IL-40. For example, a functional fragment of IL-40 is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more activity of full-length mature IL-40.

[0076] In some embodiments, the IL-40 polypeptide is immobilized on a solid support or substrate. In some embodiments, the IL-40 polypeptide is non-diffusively immobilized on a solid support (e.g., the IL-40 polypeptide does not detach from the solid support). The solid support or substrate can be any physically separable solid to which the IL-40 polypeptide can be attached directly or indirectly, including but not limited to surfaces provided by microarrays and wells, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles. The solid support can also include, for example, chips, columns, optical fibers, wipes, filters (e.g., planar filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyoxymethylene, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconducting materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylic resins, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethane, TEFLON TM , polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride (PVDF), etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials (including silicon, silica gel and modified silicon), Carbon, metals (e.g., steel, gold, silver, aluminum, silicon, and copper), inorganic glasses, conductive polymers (including polymers such as polypyrrole and polyindole); micro- or nanostructured surfaces, such as nucleic acid tile arrays, nanotubes, nanowires, or nanoparticles decorated surfaces; or porous surfaces or gels, such as methacrylates, acrylamides, sugar polymers, cellulose, silicates, or other fibers or chain polymers. In some embodiments, the solid support or substrate can be coated with any number of materials (including polymers such as dextran, acrylamide, gelatin, or agarose) using passive or chemically derived coatings. The beads and / or particles can be free or bound to each other (e.g., sintered). In some embodiments, the solid support or substrate can be a collection of particles. In some embodiments, the particles can include silica, and the silica can include silicon dioxide. In some embodiments, the silica can be porous, and in certain embodiments, the silica can be non-porous. In some embodiments, the particles also include agents that impart paramagnetism to the particles. In certain embodiments, the reagent comprises a metal, and in certain embodiments, the reagent is a metal oxide (e.g., iron or an iron oxide, wherein the iron oxide comprises Fe 2+ and Fe 3+ The IL-40 polypeptide can be attached to the solid support by a covalent bond or by a non-covalent interaction, and can be attached to the solid support directly or indirectly (e.g., via an intermediate agent such as a spacer molecule or biotin).

[0077] Stimulants and co-stimulators

[0078] Certain methods provided herein include the use of stimulants or co-stimulators. The use of stimulants and co-stimulators may be included in methods such as assessing the activity of IL-40 polypeptides, and / or in methods of inducing cell differentiation (e.g., inducing monocytes to differentiate into macrophages). Stimulants may be used in certain cases (e.g., stimulating cells before exposure to IL-40). Co-stimulators may be used in certain cases (e.g., co-stimulating cells during IL-40 exposure). In some embodiments, cells or cell groups are contacted with stimulants / co-stimulators. In some embodiments, cells or cell groups are contacted with stimulants / co-stimulators and IL-40 polypeptides simultaneously. In some embodiments, cells or cell groups are contacted with stimulants / co-stimulators and then contacted with IL-40 polypeptides.

[0079] In some cases, stimulants / co-stimulators can strengthen or enhance the effect of IL-40 on cells or cell groups. For example, stimulants / co-stimulators can enhance the production of certain cytokines, chemokines and / or growth factors in response to IL-40 stimulation. Stimulants / co-stimulators can also enhance certain types of cell differentiation in response to IL-40 stimulation. In some cases, when combined with IL-40, stimulants / co-stimulators can provide synergistic enhancement. For example, stimulants / co-stimulators can synergistically enhance the production of certain cytokines, chemokines and / or growth factors in response to IL-40 stimulation. Stimulants / co-stimulators can also synergistically enhance certain types of cell differentiation in response to IL-40 stimulation. The enhancement provided by stimulants or co-stimulators can be cumulative, multiplied or exponential.

[0080] Any suitable stimulant / co-stimulator can be used in conjunction with the methods provided herein. In some embodiments, the stimulant / co-stimulator comprises a soluble / secretory protein. In some embodiments, the stimulant / co-stimulator comprises a cytokine. In some embodiments, the stimulant / co-stimulator comprises a chemokine. Non-limiting examples of stimulatory / co-stimulatory agents include interferon-γ (IFN-γ), interferon-α (IFN-α), lipopolysaccharide (LPS), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 1β (IL-1β), interleukin 4 (IL-4), interleukin 13 (IL-13), macrophage colony stimulating factor (M-CSF), infections (e.g., fungal infections, helminth infections), immune complexes, interleukin-1 receptor (IL-1R), interleukin 10 (IL-10), transforming growth factor β (TGF-β), glucocorticoids, interleukin 6 (IL-6), leukemia inhibitory factor (LIF), tumor necrosis factor α (TNF-α), adenosine, complement components, and interleukin 32 (IL-32).

[0081] IFN-γ

[0082] Some methods provided herein include using interferon-γ (IFN-γ) as a stimulant or costimulator.Interferon-γ (also referred to as IFN-γ, IFNγ, IFN-g, IFNg, IFN-gamma, interferon gamma, immune interferon, type II interferon) is a dimerization soluble cytokine and an interferon of type II classification.IFN-γ is generally related to innate and adaptive immunity for certain viruses, bacteria and protozoa infections.IFN-γ can be used as an activator of macrophages and an inducer of expression of class II major histocompatibility complex (MHC) molecules.IFN-γ can directly inhibit viral replication, and can provide immunostimulation and immunomodulatory effects.Abnormal IFN-γ expression is generally related to many autoinflammatory diseases and autoimmune diseases. IFN-γ is typically produced by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response; CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops; and non-cytotoxic innate lymphoid cells (ILCs).

[0083] Any suitable IFN-γ, or its functional fragment, or modified form can be used in combination with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator before IL-40 exposure). In some embodiments, IFN-γ is human IFN-γ. Examples of human IFN-γ nucleic acid sequences are provided herein as SEQ ID NO: 6 (GENBANK Accession No. NM_000619.3), and examples of human IFN-γ amino acid sequences are provided herein as SEQ ID NO: 5 (GENBANK Accession No. NP_000610.2). In some embodiments, IFN-γ is mouse IFN-γ. Examples of mouse IFN-γ nucleic acid sequences are provided herein as SEQ ID NO: 8 (GENBANK Accession No. NM_008337.4), and examples of mouse IFN-γ amino acid sequences are provided herein as SEQ ID NO: 7 (GENBANK Accession No. NP_032363.1).

[0084] IFN-γ may refer to a precursor IFN-γ polypeptide (including a signal peptide) or a mature IFN-γ polypeptide (excluding a signal peptide). In some embodiments, IFN-γ is a precursor IFN-γ polypeptide (e.g., a precursor human IFN-γ polypeptide comprising amino acids 1-166 of SEQ ID NO: 5; a precursor human IFN-γ polypeptide comprising amino acids 1-161 of SEQ ID NO: 5; a precursor mouse IFN-γ polypeptide comprising amino acids 1-155 of SEQ ID NO: 7). In some embodiments, the IFN-γ polypeptide is a mature IFN-γ polypeptide (e.g., a mature human IFN-γ polypeptide comprising amino acids 24-166 of SEQ ID NO: 5; a mature human IFN-γ polypeptide comprising amino acids 24-161 of SEQ ID NO: 5; a mature mouse IFN-γ polypeptide comprising amino acids 23-155 of SEQ ID NO: 7).

[0085] In some embodiments, IFN-γ is a recombinant IFN-γ polypeptide. Recombinant IFN-γ polypeptides are typically IFN-γ polypeptides encoded by DNA (i.e., IFN-γ nucleic acid sequences) cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, IFN-γ is a recombinant human IFN-γ polypeptide (rhIFN-γ). In some embodiments, IFN-γ is a recombinant mouse IFN-γ polypeptide (rmIFN-γ). In some embodiments, IFN-γ is a commercially available recombinant human IFN-γ (e.g., BioLegend catalog number 570202). In some embodiments, IFN-γ is a commercially available recombinant mouse IFN-γ (e.g., BioLegend catalog number 575302).

[0086] IFN-γ may refer to an unmodified IFN-γ polypeptide, a modified IFN-γ polypeptide, an IFN-γ variant, an IFN-γ mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, IFN-γ refers to a polypeptide comprising an amino acid sequence at least about 75% identical to SEQ ID NO:5 or SEQ ID NO:7. For example, IFN-γ may refer to a polypeptide comprising an amino acid sequence at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5 or SEQ ID NO:7.

[0087] In some embodiments, IFN-γ refers to a fragment of an IFN-γ polypeptide. Typically, an IFN-γ fragment contains fewer amino acids than a full-length mature IFN-γ. For example, an IFN-γ fragment may include a portion of a mature human IFN-γ polypeptide (e.g., a portion of amino acids 24-166 of SEQ ID NO:5; a portion of amino acids 24-161 of SEQ ID NO:5), or a portion of a mature mouse IFN-γ polypeptide (i.e., a portion of amino acids 23-155 of SEQ ID NO:7). An example of full-length mature human IFN-γ has a length of 143 amino acids. Therefore, the length of a fragment of human IFN-γ may be 142 amino acids or shorter. Another example of full-length mature human IFN-γ has a length of 138 amino acids. Therefore, the length of a fragment of human IFN-γ may be 137 amino acids or shorter. The length of a full-length mature mouse IFN-γ is 133 amino acids. Therefore, the length of a fragment of mouse IFN-γ may be 132 amino acids or shorter.

[0088] In some embodiments, IFN-γ refers to the functional fragment of IFN-γ polypeptide. Any suitable method for evaluating the activity of the functional fragment of IFN-γ and IFN-γ can be used to determine whether the IFN-γ fragment is a functional fragment. For example, a determination for evaluating the activity of IFN-γ is to induce an antiviral state in target cells, sometimes referred to as a cytopathic protective effect (CPE) determination. The example of CPE determination uses A549 human lung cancer cells attacked by encephalomyocarditis virus (encephalomyocarditis virus) (EMCV), and the effect is compared with the standard of human IFN-γ (e.g., Gxg01-902-535, BEI Resources). In some embodiments, the functional fragment of IFN-γ is a fragment showing at least 50% activity of IFN-γ standard or full-length mature IFN-γ. For example, the functional fragment of IFN-γ is a fragment showing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more active of IFN-γ standard or full-length mature IFN-γ.

[0089] IL-4

[0090] Certain methods provided herein include the use of interleukin 4 (IL-4) as a stimulator or co-stimulator. Interleukin 4 (also known as IL-4, B cell growth factor 1 (BCGF-1), B-cell stimulating factor 1 (BSF-1), interleukin-4, lymphocyte stimulating factor 1, MGC79402) is a major cytokine involved in the development of Th2-mediated responses, which is associated with allergies and asthma. Type I receptors include IL-4Rα and public γ-chain (γc), which are also shared by cytokines IL-2, -7, -9, -15 and -21 and are present in hematopoietic cells. IL-4 can utilize a type II complex comprising IL-4Rα and IL-13Rα1, which is present in non-hematopoietic cells. The second receptor complex is a functional receptor for IL-13, which shares about 25% homology with IL-4. Type I receptor complexes can be formed only by IL-4 and are active in Th2 development. In contrast, the type II receptor complex formed by IL-4 or IL-13 is more active during airway hypersensitivity and mucus secretion and is not present in T cells.

[0091] Any suitable IL-4, or its functional fragment, or modified form can be used in combination with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator before IL-40 exposure). In some embodiments, IL-4 is human IL-4. Examples of human IL-4 nucleic acid sequences are provided herein as SEQ ID NO: 13 (full mRNA sequence, provided as GENBANK accession number NM_000589), and examples of human IL-4 amino acid sequences are provided herein as SEQ ID NO: 12 (GENBANK accession number NP_000580.1). In some embodiments, IL-4 is mouse IL-4. Examples of mouse IL-4 nucleic acid sequences are provided herein as SEQ ID NO: 15 (full mRNA sequence, provided as GENBANK accession number NM_021283), and examples of mouse IL-4 amino acid sequences are provided herein as SEQ ID NO: 14 (GENBANK accession number NP_067258.1).

[0092] IL-4 may refer to a precursor IL-4 polypeptide (including a signal peptide) or a mature IL-4 polypeptide (excluding a signal peptide). In some embodiments, IL-4 is a precursor IL-4 polypeptide (e.g., a precursor human IL-4 polypeptide comprising amino acids 1-153 of SEQ ID NO: 12; a precursor mouse IL-4 polypeptide comprising amino acids 1-140 of SEQ ID NO: 14). In some embodiments, the IL-4 polypeptide is a mature IL-4 polypeptide (e.g., a mature human IL-4 polypeptide comprising amino acids 25-153 of SEQ ID NO: 12; a mature mouse IL-4 polypeptide comprising amino acids 21-140 of SEQ ID NO: 14; a mature mouse IL-4 polypeptide comprising amino acids 23-140 of SEQ ID NO: 14).

[0093] In some embodiments, IL-4 is a recombinant IL-4 polypeptide. A recombinant IL-4 polypeptide is typically an IL-4 polypeptide encoded by a DNA (i.e., an IL-4 nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, IL-4 is a recombinant human IL-4 polypeptide (rhIL-4). In some embodiments, IL-4 is a recombinant mouse IL-4 polypeptide (rmIL-4). In some embodiments, IL-4 is a commercially available recombinant human IL-4 (e.g., BioLegend catalog number 574002). In some embodiments, IL-4 is a commercially available recombinant mouse IL-4 (e.g., BioLegend catalog number 574302).

[0094] IL-4 may refer to an unmodified IL-4 polypeptide, a modified IL-4 polypeptide, an IL-4 variant, an IL-4 mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, IL-4 refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO: 12 or SEQ ID NO: 14. For example, IL-4 may refer to a polypeptide comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12 or SEQ ID NO: 14.

[0095] In some embodiments, IL-4 refers to a fragment of an IL-4 polypeptide. Typically, an IL-4 fragment contains fewer amino acids than a full-length mature IL-4. For example, an IL-4 fragment may include a portion of a mature human IL-4 polypeptide (i.e., a portion of amino acids 25-153 of SEQ ID NO: 12), or a portion of a mature mouse IL-4 polypeptide (i.e., a portion of amino acids 21-140 of SEQ ID NO: 14). The length of an example of full-length mature human IL-4 is 129 amino acids. Therefore, the length of a fragment of human IL-4 may be 128 amino acids or shorter. The length of an example of full-length mature mouse IL-4 is 120 amino acids. Therefore, the length of a fragment of mouse IL-4 may be 119 amino acids or shorter. The length of another example of full-length mature mouse IL-4 is 118 amino acids. Therefore, the length of a fragment of mouse IL-4 may be 117 amino acids or shorter.

[0096] In some embodiments, IL-4 refers to a functional fragment of an IL-4 polypeptide. Any suitable method for evaluating the activity of IL-4 and functional fragments of IL-4 can be used to determine whether an IL-4 fragment is a functional fragment. For example, one assay for evaluating the activity of IL-4 is a cell proliferation assay. An example of a cell proliferation assay measures the ED of IL-4 based on dose-dependent stimulation of TF-1 cell proliferation. 50 Another example of a cell proliferation assay measures the ED of IL-4 based on dose-dependent stimulation of CTLL-2 cell proliferation. 50 In some embodiments, the functional fragment of IL-4 is a fragment that exhibits at least 50% of the activity of an IL-4 standard (e.g., the WHO International Standard for Human IL-4 (NIBSC code: 88 / 656)) or full-length mature IL-4. For example, a functional fragment of IL-4 is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the activity of an IL-4 standard or full-length mature IL-4.

[0097] IL-10

[0098] Some methods provided herein include using interleukin 10 (IL-10) as a stimulant or costimulator. Interleukin 10 (also referred to as IL-10, B-TCGF, ​​CSIF, TGIF) was initially described as a cytokine produced by helper T cell 2 (Th2) cell clones. It inhibits the synthesis of interferon (IFN)-γ in Th1 cells, so it was originally called cytokine synthesis inhibitory factor (CSIF). Macrophages are the main source of IL-10, and their secretion can be stimulated by endotoxin (via Toll-like receptor 4, NF-κB dependence), tumor necrosis factor TNF-α (via TNF receptor p55, NF-κB dependence), catecholamines and IL-1. IL-10 controls the inflammatory process by inhibiting the expression of proinflammatory cytokines, chemokines, adhesion molecules, and antigen presentation and costimulatory molecules in monocytes / macrophages, neutrophils, and T cells. IL-10 inhibits the production of pro-inflammatory mediators by monocytes and macrophages, such as endotoxin- and IFN-γ-induced IL-1α, IL-6, IL-8, G-CSF, GM-CSF and TNF-α release. In addition, it enhances the production of anti-inflammatory mediators such as IL-1RA and soluble TNFα receptors. IL-10 inhibits the ability of monocytes and macrophages to present antigens to T cells. This is achieved by downregulating the cell surface levels of constitutive and IFNγ-induced class II MHC, costimulatory molecules such as CD86, and some adhesion molecules such as CD58.

[0099] Any suitable IL-10, or a functional fragment thereof, or a modified form thereof can be used in conjunction with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator before IL-40 exposure). In some embodiments, IL-10 is human IL-10. Examples of human IL-10 nucleic acid sequences are provided herein as SEQ ID NO: 17 (full mRNA sequence, provided as GENBANK Accession No. NM_000572), and examples of human IL-10 amino acid sequences are provided herein as SEQ ID NO: 16 (GENBANK Accession No. NP_000563.1). In some embodiments, IL-10 is mouse IL-10. Examples of mouse IL-10 nucleic acid sequences are provided herein as SEQ ID NO: 19 (full mRNA sequence, provided as GENBANK Accession No. NM_010548), and examples of mouse IL-10 amino acid sequences are provided herein as SEQ ID NO: 18 (GENBANK Accession No. NP_034678.1).

[0100] IL-10 may refer to a precursor IL-10 polypeptide (including a signal peptide) or a mature IL-10 polypeptide (excluding a signal peptide). In some embodiments, IL-10 is a precursor IL-10 polypeptide (e.g., a precursor human IL-10 polypeptide comprising amino acids 1-178 of SEQ ID NO: 16; a precursor mouse IL-10 polypeptide comprising amino acids 1-178 of SEQ ID NO: 18). In some embodiments, the IL-10 polypeptide is a mature IL-10 polypeptide (e.g., a mature human IL-10 polypeptide comprising amino acids 19-178 of SEQ ID NO: 16; a mature mouse IL-10 polypeptide comprising amino acids 19-178 of SEQ ID NO: 18).

[0101] In some embodiments, IL-10 is a recombinant IL-10 polypeptide. A recombinant IL-10 polypeptide is typically an IL-10 polypeptide encoded by a DNA (i.e., an IL-10 nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, IL-10 is a recombinant human IL-10 polypeptide (rhIL-10). In some embodiments, IL-10 is a recombinant mouse IL-10 polypeptide (rmIL-10). In some embodiments, IL-10 is a commercially available recombinant human IL-10 (e.g., BioLegend Catalog No. 715602; BioLegend Catalog No. 571002). In some embodiments, IL-10 is a commercially available recombinant mouse IL-10 (e.g., BioLegend Catalog No. 575802).

[0102] IL-10 may refer to an unmodified IL-10 polypeptide, a modified IL-10 polypeptide, an IL-10 variant, an IL-10 mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, IL-10 refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO: 16 or SEQ ID NO: 18. For example, IL-10 may refer to a polypeptide comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16 or SEQ ID NO: 18.

[0103] In some embodiments, IL-10 refers to a fragment of an IL-10 polypeptide. Typically, an IL-10 fragment contains fewer amino acids than a full-length mature IL-10. For example, an IL-10 fragment may include a portion of a mature human IL-10 polypeptide (i.e., a portion of amino acids 19-178 of SEQ ID NO: 16), or a portion of a mature mouse IL-10 polypeptide (i.e., a portion of amino acids 19-178 of SEQ ID NO: 18). An example of full-length mature human IL-10 is 160 amino acids in length. Thus, a fragment of human IL-10 may be 159 amino acids or shorter in length. An example of full-length mature mouse IL-10 is 160 amino acids in length. Thus, a fragment of mouse IL-10 may be 159 amino acids or shorter in length.

[0104] In some embodiments, IL-10 refers to a functional fragment of an IL-10 polypeptide. Any suitable method for evaluating the activity of IL-10 and a functional fragment of IL-10 can be used to determine whether an IL-10 fragment is a functional fragment. For example, a determination for evaluating the activity of IL-10 is an IFN-γ inhibition assay. An example of an IFN-γ inhibition assay measures the extent to which IL-10 inhibits the induction of INF-γ in human PBMCs activated by PHA. Another determination for evaluating the activity of IL-10 involves dose-dependent stimulation of MC / 9 cell proliferation. In some embodiments, a functional fragment of IL-10 is a fragment that exhibits at least 50% activity of an IL-10 standard or full-length mature IL-10. For example, a functional fragment of IL-10 is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more activity of an IL-10 standard or full-length mature IL-10.

[0105] TGF-β

[0106] Some methods provided herein include using transforming growth factor β (TGF-β) as a stimulator or costimulator. Transforming growth factor β (also known as TGF-β, TGF-β1, TGFB, DPD1, transforming growth factor, transforming growth factor β1, TGF-β-1) is synthesized in cells as a 390-amino acid polypeptide. Furin cleaves the protein at residue 278 to obtain an N-terminal cleavage product corresponding to the potential associated polypeptide (LAP), and the 25-kD C-terminal portion of the precursor constitutes mature TGF-β1. TGF-β activators can release TGF-β from LAP. These activators include proteases that degrade LAP, thrombospondin-1, reactive oxygen species, and integrins avb6 and avb8. Mouse TGF-β converts naive T cells into regulatory T (Treg) cells that prevent autoimmunity. Although human TGF-β1 is widely used to induce FOXP3+ in vitro, it may not be a necessary factor for human Treg differentiation. Mouse Th17 can be induced by naive CD4+T cells by a combination of TGF-β1 and IL-6 or IL-21. However, the regulation of human Th17 differentiation is different. TGF-β1 appears to have a dual effect on human Th17 differentiation in a dose-dependent manner. Although TGF-β1 is essential for the expression of RORγt in human naive CD4+T cells from umbilical cord blood, TGF-β1 can inhibit the function of RORγt at high doses. By using serum-free medium, it has been clarified that the optimal conditions for human Th17 differentiation are a combination of TGF-β1, IL-1β and IL-2 with IL-6, IL-21 or IL-23.

[0107] Any suitable TGF-β, or its functional fragment, or modified form can be used in combination with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator before IL-40 exposure). In some embodiments, TGF-β is human TGF-β. An example of a human TGF-β nucleic acid sequence is provided herein as SEQ ID NO: 21 (full mRNA sequence, provided as GENBANK accession number NM_000660.7), and an example of a human TGF-β amino acid sequence is provided herein as SEQ ID NO: 20 (GENBANK accession number NP_000651.3). Another example of a human TGF-β nucleic acid sequence is provided as GENBANK accession number BC000125.1, and another example of a human TGF-β amino acid sequence is provided as GENBANK accession number P01137. In some embodiments, TGF-β is mouse TGF-β. An example of a mouse TGF-β nucleic acid sequence is provided herein as SEQ ID NO: 23 (full mRNA sequence provided as GENBANK Accession No. NM_011577.2), and an example of a mouse TGF-β amino acid sequence is provided herein as SEQ ID NO: 22 (GENBANK Accession No. NP_035707).

[0108] TGF-β may refer to a precursor TGF-β polypeptide (including a signal peptide) or a mature TGF-β polypeptide (excluding a signal peptide; or excluding a signal peptide and a potentially associated polypeptide). In some embodiments, TGF-β is a precursor TGF-β polypeptide (e.g., a precursor human TGF-β polypeptide comprising amino acids 1-390 of SEQ ID NO: 20; a precursor mouse TGF-β polypeptide comprising amino acids 1-390 of SEQ ID NO: 22). In some embodiments, the TGF-β polypeptide is a mature TGF-β polypeptide (e.g., a mature human TGF-β polypeptide comprising amino acids 30-390 of SEQ ID NO: 20; a mature human TGF-β polypeptide comprising amino acids 279-390 of SEQ ID NO: 20; a mature mouse TGF-β polypeptide comprising amino acids 29-390 of SEQ ID NO: 22; a mature mouse TGF-β polypeptide comprising amino acids 279-390 of SEQ ID NO: 22).

[0109] In some embodiments, TGF-β is a recombinant TGF-β polypeptide. A recombinant TGF-β polypeptide is typically a TGF-β polypeptide encoded by DNA (i.e., a TGF-β nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, TGF-β is a recombinant human TGF-β polypeptide (rhTGF-β). In some embodiments, TGF-β is a recombinant mouse TGF-β polypeptide (rmTGF-β). In some embodiments, TGF-β is a commercially available recombinant human TGF-β (e.g., BioLegend Catalog No. 580704; BioLegend Catalog No. 781802). In some embodiments, TGF-β is a commercially available recombinant mouse TGF-β (e.g., BioLegend Catalog No. 763102).

[0110] TGF-β may refer to an unmodified TGF-β polypeptide, a modified TGF-β polypeptide, a TGF-β variant, a TGF-β mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, TGF-β refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO: 20 or SEQ ID NO: 22. For example, TGF-β may refer to a polypeptide comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or SEQ ID NO: 22.

[0111] In some embodiments, TGF-β refers to a fragment of a TGF-β polypeptide. Typically, a TGF-β fragment contains fewer amino acids than a full-length mature TGF-β. For example, a TGF-β fragment may include a portion of a mature human TGF-β polypeptide (e.g., a portion of amino acids 30-390 of SEQ ID NO: 20; a portion of amino acids 279-390 of SEQ ID NO: 20), or a portion of a mature mouse TGF-β polypeptide (e.g., a portion of amino acids 29-390 of SEQ ID NO: 22; a portion of amino acids 279-390 of SEQ ID NO: 22). An example of full-length mature human TGF-β is 112 amino acids in length. Thus, a fragment of human TGF-β may be 111 amino acids or shorter in length. An example of full-length mature mouse TGF-β is 112 amino acids in length. Thus, a fragment of mouse TGF-β may be 111 amino acids or shorter in length.

[0112] In some embodiments, TGF-β refers to a functional fragment of a TGF-β polypeptide. Any suitable method for evaluating the activity of a functional fragment of TGF-β and TGF-β can be used to determine whether a TGF-β fragment is a functional fragment. For example, a determination for evaluating the activity of TGF-β is a cell proliferation inhibition assay. An example of a cell proliferation inhibition assay measures the extent to which TGF-β inhibits the proliferation of mouse HT-2 cells induced by recombinant mouse IL-4. In some embodiments, a functional fragment of TGF-β is a fragment that exhibits at least 50% activity of a TGF-β standard or full-length mature TGF-β. For example, a functional fragment of TGF-β is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more activity of a TGF-β standard or full-length mature TGF-β.

[0113] GM-CSF

[0114] Some methods provided herein include using granulocyte macrophage colony stimulating factor (GM-CSF) as a stimulant or costimulator. Granulocyte macrophage colony stimulating factor (also referred to as GM-CSF, colony stimulating factor 2, CSF2, CSF-α, Pluripoietin-α, eosinophil colony stimulating factor (Eo-CSF), burst promoting active substance (BPA)) plays a role in the emergency hematopoiesis (mainly myelopoiesis) signal transduction in response to infection, including the production of granulocytes and macrophages in the bone marrow and their maintenance, survival and functional activation at the site of injury or invasion. The receptor of GM-CSF is a heterodimer comprising a major binding subunit (GMRα) and a major signaling subunit (βc). The receptor subunits are co-expressed on the surface of leukocytes, wherein βc is expressed at a level lower than GMRα. It has also been reported that certain non-hematopoietic cell types express GM-CSF receptors and respond to GM-CSF stimulation in vitro.

[0115] Any suitable GM-CSF, or a functional fragment or modified form thereof, can be used in conjunction with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator prior to IL-40 exposure). In some embodiments, GM-CSF is human GM-CSF. An example of a human GM-CSF nucleic acid sequence is provided herein as SEQ ID NO: 25 (full mRNA sequence, provided as GENBANK Accession No. NM_000758), and an example of a human GM-CSF amino acid sequence is provided herein as SEQ ID NO: 24 (GENBANK Accession No. NP_000749.2). In some embodiments, GM-CSF is mouse GM-CSF. An example of a mouse GM-CSF nucleic acid sequence is provided herein as SEQ ID NO: 27 (full mRNA sequence, provided as GENBANK Accession No. NM_009969), and an example of a mouse GM-CSF amino acid sequence is provided herein as SEQ ID NO: 26 (GENBANK Accession No. NP_034099.2).

[0116] GM-CSF can refer to a precursor GM-CSF polypeptide (including a signal peptide) or a mature GM-CSF polypeptide (excluding a signal peptide). In some embodiments, GM-CSF is a precursor GM-CSF polypeptide (e.g., a precursor human GM-CSF polypeptide comprising amino acids 1-144 of SEQ ID NO: 24; a precursor mouse GM-CSF polypeptide comprising amino acids 1-141 of SEQ ID NO: 26). In some embodiments, the GM-CSF polypeptide is a mature GM-CSF polypeptide (e.g., a mature human GM-CSF polypeptide comprising amino acids 18-144 of SEQ ID NO: 24; a mature mouse GM-CSF polypeptide comprising amino acids 18-141 of SEQ ID NO: 26).

[0117] In some embodiments, GM-CSF is a recombinant GM-CSF polypeptide. A recombinant GM-CSF polypeptide is typically a GM-CSF polypeptide encoded by a DNA (i.e., a GM-CSF nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, GM-CSF is a recombinant human GM-CSF polypeptide (rhGM-CSF). In some embodiments, GM-CSF is a recombinant mouse GM-CSF polypeptide (rmGM-CSF). In some embodiments, GM-CSF is commercially available recombinant human GM-CSF (e.g., BioLegend catalog number 572902). In some embodiments, GM-CSF is commercially available recombinant mouse GM-CSF (e.g., BioLegend catalog number 576302).

[0118] GM-CSF can refer to an unmodified GM-CSF polypeptide, a modified GM-CSF polypeptide, a GM-CSF variant, a GM-CSF mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, GM-CSF refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO:24 or SEQ ID NO:26. For example, GM-CSF can refer to a polypeptide comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24 or SEQ ID NO:26.

[0119] In some embodiments, GM-CSF refers to a fragment of a GM-CSF polypeptide. Generally, a GM-CSF fragment contains fewer amino acids than full-length mature GM-CSF. For example, a GM-CSF fragment may include a portion of a mature human GM-CSF polypeptide (i.e., a portion of amino acids 18-144 of SEQ ID NO: 24), or a portion of a mature mouse GM-CSF polypeptide (i.e., a portion of amino acids 18-141 of SEQ ID NO: 26). An example of full-length mature human GM-CSF is 127 amino acids in length. Thus, a fragment of human GM-CSF may be 126 amino acids in length or shorter. An example of full-length mature mouse GM-CSF is 124 amino acids in length. Thus, a fragment of mouse GM-CSF may be 123 amino acids in length or shorter.

[0120] In some embodiments, GM-CSF refers to a functional fragment of a GM-CSF polypeptide. Any suitable method for evaluating the activity of GM-CSF and functional fragments of GM-CSF can be used to determine whether a GM-CSF fragment is a functional fragment. For example, one assay for evaluating the activity of GM-CSF is a cell proliferation assay. An example of a cell proliferation assay is to determine the EC of GM-CSF by dose-dependent stimulation of TF-1 cell proliferation. 50 In some embodiments, the functional fragment of GM-CSF is a fragment that exhibits at least 50% of the activity of the GM-CSF standard (e.g., the 1st WHO International Standard for Human GM-CSF (NIBSC code: 88 / 646)) or full-length mature GM-CSF. For example, the functional fragment of GM-CSF is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the activity of the GM-CSF standard or full-length mature GM-CSF.

[0121] M-CSF

[0122] Some methods provided herein include using macrophage colony stimulating factor (M-CSF) as a stimulant or costimulator. Macrophage colony stimulating factor (also referred to as M-CSF, CSF1, CSF-1, MCSF) was initially characterized as a glycoprotein, which induces the formation of monocytes and macrophage colonies by precursors in mouse bone marrow cultures. M-CSF is present in human serum in a biologically active concentration. It binds CD14+ monocytes and promotes the survival / proliferation of human peripheral blood mononuclear cells. In addition, M-CSF enhances the inducible monocyte function, including phagocytic activity in monocytes, microbial killing, cytotoxicity to tumor cells, and inflammatory cytokines such as IL-1, TNFα and IFN-γ synthesis. M-CSF induces RANKL production in mature human osteoclasts; therefore, M-CSF is an effective stimulator of mature osteoclast resorption activity. Moreover, M-CSF induces VEGF in human monocytes of human tumors; high levels of M-CSF, mononuclear phagocytes, and VEGF are associated with poor prognosis in patients with cancer. High levels of M-CSF may be associated with different pathologies such as pulmonary fibrosis and atherosclerosis. M-CSF binds to its receptor M-CSFR, and this receptor is shared by a second ligand, IL-34. Human M-CSF and IL-34 show species specificity - both bind to human and mouse M-CSF receptors.

[0123] Any suitable M-CSF, or functional fragments thereof, or modified forms thereof can be used in conjunction with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator prior to IL-40 exposure). In some embodiments, the M-CSF is human M-CSF. An example of a human M-CSF nucleic acid sequence is provided herein as SEQ ID NO: 29 (full mRNA sequence, provided as GENBANK Accession Nos. NM_172212.2 and NM_172212.3), and an example of a human M-CSF amino acid sequence is provided herein as SEQ ID NO: 28 (GENBANK Accession No. NP_757351.2). In some embodiments, the M-CSF is mouse M-CSF. An example of a mouse M-CSF nucleic acid sequence is provided herein as SEQ ID NO:31 (full mRNA sequence provided as GENBANK Accession No. NM_001113530.1), and an example of a mouse M-CSF amino acid sequence is provided herein as SEQ ID NO:30 (GENBANK Accession No. NP_001107002.1).

[0124] M-CSF can refer to a precursor M-CSF polypeptide (including a signal peptide) or a mature M-CSF polypeptide (excluding a signal peptide). In some embodiments, M-CSF is a precursor M-CSF polypeptide (e.g., a precursor human M-CSF polypeptide comprising amino acids 1-190 of SEQ ID NO: 28; a precursor human M-CSF polypeptide comprising amino acids 1-450 of SEQ ID NO: 28; a precursor human M-CSF polypeptide comprising amino acids 1-554 of SEQ ID NO: 28; a precursor mouse M-CSF polypeptide comprising amino acids 1-262 of SEQ ID NO: 30; a precursor mouse M-CSF polypeptide comprising amino acids 1-552 of SEQ ID NO: 30). In some embodiments, the M-CSF polypeptide is a mature M-CSF polypeptide (e.g., a mature human M-CSF polypeptide comprising amino acids 33-190 of SEQ ID NO:28; a mature human M-CSF polypeptide comprising amino acids 33-450 of SEQ ID NO:28; a mature human M-CSF polypeptide comprising amino acids 33-554 of SEQ ID NO:28; a mature mouse M-CSF polypeptide comprising amino acids 33-262 of SEQ ID NO:30; a mature mouse M-CSF polypeptide comprising amino acids 33-552 of SEQ ID NO:30).

[0125] In some embodiments, M-CSF is a recombinant M-CSF polypeptide. A recombinant M-CSF polypeptide is typically an M-CSF polypeptide encoded by a DNA (i.e., an M-CSF nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, M-CSF is a recombinant human M-CSF polypeptide (rhM-CSF). In some embodiments, M-CSF is a recombinant mouse M-CSF polypeptide (rmM-CSF). In some embodiments, M-CSF is commercially available recombinant human M-CSF (e.g., BioLegend catalog number 574802). In some embodiments, M-CSF is commercially available recombinant mouse M-CSF (e.g., BioLegend catalog number 576402).

[0126] M-CSF can refer to an unmodified M-CSF polypeptide, a modified M-CSF polypeptide, an M-CSF variant, an M-CSF mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, M-CSF refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO:28 or SEQ ID NO:30. For example, M-CSF can refer to a polypeptide comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:28 or SEQ ID NO:30.

[0127] In some embodiments, M-CSF refers to a fragment of an M-CSF polypeptide. Typically, an M-CSF fragment contains fewer amino acids than full-length mature M-CSF. For example, an M-CSF fragment may include a portion of a mature human M-CSF polypeptide (e.g., a portion of amino acids 33-190 of SEQ ID NO: 28; a portion of amino acids 33-450 of SEQ ID NO: 28; a portion of amino acids 33-554 of SEQ ID NO: 28), or a portion of a mature mouse M-CSF polypeptide (e.g., a portion of amino acids 33-262 of SEQ ID NO: 30; a portion of amino acids 33-552 of SEQ ID NO: 30). An example of full-length mature human M-CSF is 158 amino acids in length. Thus, a fragment of human M-CSF may be 157 amino acids in length or less. An example of full-length mature mouse M-CSF is 230 amino acids in length. Thus, a fragment of mouse M-CSF may be 229 amino acids in length or less.

[0128] In some embodiments, M-CSF refers to a functional fragment of an M-CSF polypeptide. Any suitable method for evaluating the activity of M-CSF and a functional fragment of M-CSF can be used to determine whether an M-CSF fragment is a functional fragment. For example, one assay for evaluating the activity of M-CSF is a cell proliferation assay. An example of a cell proliferation assay is to determine the EC activity of M-CSF by dose-dependent stimulation of M-NFS60 cell proliferation. 50 In some embodiments, a functional fragment of M-CSF is a fragment that exhibits at least 50% of the activity of standard M-CSF or full-length mature M-CSF. For example, a functional fragment of M-CSF is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the activity of standard M-CSF or full-length mature M-CSF.

[0129] IFN-α

[0130] Some methods provided herein include using interferon alpha (IFN-α) as a stimulant or costimulator. Interferon alpha may also be referred to as IFN-α, IFN-α2, IFN-α2B, IFN-αA, IFNA2 or IFNA2B. Interferon is divided into type I, type II and type III. Type I IFN (IFN-α and IFN-β) is the most abundant in quantity, distribution and expression. Moreover, they are highly conserved in structure and function in mammals. IFN-α2 has been used to treat cancer, such as bladder cancer, hepatocellular carcinoma and leukemia. IFN-α2 enhances the suppressed immune function of patients with head and neck squamous cell carcinoma (HNSCC). IFN-α2 triggers the cytotoxic effect of tumor cells mediated by T and NK cells through IFNγ-dependent and -independent mechanisms. IFN-α2 enhances suppressed T cell cytotoxicity by stimulating the perforin-granzyme B system (IFNγ-dependent). Moreover, IFN-α2 induces the expression of perforin-granzyme B in NK cells (NK-mediated cytotoxicity, IFNγ-independent). IFN-α2 can be an immunostimulant and can affect the clinical outcomes of patients with tongue squamous cell carcinoma. IFN-α has been used to treat chronic hepatitis C (CHC); however, IFN-α is relatively unstable and requires frequent parenteral administration. Pegylated IFN-α (i.e., polyethylene glycol (PEG)-IFN-α) reduces in vitro activity but increases the stability and plasma half-life of IFN-α; therefore, PEG-IFN-α has replaced IFN-α in CHC treatment.

[0131] Any suitable IFN-α, or its functional fragment, or modified form can be used in combination with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator before IL-40 exposure). In some embodiments, IFN-α is human IFN-α. Examples of human IFN-α nucleic acid sequences are provided herein as SEQ ID NO: 33 (full mRNA sequence, provided as GENBANK accession number NM_000605), and examples of human IFN-α amino acid sequences are provided herein as SEQ ID NO: 32 (GENBANK accession number NP_000596.2). In some embodiments, IFN-α is mouse IFN-α. Examples of mouse IFN-α nucleic acid sequences are provided herein as SEQ ID NO: 35 (full mRNA sequence, provided as GENBANK accession number NM_206870), and examples of mouse IFN-α amino acid sequences are provided herein as SEQ ID NO: 34 (GENBANK accession number NP_996753.1).

[0132] IFN-α may refer to a precursor IFN-α polypeptide (including a signal peptide) or a mature IFN-α polypeptide (excluding a signal peptide). In some embodiments, IFN-α is a precursor IFN-α polypeptide (e.g., a precursor human IFN-α polypeptide comprising amino acids 1-188 of SEQ ID NO: 32; a precursor mouse IFN-α polypeptide comprising amino acids 1-190 of SEQ ID NO: 34). In some embodiments, the IFN-α polypeptide is a mature IFN-α polypeptide (e.g., a mature human IFN-α polypeptide comprising amino acids 24-188 of SEQ ID NO: 32; a mature mouse IFN-α polypeptide comprising amino acids 24-190 of SEQ ID NO: 34).

[0133] In some embodiments, IFN-α is a recombinant IFN-α polypeptide. A recombinant IFN-α polypeptide is typically an IFN-α polypeptide encoded by a DNA (i.e., an IFN-α nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, IFN-α is a recombinant human IFN-α polypeptide (rhIFN-α). In some embodiments, IFN-α is a recombinant mouse IFN-α polypeptide (rmIFN-α). In some embodiments, IFN-α is a commercially available recombinant human IFN-α (e.g., BioLegend catalog number 592702). In some embodiments, IFN-α is a commercially available recombinant mouse IFN-α (e.g., BioLegend catalog number 752802).

[0134] IFN-α may refer to an unmodified IFN-α polypeptide, a modified IFN-α polypeptide, an IFN-α variant, an IFN-α mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants and fragments are described herein. In some embodiments, IFN-α refers to a polypeptide comprising an amino acid sequence at least about 75% identical to SEQ ID NO: 32 or SEQ ID NO: 34. For example, IFN-α may refer to a polypeptide comprising an amino acid sequence at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 32 or SEQ ID NO: 34.

[0135] In some embodiments, IFN-α refers to a fragment of an IFN-α polypeptide. Typically, an IFN-α fragment comprises fewer amino acids than full-length mature IFN-α. For example, an IFN-α fragment may include a portion of a mature human IFN-α polypeptide (e.g., a portion of amino acids 24-188 of SEQ ID NO:32), or a portion of a mature mouse IFN-α polypeptide (e.g., a portion of amino acids 24-190 of SEQ ID NO:34). The length of an example of full-length mature human IFN-α is 165 amino acids. Therefore, the length of a fragment of human IFN-α may be 164 amino acids or shorter. The length of an example of full-length mature mouse IFN-α is 167 amino acids. Therefore, the length of a fragment of mouse IFN-α may be 166 amino acids or shorter.

[0136] In some embodiments, IFN-α refers to a functional fragment of an IFN-α polypeptide. Any suitable method for assessing the activity of a functional fragment of IFN-α and IFN-α can be used to determine whether an IFN-α fragment is a functional fragment. For example, a determination for assessing the activity of IFN-α is a cytopathic effect inhibition assay. For example, the specific activity of IFN-α can be determined in a cytopathic effect inhibition assay of L929 or A549 cells using EMC virus. In some embodiments, a functional fragment of IFN-α is a fragment showing at least 50% activity of an IFN-α standard or full-length mature IFN-α. For example, a functional fragment of IFN-α is a fragment showing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more activity of an IFN-α standard or full-length mature IFN-α.

[0137] IL-1β

[0138] Some methods provided herein include using interleukin 1β (IL-1β) as a stimulant or costimulator. Interleukin 1β may also be referred to as IL-1β, decomposition protein, pro-IL-1β, or pro-IL-1-β. IL-1β in humans and mice does not encode a typical signal peptide, so newly synthesized pre-IL-1β accumulates in the cytoplasm of activated monocytes and macrophages. The conversion of inactive pre-IL-1β into its mature form requires proteolysis of IL-1β-converting enzyme (ICE) (also referred to as caspase-1). The secretion of mature IL-1β by LPS-activated monocytes / macrophages is not a constitutive process. These cells encounter secondary stimulation of specific activation of post-translational processing events. In addition, due to its pro-inflammatory properties, IL-1β is regarded as a tumor-promoting cytokine. Tumor metastasis and angiogenesis enhancement were observed under the influence of IL-1β. IL-1β can promote tumor progression in a mouse lung cancer model. Furthermore, upregulation of metastasis and tumor angiogenesis by IL-1β was associated with increased activity of matrix metalloproteinases and expression of the proangiogenic molecule hepatocyte growth factor.

[0139] Any suitable IL-1β, or a functional fragment thereof, or a modified form thereof can be used in conjunction with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator before IL-40 exposure). In some embodiments, IL-1β is human IL-1β. Examples of human IL-1β nucleic acid sequences are provided herein as SEQ ID NO: 37 (full mRNA sequence, provided as GENBANK Accession No. NM_000576), and examples of human IL-1β amino acid sequences are provided herein as SEQ ID NO: 36 (GENBANK Accession No. NP_000567.1). In some embodiments, IL-1β is mouse IL-1β. Examples of mouse IL-1β nucleic acid sequences are provided herein as SEQ ID NO: 39 (full mRNA sequence, provided as GENBANK Accession No. NM_008361), and examples of mouse IL-1β amino acid sequences are provided herein as SEQ ID NO: 38 (GENBANK Accession No. NP_032387.1).

[0140] IL-1β may refer to a precursor IL-1β polypeptide (including a propeptide) or a mature IL-1β polypeptide (excluding a propeptide). In some embodiments, IL-1β is a precursor IL-1β polypeptide (e.g., a precursor human IL-1β polypeptide comprising amino acids 1-269 of SEQ ID NO: 36; a precursor mouse IL-1β polypeptide comprising amino acids 1-269 of SEQ ID NO: 38). In some embodiments, the IL-1β polypeptide is a mature IL-1β polypeptide (e.g., a mature human IL-1β polypeptide comprising amino acids 117-269 of SEQ ID NO: 36; a mature mouse IL-1β polypeptide comprising amino acids 118-269 of SEQ ID NO: 38).

[0141] In some embodiments, IL-1β is a recombinant IL-1β polypeptide. A recombinant IL-1β polypeptide is typically an IL-1β polypeptide encoded by a DNA (i.e., an IL-1β nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, IL-1β is a recombinant human IL-1β polypeptide (rhIL-1β). In some embodiments, IL-1β is a recombinant mouse IL-1β polypeptide (rmIL-1β). In some embodiments, IL-1β is a commercially available recombinant human IL-1β (e.g., BioLegend catalog number 579402). In some embodiments, IL-1β is a commercially available recombinant mouse IL-1β (e.g., BioLegend catalog number 575102).

[0142] IL-1β may refer to an unmodified IL-1β polypeptide, a modified IL-1β polypeptide, an IL-1β variant, an IL-1β mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, IL-1β refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO: 36 or SEQ ID NO: 38. For example, IL-1β may refer to a polypeptide comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 36 or SEQ ID NO: 38.

[0143] In some embodiments, IL-1β refers to a fragment of an IL-1β polypeptide. Typically, an IL-1β fragment contains fewer amino acids than a full-length mature IL-1β. For example, an IL-1β fragment may include a portion of a mature human IL-1β polypeptide (e.g., a portion of amino acids 117-269 of SEQ ID NO: 36), or a portion of a mature mouse IL-1β polypeptide (e.g., a portion of amino acids 118-269 of SEQ ID NO: 38). The length of an example of full-length mature human IL-1β is 153 amino acids. Therefore, the length of a fragment of human IL-1β may be 152 amino acids or shorter. The length of an example of full-length mature mouse IL-1β is 152 amino acids. Therefore, the length of a fragment of mouse IL-1β may be 151 amino acids or shorter.

[0144] In some embodiments, IL-1β refers to a functional fragment of an IL-1β polypeptide. Any suitable method for evaluating the activity of IL-1β and a functional fragment of IL-1β can be used to determine whether an IL-1β fragment is a functional fragment. For example, one assay for evaluating the activity of IL-1β is a cell proliferation assay. For example, the ED of IL-1β can be determined by dose-dependent stimulation of D10.G4.1 cell proliferation. 50 In some embodiments, the functional fragment of IL-1β is a fragment that exhibits at least 50% of the activity of IL-1β standard or full-length mature IL-1β. For example, the functional fragment of IL-1β is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the activity of IL-1β standard or full-length mature IL-1β.

[0145] IL-13

[0146] Some methods provided herein include using interleukin 13 (IL-13) as a stimulator or costimulator. Interleukin 13 may also be referred to as IL-13, ALRH, BHR1, P600, IL-13, MGC116786, MGC116788, MGC116789, NC30 or IL13. Human IL-13 was initially cloned from a cDNA library of activated T cells. IL-13 is an immunomodulatory cytokine secreted primarily by activated T (H) 2 cells, and is a mediator in the pathogenesis of allergic inflammation. IL-13 shares many functional properties with IL-4, and they share a common receptor subunit, i.e., the α subunit (IL-4Rα) of the IL-4 receptor. IL-13 mediates its effect by interacting with a composite receptor system consisting of IL-4Rα and two IL-13 binding proteins (i.e., IL-13Rα1 and IL-13Rα2). Ligation of the IL-13 receptor complex results in signaling via the insulin receptor substrate (IRS)-1 and 2 and STAT-6 pathways. Similar to IL-4, IL-13 is a cytokine produced by T(H)2 type helper T cells in response to signaling through the T cell antigen receptor, and is a cytokine produced by mast cells and basophils when the high affinity receptor is cross-linked with immunoglobulin E (IgE). IL-13 is associated with airway hypersensitivity and mucus hypersecretion, inflammatory bowel disease, and expulsion of parasitic nematodes.

[0147] Any suitable IL-13, or a functional fragment thereof, or a modified form thereof can be used in conjunction with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator before IL-40 exposure). In some embodiments, IL-13 is human IL-13. An example of a human IL-13 nucleic acid sequence is provided herein as SEQ ID NO: 41 (full mRNA sequence, provided as GENBANK accession number X69079), and an example of a human IL-13 amino acid sequence is provided herein as SEQ ID NO: 40 (GENBANK accession number CAA48823.1 or P35225.2). Another example of a human IL-13 amino acid sequence is provided as GENBANK accession number NP_002179.2. In some embodiments, IL-13 is mouse IL-13. An example of a mouse IL-13 nucleic acid sequence is provided herein as SEQ ID NO:43 (full mRNA sequence provided as GENBANK Accession No. NM_008355), and an example of a mouse IL-13 amino acid sequence is provided herein as SEQ ID NO:42 (GENBANK Accession No. NP_032381.1).

[0148] IL-13 may refer to a precursor IL-13 polypeptide (including a signal peptide) or a mature IL-13 polypeptide (excluding a signal peptide). In some embodiments, IL-13 is a precursor IL-13 polypeptide (e.g., a precursor human IL-13 polypeptide comprising amino acids 1-132 of SEQ ID NO: 40; a precursor human IL-13 polypeptide comprising amino acids 1-146 of SEQ ID NO: 40; a precursor mouse IL-13 polypeptide comprising amino acids 1-131 of SEQ ID NO: 42). In some embodiments, the IL-13 polypeptide is a mature IL-13 polypeptide (e.g., a mature human IL-13 polypeptide comprising amino acids 21-132 of SEQ ID NO: 40; a mature mouse IL-13 polypeptide comprising amino acids 26-131 of SEQ ID NO: 42).

[0149] In some embodiments, IL-13 is a recombinant IL-13 polypeptide. A recombinant IL-13 polypeptide is typically an IL-13 polypeptide encoded by a DNA (i.e., an IL-13 nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, IL-13 is a recombinant human IL-13 polypeptide (rhIL-13). In some embodiments, IL-13 is a recombinant mouse IL-13 polypeptide (rmIL-13). In some embodiments, IL-13 is a commercially available recombinant human IL-13 (e.g., BioLegend catalog number 571102). In some embodiments, IL-13 is a commercially available recombinant mouse IL-13 (e.g., BioLegend catalog number 575902).

[0150] IL-13 may refer to an unmodified IL-13 polypeptide, a modified IL-13 polypeptide, an IL-13 variant, an IL-13 mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, IL-13 refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO:40 or SEQ ID NO:42. For example, IL-13 may refer to a polypeptide comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:40 or SEQ ID NO:42.

[0151] In some embodiments, IL-13 refers to a fragment of an IL-13 polypeptide. Typically, an IL-13 fragment contains fewer amino acids than a full-length mature IL-13. For example, an IL-13 fragment may include a portion of a mature human IL-13 polypeptide (e.g., a portion of amino acids 21-132 of SEQ ID NO: 40), or a portion of a mature mouse IL-13 polypeptide (e.g., a portion of amino acids 26-131 of SEQ ID NO: 42). An example of full-length mature human IL-13 has a length of 112 amino acids. Thus, a fragment of human IL-13 may have a length of 111 amino acids or less. An example of full-length mature mouse IL-13 has a length of 106 amino acids. Thus, a fragment of mouse IL-13 may have a length of 105 amino acids or less.

[0152] In some embodiments, IL-13 refers to a functional fragment of an IL-13 polypeptide. Any suitable method for evaluating the activity of IL-13 and functional fragments of IL-13 can be used to determine whether an IL-13 fragment is a functional fragment. For example, one assay for evaluating the activity of IL-13 is a cell proliferation assay. For example, the ED value of IL-13 can be determined by dose-dependent stimulation of TF-1 cell proliferation. 50 In some embodiments, the functional fragment of IL-13 is a fragment that exhibits at least 50% of the activity of IL-13 standard or full-length mature IL-13. For example, the functional fragment of IL-13 is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the activity of IL-13 standard or full-length mature IL-13.

[0153] TNF-α

[0154] Some methods provided herein include using tumor necrosis factor alpha (TNF-α) as a stimulant or costimulator. Tumor necrosis factor alpha (TNF-α) may also be referred to as cachectin, necrosis factor, macrophage cytotoxic factor (MCF), differentiation inducing factor (DIF) or TNFSF2. TNF-α is released from macrophages, monocytes, neutrophils, T-cells (mainly CD4+), NK-cells, and a variety of transformed cell lines. Soluble homotrimeric TNF-α is released from cells by proteolysis of the integral membrane precursor form of TNF-α. TNF-α that is bound to some TNF-α receptors induces apoptosis, and depending on cell type, receptor expression, and signal transduction status, other responses may be induced. TNF-α participates in inflammatory responses.

[0155] Any suitable TNF-α, or its functional fragment, or modified form can be used in conjunction with the methods described herein (e.g., as a co-stimulator with IL-40; as a stimulator before IL-40 exposure). In some embodiments, TNF-α is human TNF-α. Examples of human TNF-α nucleic acid sequences are provided herein as SEQ ID NO: 45 (full mRNA sequence, provided as GENBANK Accession No. NM_000594), and examples of human TNF-α amino acid sequences are provided herein as SEQ ID NO: 44 (GENBANK Accession No. NP_000585.2). In some embodiments, TNF-α is mouse TNF-α. Examples of mouse TNF-α nucleic acid sequences are provided herein as SEQ ID NO: 47 (full mRNA sequence, provided as GENBANK Accession No. NM_013693), and examples of mouse TNF-α amino acid sequences are provided herein as SEQ ID NO: 46 (GENBANK Accession No. NP_038721.1).

[0156] TNF-α can refer to a precursor TNF-α polypeptide (an integral membrane precursor form) or a mature TNF-α polypeptide (a soluble homotrimeric form). In some embodiments, TNF-α is a precursor TNF-α polypeptide (e.g., a precursor human TNF-α polypeptide comprising amino acids 1-233 of SEQ ID NO: 44; a precursor mouse TNF-α polypeptide comprising amino acids 1-235 of SEQ ID NO: 46). In some embodiments, the TNF-α polypeptide is a mature TNF-α polypeptide (e.g., a mature human TNF-α polypeptide comprising amino acids 77-233 of SEQ ID NO: 44; a mature mouse TNF-α polypeptide comprising amino acids 80-235 of SEQ ID NO: 46).

[0157] In some embodiments, TNF-α is a recombinant TNF-α polypeptide. A recombinant TNF-α polypeptide is typically a TNF-α polypeptide encoded by a DNA (i.e., a TNF-α nucleic acid sequence) that has been cloned into a vector or system that supports DNA expression and messenger RNA translation. In some embodiments, TNF-α is a recombinant human TNF-α polypeptide (rhTNF-α). In some embodiments, TNF-α is a recombinant mouse TNF-α polypeptide (rmTNF-α). In some embodiments, TNF-α is a commercially available recombinant human TNF-α (e.g., BioLegend catalog number 570102). In some embodiments, TNF-α is a commercially available recombinant mouse TNF-α (e.g., BioLegend catalog number 575202).

[0158] TNF-α may refer to an unmodified TNF-α polypeptide, a modified TNF-α polypeptide, a TNF-α variant, a TNF-α mutant; or a fragment thereof or a functional fragment thereof. Unmodified polypeptides, modified polypeptides, mutants, variants, and fragments are described herein. In some embodiments, TNF-α refers to a polypeptide comprising an amino acid sequence at least about 75% identical to SEQ ID NO:44 or SEQ ID NO:46. For example, TNF-α may refer to a polypeptide comprising an amino acid sequence at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:44 or SEQ ID NO:46.

[0159] In some embodiments, TNF-α refers to a fragment of a TNF-α polypeptide. Typically, a TNF-α fragment comprises less amino acids than a full-length mature TNF-α. For example, a TNF-α fragment may include a portion of a mature human TNF-α polypeptide (e.g., a portion of amino acids 77-233 of SEQ ID NO:44), or a portion of a mature mouse TNF-α polypeptide (e.g., a portion of amino acids 80-235 of SEQ ID NO:46). The length of an example of full-length mature human TNF-α is 157 amino acids. Therefore, the length of a fragment of human TNF-α may be 156 amino acids or shorter. The length of an example of full-length mature mouse TNF-α is 156 amino acids. Therefore, the length of a fragment of mouse TNF-α may be 155 amino acids or shorter.

[0160] In some embodiments, TNF-α refers to a functional fragment of a TNF-α polypeptide. Any suitable method for evaluating the activity of TNF-α and functional fragments of TNF-α can be used to determine whether a TNF-α fragment is a functional fragment. For example, one assay for evaluating the activity of TNF-α is a cytotoxicity assay. For example, the ED value of TNF-α can be determined by the dose-dependent cytotoxicity of L929 cells stimulated with actinomycin D. 50 In some embodiments, the functional fragment of TNF-α is a fragment that exhibits at least 50% of the activity of the TNF-α standard (e.g., the 3rd WHO International Standard for Human TNF-α (NIBSC code: 12 / 154)) or full-length mature TNF-α. For example, the functional fragment of TNF-α is a fragment that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more activity of the TNF-α standard or full-length mature TNF-α.

[0161] cell

[0162] Some methods described herein include stimulating cells or cell groups (e.g., with IL-40; with stimulants; with IL-40+co-stimulators). Cells can be obtained from subjects and / or cell sources, or can be obtained as established cell lines. Cell sources may include groups of embryonic stem cells (ES), induced pluripotent stem cells (iPSC), etc. Cells can be separated from embryos or stem cell cultures derived from embryos. Cells can be separated from induced pluripotent stem cell (iPSC) cultures. Cells can be obtained from subjects in various ways (e.g., harvested from living tissues such as biopsies, extracted hair follicles, body fluids such as urine or coelomic fluids, or isolated from circulation). Subjects may include any animal, including but not limited to any mammal, such as mice, rats, dogs, cats, cattle, horses, pigs, non-human primates, and humans. In certain embodiments, the subject is a human. In some embodiments, the subject is an animal or human whose pregnancy is longer than that of an embryo in an intrauterine environment, and is typically a human or animal after birth (e.g., a newborn, a newborn animal, an adult, or an adult animal). The subject is sometimes a juvenile animal, a juvenile human, an adult animal, or an adult human.

[0163] In some embodiments, cells are separated from the sample of the subject. The separated cells refer to cells that have been separated from the components of their original environment (e.g., separated from the host and / or purified from the sample) and therefore changed from their original environment by human intervention (e.g., "by artificial"). The sample may include any specimen separated or obtained from the subject or its part. The non-limiting examples of specimens include fluids or tissues derived from the subject, including but not limited to blood or blood products (e.g., serum, plasma, etc.), cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, stomach, peritoneum, catheter, ear, arthroscopy), biopsy samples or biopsy tissue sections, oral swabs, peritoneal puncture samples, female reproductive tract washings, urine, feces, sputum, saliva, nasal mucosa, prostatic fluid, lavage fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, hard tissue (e.g., liver, spleen, kidney, lung or ovary), etc., or combinations thereof. The term blood encompasses whole blood, blood products or any fraction of blood, such as conventionally defined serum, plasma, buffy coat, etc. Plasma refers to the fraction of whole blood obtained by centrifugation of blood treated with an anticoagulant. Serum refers to the watery portion of the fluid remaining after a blood sample has coagulated.

[0164] In some embodiments, cells include normal healthy cells (e.g., non-lesional cells). In some embodiments, cells include genetically altered cells. In some embodiments, cells include genetically unaltered cells. In some embodiments, cells include pathological cells. Pathological cells may include cells from subjects carrying pathogenic mutations. Pathological cells may include cells from abnormal tissues, such as from neoplasia, hyperplasia, malignant tumors or benign tumors. In certain embodiments, pathological cells include cells that are not tumor cells. In certain embodiments, pathological cells may include cells separated from the circulation (e.g., circulating tumor cells (CTCs)) of a subject. In certain embodiments, pathological cells may include cells separated from, for example, body samples, such as urine, semen, stool (feces), etc.

[0165] In some embodiments, cells include primary cells.Primary cells are usually directly obtained from living tissues such as biopsy, extracted hair follicles, body samples such as stool samples, body fluids such as urine, semen or body cavity fluids, or separated from circulation.In some cases, primary cells have not yet been passaged.In some cases, primary cells have been passaged once.Primary cells can be separated from differentiated tissues.Usually, primary cells are freshly separated, for example, by tissue digestion and plating.Primary cells may or may not be frozen, and then thawed later.In addition, the tissue from which primary cells are isolated may or may not be frozen or preserved in some other ways before treatment.Usually, after cells have been passaged more than once, cells are no longer primary cells.Cells that are passaged once or multiple times and frozen immediately after passage are not considered as primary cells when thawed.In some embodiments, cells are initially primary cells, and become non-primary cells after passage.In some embodiments, after separating cells from differentiated tissues and before using in the methods described herein, cells are maintained or propagated in cell culture.

[0166] In some embodiments, the cells include non-primary cells, such as cells from an established cell line, transformed cells, thawed cells from a previously frozen collection, etc. Any suitable cell line can be used in conjunction with the methods described herein. Examples of established cell lines include, e.g., THP-1 (acute myeloid leukemia), DU145 (prostate cancer), H295R (adrenocortical cancer), HeLa (cervical cancer), KBM-7 (chronic myelogenous leukemia), LNCaP (prostate cancer), MCF-7 (breast cancer), MDA-MB-468 (breast cancer), PC3 (prostate cancer), SaOS-2 (bone cancer), SH-SY5Y (neuroblastoma, cloned from myeloma), T-47D (breast cancer), U87 (glioblastoma), Vero (African green monkey Chlorocebus) renal epithelial cell line), MC3T3 (embryonic calvarium), GH3 (pituitary tumor), PC12 (pheochromocytoma), CHO (Chinese hamster ovary), MDCK (renal epithelial), A6 (renal epithelial), and AB9. In some embodiments, the cell comprises a THP-1 cell.

[0167] In some embodiments, cell includes immune cell.Immune cell may include, for example, lymphocyte, leukocyte, agranulocyte, monocyte, macrophage, B cell, dendritic cell, granulocyte, neutrophil, innate lymphocyte (ILC), megakaryocyte, myeloid-derived suppressor cell (MDSC), natural killer cell (NK cell), platelet, red blood cell (RBC), T cell, mast cell, eosinophil, basophil and thymocyte.In some embodiments, cell includes or is derived from peripheral blood mononuclear cell (PBMC), it may include, for example, T cell, B cell, natural killer cell and monocyte.

[0168] In some embodiments, the cell includes a monocyte. A monocyte is a type of leukocyte or white blood cell, and can be differentiated into a macrophage and a myeloid dendritic cell. Monocytes are part of the innate immune system of vertebrates, and can affect the process of adaptive immunity. There are at least three subclasses of monocytes in human blood, which can be characterized according to certain markers. For example, typical monocytes are characterized by high levels of expression of CD14 cell surface receptors (CD14++CD16-monocytes); atypical monocytes show low levels of expression of CD14 and additional co-expression of CD16 receptors (CD14+CD16++monocytes); and intermediate monocytes are characterized by high levels of expression of CD14 and low levels of expression of CD16 (CD14++CD16+monocytes).

[0169] In some embodiments, the cell includes a macrophage. A macrophage is a type of white blood cell that engulfs and digests foreign proteins and other substances, cell debris, microorganisms, and cancer cells. Macrophages may be referred to as phagocytes, tissue cells, Kupffer cells, alveolar macrophages, or microglia, and may be present in almost all body tissues. Macrophages participate in nonspecific defense (innate immunity), and can help induce specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. Macrophages also have anti-inflammatory effects and can reduce immune responses by the release of cytokines. Macrophages that promote inflammation are commonly referred to as M1 macrophages, while macrophages that reduce inflammation and promote tissue repair are commonly referred to as M2 macrophages. Macrophages are produced by the differentiation of monocytes. Macrophages can be identified using flow cytometry and / or immunohistochemical staining by their specific expression of proteins such as CD14, CD40, CD11b, CD64, F4 / 80, EMR1, lysozyme M, MAC-1 / MAC-3, and CD68. Dysfunctional macrophages can cause diseases such as chronic granulomatous disease that leads to frequent infections.

[0170] In some embodiments, the cell includes non-immune cells. Non-immune cells may include, for example, epithelial cells (e.g., body cavity lining cells), cells derived from the central nervous system (e.g., nerve cells, neurons, glial cells), matrix cells (e.g., connective tissue cells, fibroblasts, pericytes), stem cells (e.g., embryonic stem cells, adult stem cells), muscle cells (e.g., bones, heart, smooth muscle), cartilage bodies (e.g., chondrocytes), bone cells (e.g., osteoblasts, osteoclasts, osteocytes, bone lining cells), skin cells (e.g., keratinocytes, melanocytes, Merkel cells, Langerhans cells), endothelial cells (e.g., blood vessel lining cells), adipocytes (e.g., white adipocytes, brown adipocytes), and germ cells (sperm cells, egg cells).

[0171] In some embodiments, the cell includes an epithelial cell. Epithelial cells or epithelium generally refer to one or more cells arranged in a hollow organ, and those constituting glands and the outer surface of the body. Epithelial cells may include squamous epithelial cells, columnar epithelial cells, adenoma epithelial cells or transitional epithelial cells. Depending on the organ and position, epithelial cells may be arranged in a single layer or in multiple layers, and may include keratinocyte (KE) epithelial cells or non-keratinocyte (NKE) epithelial cells.

[0172] Keratinocytes form squamous epithelia present in anatomical sites such as the skin, ocular surface, oral mucosa, esophagus, and cervix. Keratinocytes eventually differentiate into flat, highly keratinized, non-viable cells that help defend against the environment and infection by forming a protective barrier. Examples of keratinocyte epithelial cells include, but are not limited to, dermal keratinocytes, ocular epithelial cells, corneal epithelial cells, oral mucosal epithelial cells, esophageal epithelial cells, and cervical epithelial cells.

[0173] Non-keratinocyte (NKE) epithelial cells form the epithelium of the body, such as being present in the breast, prostate, liver, respiratory tract, retina and gastrointestinal tract. NKE cells are usually differentiated into functional living cells that, for example, work in absorption and / or secretion. These cells do not usually form the highly keratinized structure characteristic of squamous epithelial cells. The example of NKE cells includes but is not limited to prostate cells, breast cells, hepatocytes, hepatic epithelial cells, bile duct epithelial cells, gallbladder cells, islet cells, pancreatic β cells, pancreatic duct epithelial cells, lung epithelial cells, airway epithelial cells, nasal epithelial cells, kidney cells, bladder cells, urethral epithelial cells, gastric epithelial cells, large intestine epithelial cells, small intestine epithelial cells, testicular epithelial cells, ovarian epithelial cells, fallopian tube epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymocytes, pituitary cells, glandular cells, amniotic epithelial cells, retinal pigment epithelial cells, sweat gland epithelial cells, sebaceous gland epithelial cells and hair follicle cells.

[0174] Cytokine, chemokine, and growth factor production

[0175] Certain methods described herein include measuring the production of one or more cytokines and / or chemokines. Certain methods described herein include measuring the production of one or more cytokines, chemokines and / or growth factors.

[0176] Cytokines generally refer to small proteins (usually about 5–20 kDa) that are involved in cell signaling, and the release of cytokines can have an effect on the behavior of nearby cells. Cytokines are generally considered to be immunomodulators, and may be involved in autocrine signaling, paracrine signaling, and endocrine signaling. Cytokines may include chemokines, interferons, interleukins, lymphokines, monokines, colony stimulating factors, and tumor necrosis factors. Cytokines can be produced by immune cells (e.g., monocytes, macrophages, B lymphocytes, T lymphocytes, and mast cells), endothelial cells, fibroblasts, and stromal cells.

[0177] In some embodiments, the method herein includes measuring one or more cytokines produced by cells. In some embodiments, the method herein includes measuring one or more interleukins produced by cells. In some embodiments, the method herein includes measuring one or more IL-6 family cytokines produced by cells. In some embodiments, the method herein includes measuring one or more IL-1 family cytokines produced by cells. The method herein may include measuring the production of one or more cytokines provided in Table 1 below. Table 1 also provides corresponding human genes and human receptors, however, the cytokines, genes and receptors listed in Table 1 are not limited to human cytokines, genes and receptors. In some embodiments, the method herein includes measuring the cytokines produced by cells that are bound to one or more receptors provided in Table 1.

[0178]

[0179]

[0180] Chemokine generally refers to the subfamily of cytokines (signal proteins secreted by cells).Chemokines can induce directional chemotaxis in nearby reactive cells, and can be referred to as chemotactic cytokines.Chemokines are relatively small (usually about 8-10kDa) and usually have 4 cysteine ​​residues in conservative positions to form their three-dimensional shapes.Some chemokines are proinflammatory and can be induced during the immune response of the cells of the immune system recruited to the site of infection, and some chemokines are homeostatic and participate in the migration of control cells during the normal process of tissue maintenance or development.Chemokines can be divided into subfamily (i.e. C, CX3C, CC and CXC), and exert their biological effects by interacting with the transmembrane receptors (chemokine receptors) connected with the G protein present on the target cell surface.

[0181] In some embodiments, the method herein includes measuring one or more chemokines produced by cells. In some embodiments, the method herein includes measuring one or more C family chemokines produced by cells. In some embodiments, the method herein includes measuring one or more CX3C family chemokines produced by cells. In some embodiments, the method herein includes measuring one or more CC family chemokines produced by cells. In some embodiments, the method herein includes measuring one or more CXC family chemokines produced by cells. The method herein may include measuring the production of one or more chemokines provided in Table 2 below. Table 2 also provides corresponding human genes and human receptors, however, the chemokines, genes and receptors herein are not limited to human chemokines, genes and receptors. In some embodiments, the method herein includes measuring the chemokines produced by cells that are bound to one or more receptors provided in Table 2.

[0182]

[0183]

[0184] Growth factors generally refer to substances that can stimulate cell growth, proliferation, healing, cell differentiation, etc. Growth factors can be proteins or hormones (e.g., steroid hormones). In some cases, cytokines can be classified as growth factors. Growth factors can include, for example, erythropoietin, platelet-derived growth factor (PDGF), PDGF-AA, and vascular endothelial growth factor (VEGF).

[0185] In some embodiments, the method herein includes measuring one or more cytokines and / or chemokines selected from CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, IL-1RA and IL-6 produced by the cell. In some embodiments, the method herein includes measuring one or more cytokines, chemokines and / or growth factors selected from CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, IL-1RA, IL-6, erythropoietin, PDGF-AA and VEGF produced by the cell. In some embodiments, the method herein includes measuring one or more cytokines and / or chemokines selected from CCL2, CCL3, CCL4, CCL5, CCL11, CXCL8, CXCL10 and IL-1RA produced by the cell. In some embodiments, the method herein includes measuring one or more cytokines, chemokines and / or growth factors selected from CCL2, CCL3, CCL4, CCL5, CCL11, CXCL8, CXCL10, IL-1RA, erythropoietin, PDGF-AA and VEGF produced by the cell. In some embodiments, the method herein includes measuring CCL2 produced by the cell. In some embodiments, the method herein includes measuring CCL3 produced by the cell. In some embodiments, the method herein includes measuring CCL4 produced by the cell. In some embodiments, the method herein includes measuring CCL5 produced by the cell. In some embodiments, the method herein includes measuring CCL11 produced by the cell. In some embodiments, the method herein includes measuring CCL17 produced by the cell. In some embodiments, the method herein includes measuring CCL20 produced by the cell. In some embodiments, the method herein includes measuring CXCL1 produced by the cell. In some embodiments, the methods herein include measuring CXCL5 produced by cells. In some embodiments, the methods herein include measuring CXCL8 produced by cells. In some embodiments, the methods herein include measuring CXCL9 produced by cells. In some embodiments, the methods herein include measuring CXCL10 produced by cells. In some embodiments, the methods herein include measuring CXCL11 produced by cells. In some embodiments, the methods herein include measuring IL-1RA produced by cells. In some embodiments, the methods herein include measuring IL-6 produced by cells. In some embodiments, the methods herein include measuring erythropoietin produced by cells. In some embodiments, the methods herein include measuring PDGF-AA produced by cells.In some embodiments, the methods herein comprise measuring VEGF production by cells.

[0186] The production of cytokines, chemokines and / or growth factors can be measured using any suitable method, device or machine for measuring protein secretion and / or DNA expression (e.g., mRNA). For example, the production of cytokines, chemokines and / or growth factors can be measured by immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, Western blotting, protein immunostaining), spectrometry (e.g., high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS)), flow cytometry, quantitative polymerase chain reaction (qPCR), gel electrophoresis, photometer, fluorometer, spectrophotometer, suitable gene chip or microarray analysis, mass spectrometry, chromatography, cell fluorescence analysis, fluorescence microscopy, suitable fluorescence or digital imaging methods, confocal laser scanning microscopy, laser scanning cytometry, affinity chromatography, manual batch mode separation, electric field suspension, suitable nucleic acid sequencing methods and / or nucleic acid sequencing equipment, etc. and combinations thereof. Examples of immunoassays include LEGEND MAX with pre-coated plates. TM ELISA kit (BioLegend), Macrophage / Microglia LEGENDplex TM Plate (BioLegend) and ChemokineInflammatory LEGENDplex TM Plate (BioLegend).

[0187] Methods for Assessing IL-40 Activity

[0188] Provided herein are methods for assessing IL-40 activity. Methods for assessing IL-40 activity are generally performed ex vivo (i.e., outside the organism) or in vitro (i.e., performed or occurring in a test tube, culture dish, or other container outside the organism). The terms ex vivo and in vitro are used interchangeably and are generally used in conjunction with methods performed in an artificial environment or artificial system. The methods herein generally include contacting cells with IL-40 or IL-40 and a co-stimulator, and such contact is generally performed ex vivo / in vitro (e.g., in cell culture).

[0189] In some embodiments, the method includes contacting the cell with IL-40, measuring the production of one or more cytokines, chemokines and / or growth factors described herein, and detecting the activity of IL-40 according to cytokine production. In some embodiments, detecting the activity of IL-40 includes comparing the production of cytokines / chemokines / growth factors under test conditions (IL-40 stimulation) with the production of cytokines / chemokines / growth factors under control conditions (no IL-40 stimulation). In some embodiments, detecting the activity of IL-40 includes comparing the production of cytokines / chemokines / growth factors under test conditions with the production of cytokines / chemokines / growth factors in a standard curve (e.g., cytokines / chemokines / growth factors measured for a variety of different amounts of active IL-40). Active IL-40 may include IL-40 standards (e.g., unmodified full-length mature IL-40; full-length mature IL-40 comprising a modification that enhances activity). In some embodiments, detecting the activity of IL-40 includes comparing the production of cytokines / chemokines / growth factors under the test conditions with the production of cytokines / chemokines / growth factors measured by the control, thereby providing a comparison. The control may include, for example, the production of cytokines / chemokines / growth factors measured in the absence of IL-40. Typically, for the control, or for generating a standard curve, the production of cytokines of the same cells (e.g., the same cell type, the same cell source, and / or the same cell population) used to assess the activity of IL-40 (test conditions) is measured. Typically, for the control, or for generating a standard curve, the production of cytokines / chemokines / growth factors can be the measured level of cytokines / chemokines / growth factors, or the cytokines / chemokines / growth factors in the combination of cytokines, chemokines, and growth factors; or cytokines and chemokines; or cytokines and growth factors; or chemokines and growth factors; or the measured levels of cytokines, chemokines, and growth factors. Typically, the levels of the same cytokine / chemokine / growth factor, or the levels of the same cytokine, chemokine, or cytokine and chemokine, or cytokine and growth factor, or chemokine and growth factor, or cytokine, chemokine and growth factor in the same cytokine / chemokine / growth factor combination, are measured for the test condition and the control condition or standard curve condition.

[0190] In some embodiments, the method includes contacting the cell with IL-40 and a co-stimulator (e.g., IFN-γ, GM-CSF, IFN-α, IL-1β, IL-4, IL-10, IL-13, M-CSF, TGF-β, TNF-α), measuring the production of one or more cytokines, chemokines and / or growth factors described herein, and detecting the activity of IL-40 based on the production of cytokines, chemokines and / or growth factors. In some embodiments, detecting the activity of IL-40 includes comparing the production of cytokines / chemokines / growth factors under test conditions (IL-40+co-stimulators) with the production of cytokines / chemokines / growth factors under control conditions (no IL-40, no co-stimulators, or no IL-40+co-stimulators). In some embodiments, detecting the activity of IL-40 includes comparing the production of cytokines / chemokines / growth factors under test conditions with the production of cytokines / chemokines / growth factors in a standard curve (e.g., cytokines / chemokines / growth factors measured for a variety of different amounts of active IL-40 or active IL-40+co-stimulators). Active IL-40 may include IL-40 standards (e.g., unmodified full-length mature IL-40; full-length mature IL-40 comprising a modification that enhances activity). In some embodiments, detecting the activity of IL-40 includes comparing the production of cytokines / chemokines / growth factors under test conditions with the production of cytokines / chemokines / growth factors measured by controls, thereby providing a comparison. Controls may include, for example, cytokines / chemokines / growth factor production measured in the absence of IL-40; cytokines / chemokines / growth factor production measured in the absence of co-stimulators; and cytokines / chemokines / growth factor production measured in the absence of IL-40 and co-stimulators. Typically, for a control, or for generating a standard curve, the cytokine / chemokine / growth factor production of the same cells (e.g., the same cell type, the same cell source, and / or the same cell population) used to assess IL-40 activity (test conditions) is measured. Typically, for a control, or for generating a standard curve, cytokine / chemokine / growth factor production can be the measured level of a cytokine / chemokine / growth factor, or a cytokine / chemokine / growth factor in a combination of a cytokine, a chemokine, a growth factor, or a cytokine and a chemokine, or a cytokine and a growth factor, or a chemokine and a growth factor, or a cytokine, a chemokine, and a growth factor.Typically, the levels of the same cytokine / chemokine / growth factor, or the levels of the same cytokine, chemokine, growth factor, or cytokine and chemokine, or cytokine and growth factor, or chemokine and growth factor, or cytokine, chemokine and growth factor in the same cytokine / chemokine / growth factor combination, are measured for the test condition and the control condition or standard curve condition.

[0191] In some embodiments, compared with the production under control conditions (i.e., the absence of IL-40 or IL-40+ co-stimulator), the production of one or more cytokines, chemokines and / or growth factors under test conditions (i.e., under IL-40 stimulation or under IL-40+ co-stimulator stimulation) increases. In some embodiments, compared with the production under control conditions, the production of one or more cytokines, chemokines and / or growth factors under test conditions increases by at least about 10%. For example, compared with the production under control conditions, the production of one or more cytokines, chemokines and / or growth factors under test conditions can increase by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more. In some embodiments, compared with the production under control conditions, the production of one or more cytokines, chemokines and / or growth factors under test conditions increases by at least about 2 times. For example, production of one or more cytokines, chemokines and / or growth factors under test conditions can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more compared to production under control conditions.

[0192] In some embodiments, the method includes contacting a cell or a cell population with IL-40, detecting the differentiation of the cell or the differentiation of one or more cells in the population, and evaluating the activity of IL-40 according to the differentiation. In some embodiments, the method includes contacting an immune cell population with IL-40, detecting the differentiation of one or more immune cells in the population, and evaluating the activity of IL-40 according to the differentiation. In some embodiments, the method includes contacting a monocyte population with IL-40, detecting the differentiation of monocytes in the population into macrophages, and evaluating the activity of IL-40 according to the differentiation of monocytes into macrophages. Macrophage differentiation may refer to any stage of macrophage differentiation, including but not limited to M1, M2a, M2b, M2c, and M2d.

[0193] The differentiation of monocytes to macrophages can be detected according to changes in cell morphology. For example, the differentiation of monocytes to macrophages may be characterized by attachment / adhesion to a culture plate, cells with a flat and / or stretched appearance, cells with tentacles / extensions / protrusions, and / or cells with irregular shapes (compared to spherical or substantially spherical monocytes). In some embodiments, compared with the differentiation of monocytes to macrophages under control conditions (i.e., the absence of IL-40 or IL-40+co-stimulators), there is no or substantially no (e.g., less than 1%) monocytes to macrophages, under test conditions (i.e., under IL-40 stimulation or under IL-40+co-stimulators stimulation), it is observed that a portion of cells in the group undergo monocyte differentiation to macrophages. For example, it can be observed under test conditions that at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of cells in the group undergo monocyte differentiation to macrophages. In some embodiments, at least about 10% of cells in the population are observed to differentiate into macrophages from monocytes under IL-40 stimulation. In some embodiments, at least about 30% of cells in the population are observed to differentiate into macrophages from monocytes under IL-40+co-stimulator stimulation.

[0194] The differentiation of monocytes to macrophages can be detected according to the expression and / or generation change of one or more markers, cytokines, chemokines and / or growth factors. In some embodiments, the differentiation of monocytes to macrophages is detected according to the expression (presence or absence, increase or decrease) of one or more markers (such as cell surface markers). Any suitable method for detecting cell surface proteins (for example, immunoassay (ELISA), flow cytometry) and / or mRNA expression (for example, reverse transcriptase quantitative PCR) can be used to detect and / or the expression of quantitative markers. In some embodiments, the differentiation of monocytes to macrophages is detected according to the generation (presence or absence, increase or decrease) of one or more cytokines, chemokines and / or growth factors. The generation of cytokines, chemokines and / or growth factors can be detected and / or quantitative using any suitable method (for example, detection method described herein) for detecting secretory proteins. The presence, absence, increase and decrease of markers, cytokines, chemokines and / or growth factors can be determined by measuring the expression or production level under test conditions (i.e., stimulation by IL-40 or stimulation by IL-40 + a co-stimulator) compared to the expression or production level under control conditions (i.e., in the absence of IL-40 or in the absence of IL-40 + a co-stimulator).

[0195] Examples of markers that can be used to identify monocyte differentiation into macrophages are provided in Table 3 below.

[0196]

[0197] Examples of cytokines and chemokines that can be used to identify monocytes to macrophage differentiation are provided in Table 4 below.

[0198]

[0199] In some embodiments, the differentiation of monocytes into macrophages is detected according to the expression of one or more markers selected from CD14, Ly6C, CD115, CD15s, CD33, CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R. In some embodiments, the differentiation of monocytes into macrophages is detected according to the increased expression of one or more markers selected from CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R. In some embodiments, the differentiation of monocytes into macrophages is detected according to the decreased expression of one or more markers selected from CD14, Ly6C, CD115, CD15s and CD33. In some embodiments, the differentiation of monocytes into macrophages is detected according to the increased production of one or more chemokines selected from CCL10, CCL11, CCL5, CCL8, CCL9, CCL2, CCL3, CCL4, CCL17, CCL22, CCL24, CCL1, CCR2, CCL5, CXCL10, CXCL8, CXCL9 and CXCL16. In some embodiments, the differentiation of monocytes into macrophages is detected according to the increased production of one or more cytokines selected from TNF, IL-1β, IL-6, IL-12, IL-23, IL-10, TGF-β, IL-1RA, IL-1, TNF-α and IL-10.

[0200] Methods for inducing cell differentiation

[0201] Some methods herein include inducing cell differentiation. Cell differentiation can be induced in vitro or in vitro according to the methods herein. In some embodiments, cell differentiation is induced by contacting cells or cell groups with IL-40. Some methods herein include inducing immune cell differentiation. In some embodiments, immune cell differentiation is induced by contacting immune cells or immune cell groups with IL-40. Some methods herein include inducing monocytes to differentiate into macrophages. In some embodiments, monocytes are induced to differentiate into macrophages by contacting monocytes or monocyte groups with IL-40. Macrophage differentiation may refer to any stage of macrophage differentiation, including but not limited to M1, M2a, M2b, M2c and M2d.

[0202] The differentiation of monocytes into macrophages can be detected based on changes in cell morphology. Methods for evaluating changes in cell morphology are described herein. The differentiation of monocytes into macrophages can be detected based on changes in the expression and / or production of one or more markers, cytokines, chemokines, and / or growth factors. Markers, cytokines, chemokines, and / or growth factors that can be used to detect the differentiation of monocytes into macrophages, as well as methods for detecting / quantifying their expression / production are described herein.

[0203] Reagent test kit

[0204] In certain embodiments, a kit is provided. The kit may include any component and composition described herein that can be used to perform any method described herein in any suitable combination (e.g., IFN-γ, GM-CSF, IFN-α, IL-1β, IL-4, IL-10, IL-13, M-CSF, TGF-β and / or TNF-α; one or more components, cells or cell groups for measuring cytokines, chemokines and / or growth factors). In some embodiments, the kit also includes IL-40 (e.g., used as a standard / control and / or for users to generate a standard curve). The IL-40 for testing can be provided by the user / purchaser of the kit. The kit may also include any reagent, buffer or other component that can be used to implement any method described herein. For example, the kit may include one or more binding molecules that immunospecifically bind to one or more cytokines, chemokines and / or growth factors under binding conditions.

[0205] The components of the kit may be present in separate containers, or multiple components may be present in a single container. Suitable containers include single tubes (e.g., vials), cell culture plates, one or more wells of a plate (e.g., 6-well plates, 12-well plates, 24-well plates, 96-well plates, 384-well plates, etc.), etc.

[0206] The kit may also include instructions for performing one or more methods described herein and / or the description of one or more components described herein. The instructions and / or description may be in printed form and may be included in a kit insert. In some embodiments, the instructions and / or description are provided as electronic storage data files present in a suitable computer-readable storage medium (e.g., a portable flash drive, a DVD, a CD-ROM, a disk, etc.). The kit may also include a written description of an internet location providing such instructions or descriptions.

[0207] Example

[0208] The examples set forth below illustrate certain implementations without limiting the technology.

[0209] Example 1: Cytokine and chemokine production and cell morphological changes of THP-1 cells under IL-40 stimulation

[0210] In this example, certain cytokines and chemokines produced by THP-1 cells under IL-40 stimulation and IFN-γ+IL-40 co-stimulation were measured. Changes in cell morphology of THP-1 cells under IL-40 stimulation and IFN-γ+IL-40 co-stimulation were also observed.

[0211] Materials and methods

[0212] Recombinant protein

[0213] Nucleic acid encoding c17orf99 (chromosome 17 open reading frame 99) [Homo sapiens (human) NCBI reference sequence: NP_001156547], also known as IL-40 or UNQ464, was used to produce recombinant human protein I (rhIL-40). The protein contains an Fc portion at the C-terminus for recovery and purification purposes. The rhFc portion from IgG1 protein was used as a control. Recombinant proteins, rhFc, rhIL-40, IFN-γ (BioLegend Catalog No. 570202), IFN-α (BioLegend Catalog No. 592702), IL-4 (BioLegend Catalog No. 592702), IL-13 (BioLegend Catalog No. 571102), TGF-β (BioLegend Catalog No. 580704), M-CSF (BioLegend Catalog No. 574802), GM-CSF (BioLegend Catalog No. 572902), IL-10 (BioLegend Catalog No. 715602), TNF-α (BioLegend Catalog No. 570102), and IL-1β (BioLegend Catalog No. 579402) were obtained from BioLegend.

[0214] Cell culture

[0215] The THP-1 cell line was cultured and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2 in a humidified atmosphere. For stimulation experiments, THP-1 cells were cultured in RPMI-1640 medium supplemented with 1.1% FBS. 30,000 THP-1 cells were seeded in 96-well plates and stimulated with the same micromolar concentration of rhIL-40 (BioLegend, CA) or rhFc part (BioLegend, CA) in the presence or absence of IFN-γ (250 or 50 ng / mL), IFN-α, IL-4, Il-13, TGF-β, M-CSF, GM-CSF, IL-10, TNF-α or IL-1β (50 ng / mL) for 24, 48 or 72 hours in a final volume of 200 μL.

[0216] Healthy human PBMCs were isolated from whole blood by density centrifugation. Cells were cultured in RPMI-1640 medium supplemented with 1.1% FBS. 30,000 PBMCs were seeded in 96-well plates and stimulated with the same micromolar concentration of rhIL-40 (BioLegend, CA) or rhFc fraction (control) for 24 hours in a final volume of 200 μL.

[0217] Cytokine and chemokine quantification

[0218] Concentrations of CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, IL-12p70, IL-12p40, TNF-α, IL-6, IL-4, IL-10, IL-1β, arginase, IL-1RA, and IL-23 were measured in cell culture supernatants using LEGEND MAX ELISA kits with pre-coated plates, or Human Macrophage / Microglia and Chemokine Inflammatory LEGENDplex plates. The protocol followed the supplier's recommendations.

[0219] Cell phenotype

[0220] The morphological changes of THP-1 cells after rhIL-40 stimulation for 24 hours were recorded using a Keyence BZ-X700 microscope in phase contrast mode and 10X lens resolution.

[0221] Data analysis

[0222] The effects of the different stimuli were normalized. To this end, the value in pg / mL for each molecule was divided by the value obtained under control conditions (unstimulated cells).

[0223] To quantify the effect of rhIL-40 or rhIL40+IFN-γ on THP-1 monocyte morphology, count the total number of cells and cells attached to the plate surface in several fields of view. Determine the percentage of cells with morphological changes using the following calculation:

[0224] (Number of attached cells x 100%) / total number of cells.

[0225] result

[0226] CXCL8 production

[0227] The production of CXCL8 was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (0.29 μM, 0.58 μM, 1.16 μM, 2.31 μM, 4.63 μM, 9.25 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 h, and the results are shown in Figure 1 When cells were stimulated with rhIL-40 or rhIL-40+IFN-γ, a dose-dependent production of CXCL8 was observed. A higher effect was observed after stimulation with rhIL-40+IFN-γ.

[0228] CXCL9 production

[0229] The production of CXCL9 was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (1.16 μM, 4.63 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 hours, and the results are shown in Figure 2 When THP-1 cells were stimulated with rhIL-40+IFN-γ, a significant increase in CXCL9 production was observed. In addition, a lower effect of CXCL9 secretion was observed in cells stimulated with rhFc+IFN-γ.

[0230] CCL2 production

[0231] The production of CCL2 was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (1.16 μM, 4.63 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 hours, and the results are shown in Figure 3When stimulated with rhIL-40, a dose-dependent production of CCL2 was observed. The maximum detection level of CCL2 was reached when cells were stimulated with rhIL-40+IFN-γ, regardless of the rhIL-40 concentration. In addition, a lower effect of CCL2 secretion was observed in cells stimulated with rhFc+IFN-γ.

[0232] CCL3 production

[0233] The production of CCL3 was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (1.16 μM, 4.63 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 hours, and the results are shown in Figure 4 After stimulation with rhIL-40, a dose-dependent production of CCL3 was observed. A synergistic effect was observed when stimulating with rhIL-40+IFN-γ. Similar effects were observed using the two highest concentrations evaluated for rhIL-40. Effects of CCL3 secretion were also observed in cells stimulated with rhFc+IFN-γ.

[0234] CCL4 production

[0235] The production of CCL4 was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (1.16 μM, 4.63 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 hours, and the results are shown in Figure 5 When stimulated with rhIL-40, a dose-dependent production of CCL4 was observed. A synergistic effect was observed when stimulated with rhIL-40+IFN-γ. Similar effects were observed using the two highest concentrations evaluated for rhIL-40. Effects of CCL4 secretion were also observed in cells stimulated with rhFc+IFN-γ.

[0236] CCL5 production

[0237] The production of CCL5 was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (1.16 μM, 4.63 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 hours, and the results are shown in Figure 6 When stimulated with rhIL-40 or rhIL-40+IFN-γ, an increase in CCL5 production was observed. The effect of CCL5 secretion was also observed in cells stimulated with rhFc and rhFc+IFN-γ.

[0238] CXCL10 production

[0239] The production of CXCL10 was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (1.16 μM, 4.63 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 hours, and the results are shown in Figure 7 When THP-1 cells were stimulated with rhIL40, rhFc+IFN-γ or rhIL-40+IFN-γ, a significant increase in CXCL10 production was observed. rhIL-40 alone was able to induce the production of CXCL10. The production of CXCL10 was exacerbated in the presence of IFN-γ, which showed higher production when combined with rhIL-40.

[0240] IL1RA production

[0241] The production of IL1RA was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (0.58 μM, 2.31 μM, 4.63 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 hours, and the results are shown in Figure 8 When stimulated with rhIL-40 or rhIL-40+IFN-γ, a dose-dependent production of IL1RA was observed. A synergistic effect was observed when stimulated with rhIL-40+IFN-γ. An effect on IL1RA secretion was also observed in cells stimulated with rhFc+IFN-γ.

[0242] IL-6 production

[0243] The production of IL-6 was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (0.58 μM, 2.31 μM, 4.63 μM or 18.5 μM) ± IFN-γ (250 ng / mL) for 24 hours, and the results are shown in Fig. 9 When THP-1 cells were stimulated with rhIL-40+IFN-γ, a significant increase in IL-6 production was observed.

[0244] Similar to the effects of chemokine and growth factor production by THP-1 cells stimulated with rhIL40 and IFN-γ, an enhanced production of several molecules was observed when cells were stimulated with rhIL40 and IL-10, GM-CSF, TNF-α, or IL-1β for 24 h.

[0245] Erythropoietin production

[0246] Erythropoietin production was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (4.63 μM) ± 50 ng / mL of IL-4, IL-13, IFN-α, TGF-β, M-CSF, IL-10, GM-CSF, IFN-γ, TNF-α, or IL-1β for 24 h, and the results are shown in Fig.10 A significant increase in erythropoietin production was observed when THP-1 cells were stimulated with rhIL-40, and its production was exacerbated when the cells were co-stimulated with rhIL40 plus TGF-β, M-CSF, IL-10, GM-CSF, IFN-γ, or IL-1β.

[0247] PDGF-AA production

[0248] The production of PDGF-AA was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL40 (4.63 μM) ± 50 ng / mL of IL-4, Il-13, IFN-α, TGF-β, M-CSF, IL-10, GM-CSF, IFN-γ, TNF-α, or IL-1β for 24 h, and the results are shown in Fig.11 A significant increase in PDGF-AA production was observed when THP-1 cells were stimulated with rhIL-40, and its production was exacerbated when cells were co-stimulated with rhIL40 plus IFN-γ, TGF-β, IL-10, or IL-1β.

[0249] VEGF production

[0250] VEGF production was measured in the cell supernatant of THP-1 cells stimulated with different concentrations of recombinant protein rhFc or rhIL-40 (4.63 μM) ± 250 ng / mL of IL-4, IL-13, IFN-α, TGF-β, M-CSF, IL-10, GM-CSF, IFN-γ, TNF-α, or IL-1β for 24 h, and the results are shown in Fig.12 A significant increase in VEGF production was observed when THP-1 cells were stimulated with rhIL-40, and its production was exacerbated when cells were co-stimulated with rhIL40 plus IL-4, IL-13, M-CSF, GM-CSF, TNF-α, or IL-1β.

[0251] Production of various cytokines, chemokines and growth factors

[0252] THP-1 cells

[0253] THP-1 cells were stimulated with recombinant protein rhFc or rhIL-40 (4.63 μM) ± IFN-γ (250 ng / mL), IFN-γ alone for 24 hours or without stimulation (control). Multiplexing technology (LEGENDplex) was used to evaluate the changes in the production of 23 cytokines, chemokines or growth factors in the supernatant by ELISA or flow cytometry. The difference was calculated by normalizing the mean value (pg / mL) of each stimulation with the value under the control condition. When cells were stimulated with rhIL-40, increased secretion of CCL3, CXCL8, CCL4, CCL2, CXCL10, CCL11, CCL5, IL-1RA, erythropoietin, PDGF-AA and VEGF was observed. It was found that when cells were stimulated with rhIL-40 + IFN-γ, the secretion of CCL3, CXCL8, CCL4, CCL2, CXCL10, CCL11, CCL5, CXCL9, CXCL1, CXCL11, CXCL5, CCL20, CCL17, IL-1RA, IL-6, erythropoietin and PDGF-AA was significantly different compared to the rest of the stimulations. IFN-γ and rhFc + IFN-γ also had an effect on several of the molecules evaluated. The results are shown in Table 5 below.

[0254]

[0255]

[0256] PBMC

[0257] PBMCs were isolated from healthy donor samples and stimulated with rhFc or IL-40 peptide at equimolar concentrations for 24 hours. The results are shown in Fig.13 The changes in the production of 13 chemokines in the supernatant were evaluated by ELISA or flow cytometry using multiplexing technology (LEGENDplex). The differences were calculated by normalizing the mean value (pg / mL) of each stimulation to the value under control conditions. When cells were stimulated with rhIL-40, a significant increase in the secretion of CXCL1, CXCL5, CXCL8, CCL2, CCL3, CCL4, CCL17, and CCL20 was observed.

[0258] Cell morphology

[0259] The morphological changes of THP-1 cells induced by rhIL-40 were observed in the presence or absence of IFN-γ. THP-1 cells were treated with IFN-γ (250 ng / mL) ( Fig.14 , Figure B), rhFc (2.31uM) ( Fig.14 , Figure C), rhFc+IFN-γ( Fig.14, Figure D), rhIL-40 (2.31uM) ( Fig.14 , Figure E), rhIL-40+IFN-γ( Fig.14 , Figure F) stimulated for 24 hours or unstimulated ( Fig.14 , Figure A). Morphological changes were recorded by optical microscopy (Keyence BZ-X700) in phase contrast mode (magnification 10X). The percentage of cells with morphological changes was calculated by counting cells and using the following formula: (number of cells attached to the plate × 100%) / total number of cells. Fig.14 Figure B (13%) Fig.14 Figure E (10%) and Fig.14 Differences in cell shape and attachment to the plate were observed in Figure F (35%). IFN-γ and IL-40 showed a synergistic effect. Fig.14 Figure A, or Fig.14 No changes in cell morphology were observed in Figure C.

[0260] Similarly, under the co-stimulation of IL-10, TNF-α and IL-1β, the morphological changes of rhIL-40-induced THP-1 cells were observed in the presence of IFN-γ. THP-1 cells were stimulated for 24 hours with each cytokine (50ng / mL), rhFc (2.31uM), rhFc+each cytokine, rhIL-40 (2.31uM), rhIL-40+each cytokine, or the cells were not stimulated. Morphological changes were recorded in phase contrast mode (magnification 10X) by optical microscopy (Keyence BZ-X700). The number of macrophages / field of view is the average value of 4 different fields of view under the same conditions.

[0261] Cell surface molecules

[0262] THP-1 cells were stimulated with rhFc, rhIL-40, IFN-γ, IFN-γ+rhFc, or IFN-γ+rh-IL-40, and the results are shown in Fig.15 Stimulation with rhIL40 plus co-stimulatory cytokines such as IFN-γ induced overexpression of co-stimulatory proteins such as HLA-A, B, C (Figure A), CD40 (Figure B), and CD11b (Figure C).

[0263] Identification of the IL-40 receptor

[0264] IL-40 receptor expression was observed in THP-1 cells and CD11b+ cells from healthy donors, and the results are shown in Fig.16To identify positive cells, cells were incubated with rhIL-40 polypeptide conjugated to an Fc tag for 30 min at 4°C, followed by incubation with a secondary antibody anti-tag conjugated to an APC fluorophore for 15 min at 4°C before flow cytometry analysis.

[0265] Example 2: Examples of amino acid and nucleic acid sequences

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278] Example 3: Example of implementation

[0279] The examples set forth below illustrate certain implementations without limiting the technology.

[0280] A1. A method for evaluating the activity of an IL-40 polypeptide, comprising:

[0281] a) contacting a cell with a first composition comprising an IL-40 polypeptide;

[0282] b) measuring the production of one or more cytokines and / or chemokines selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CCL11, CXCL8, CXCL10 and IL-1RA by the cells, thereby measuring cytokine production; and

[0283] c) detecting the activity of the IL-40 polypeptide in the first composition according to the cytokine production measured in (b).

[0284] A2. A method according to embodiment A1, wherein the production of the one or more cytokines and / or chemokines is increased compared to the production of cells not contacted with the first composition.

[0285] A3. A method according to embodiment A1 or A2, wherein (a) comprises contacting the cell with a second composition comprising a co-stimulatory agent.

[0286] A4. A method according to embodiment A3, wherein the co-stimulatory agent comprises IFN-γ.

[0287] A5. A method according to embodiment A3 or A4, wherein (a) comprises contacting the cell with the first composition and the second composition simultaneously.

[0288] A6. A method according to embodiment A3 or A4, wherein (a) comprises contacting the cell with the second composition before contacting the cell with the first composition.

[0289] A7. The method of any one of embodiments A3 to A6, wherein the production of the one or more cytokines and / or chemokines is increased compared to production in cells not contacted with the first composition and the second composition.

[0290] A8. A method for evaluating the activity of an IL-40 polypeptide, comprising:

[0291] a) contacting a cell with a first composition comprising an IL-40 polypeptide and a second composition comprising a co-stimulatory agent;

[0292] b) measuring the production of one or more cytokines and / or chemokines selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, IL-1RA and IL-6 by the cells, thereby measuring cytokine production; and

[0293] c) detecting the activity of the IL-40 polypeptide in the first composition according to the cytokine production measured in (b).

[0294] A9. A method according to embodiment A8, wherein the production of the one or more cytokines and / or chemokines is increased compared to the production of cells not contacted with the first composition and the second composition.

[0295] A10. The method of embodiment A8 or A9, wherein the co-stimulatory agent comprises IFN-γ.

[0296] A11. The method of any one of embodiments A8 to A10, wherein (a) comprises contacting the cell with the first composition and the second composition simultaneously.

[0297] A12. The method of any one of embodiments A8 to A10, wherein (a) comprises contacting the cell with the second composition before contacting the cell with the first composition.

[0298] A13. The method of any one of embodiments A1 to A12, wherein the cells are from a subject.

[0299] A14. The method of any one of embodiments A1 to A12, wherein the cell is from a cell line.

[0300] A15. The method of any one of embodiments A1 to A14, wherein the cell is an isolated cell.

[0301] A16. The method of any one of embodiments A1 to A15, wherein the cell is an immune cell.

[0302] A17. A method according to embodiment A16, wherein the cells are monocytes.

[0303] A18. A method according to embodiment A16, wherein the cell is a macrophage.

[0304] A19. The method of any one of embodiments A1 to A15, wherein the cell is a non-immune cell.

[0305] A20. A method according to embodiment A19, wherein the cell is a stromal cell or a cell derived from the central nervous system.

[0306] A21. A method according to any one of embodiments A1 to A20, wherein the method is performed ex vivo or in vitro.

[0307] A22. The method of any one of embodiments A1 to A21, wherein the IL-40 polypeptide is a recombinant IL-40 polypeptide.

[0308] A23. The method of any one of embodiments A1 to A21, wherein the IL-40 polypeptide is a human IL-40 polypeptide.

[0309] A24. A method according to embodiment A23, wherein the IL-40 polypeptide is a recombinant human IL-40 polypeptide.

[0310] A25. The method of embodiment A23 or A24, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

[0311] A26. The method of embodiment A23 or A24, wherein the IL-40 polypeptide comprises amino acids 21-265 of SEQ ID NO:1.

[0312] A27. The method of embodiment A23 or A24, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO: 1.

[0313] A28. The method of embodiment A23 or A24, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

[0314] A29. A method according to embodiment A23 or A24, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:1.

[0315] A30. The method of any one of embodiments A1 to A21, wherein the IL-40 polypeptide is a mouse IL-40 polypeptide.

[0316] A31. A method according to embodiment A30, wherein the IL-40 polypeptide is a recombinant mouse IL-40 polypeptide.

[0317] A32. A method according to embodiment A30 or A31, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO:3.

[0318] A33. The method of embodiment A30 or A31, wherein the IL-40 polypeptide comprises amino acids 19-252 of SEQ ID NO:3.

[0319] A34. A method according to embodiment A30 or A31, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:3.

[0320] A35. A method according to embodiment A30 or A31, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:3.

[0321] A36. A method according to embodiment A30 or A31, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:3.

[0322] A37. The method of any one of embodiments A1 to A36, wherein the IL-40 polypeptide comprises one or more chemical modifications.

[0323] A38. The method of any one of embodiments A1 to A37, wherein the IL-40 polypeptide comprises a tag.

[0324] A39. The method of any one of embodiments A1 to A38, wherein the IL-40 polypeptide comprises a detectable label.

[0325] A40. The method of any one of embodiments A1 to A39, wherein the IL-40 polypeptide comprises a fusion polypeptide.

[0326] B1. A method for evaluating the activity of an IL-40 polypeptide, comprising:

[0327] a) contacting a population of monocytes with a first composition comprising an IL-40 polypeptide;

[0328] b) detecting differentiation of monocytes in the population into macrophages; and

[0329] c) evaluating the activity of the IL-40 polypeptide in the first composition based on the differentiation of the monocytes into macrophages detected in (b).

[0330] B2. A method according to embodiment B1, wherein the differentiation of monocytes into macrophages is detected based on changes in cell morphology.

[0331] B3. A method according to embodiment B2, wherein at least about 10% of the cells in the population show changes in cell morphology.

[0332] B4. A method according to embodiment B1, wherein the differentiation of monocytes into macrophages is detected based on the expression of one or more markers selected from CD14, Ly6C, CD115, CD15s, CD33, CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R.

[0333] B4.1 A method according to embodiment B1, wherein the differentiation of monocytes into macrophages is detected based on the increased expression of one or more markers selected from CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R.

[0334] B4.2 A method according to embodiment B1, wherein the differentiation of monocytes into macrophages is detected based on the reduced expression of one or more markers selected from CD14, Ly6C, CD115, CD15s and CD33.

[0335] B5. A method according to embodiment B1, wherein the differentiation of monocytes into macrophages is detected based on the increased production of one or more chemokines selected from CCL10, CCL11, CCL5, CCL8, CCL9, CCL2, CCL3, CCL4, CCL17, CCL22, CCL24, CCL1, CCR2, CCL5, CXCL10, CXCL8, CXCL9 and CXCL16.

[0336] B5.1 A method according to embodiment B1, wherein the differentiation of monocytes into macrophages is detected based on the increased production of one or more cytokines selected from TNF, IL-1β, IL-6, IL-12, IL-23, IL-10, TGF-β, IL-1RA, IL-1, TNF-α and IL-10.

[0337] B6. A method according to embodiment B1, wherein (a) comprises contacting the monocyte population with a second composition comprising a co-stimulatory agent.

[0338] B7. A method according to embodiment B6, wherein the co-stimulatory agent comprises IFN-γ.

[0339] B8. A method according to embodiment B6 or B7, wherein (a) comprises contacting the monocyte population with the first composition and the second composition simultaneously.

[0340] B9. A method according to embodiment B6 or B7, wherein (a) comprises contacting the monocyte population with the second composition before contacting the cells with the first composition.

[0341] B10. A method according to any one of embodiments B6 to B9, wherein the differentiation of the monocytes into macrophages is detected based on changes in cell morphology.

[0342] B11. A method according to embodiment B10, wherein at least about 30% of the cells in the population show changes in cell morphology.

[0343] B12. A method according to any one of embodiments B6 to B9, wherein the differentiation of monocytes into macrophages is detected based on the expression of one or more markers selected from CD14, Ly6C, CD115, CD15s, CD33, CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R.

[0344] B12.1 A method according to any one of embodiments B6 to B9, wherein the differentiation of monocytes into macrophages is detected based on the increased expression of one or more markers selected from CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R.

[0345] B12.2 A method according to any one of embodiments B6 to B9, wherein the differentiation of monocytes into macrophages is detected based on the reduced expression of one or more markers selected from CD14, Ly6C, CD115, CD15s and CD33.

[0346] B13. A method according to any one of embodiments B6 to B9, wherein the differentiation of monocytes into macrophages is detected based on the increased production of one or more chemokines selected from CCL10, CCL11, CCL5, CCL8, CCL9, CCL2, CCL3, CCL4, CCL17, CCL22, CCL24, CCL1, CCR2, CCL5, CXCL10, CXCL8, CXCL9 and CXCL16.

[0347] B13.1 A method according to any one of embodiments B6 to B9, wherein the differentiation of monocytes into macrophages is detected based on the increased production of one or more cytokines selected from TNF, IL-1β, IL-6, IL-12, IL-23, IL-10, TGF-β, IL-1RA, IL-1, TNF-α and IL-10.

[0348] B14. A method according to any one of embodiments B1 to B13.1, wherein the monocyte population is from a subject.

[0349] B15. A method according to any one of embodiments B1 to B13.1, wherein the monocyte population is from a cell line.

[0350] B16. A method according to any one of embodiments B1 to B15, wherein the monocyte population comprises isolated monocytes.

[0351] B17. A method according to any one of embodiments B1 to B16, wherein the method is performed ex vivo or in vitro.

[0352] B18. The method of any one of embodiments B1 to B17, wherein the IL-40 polypeptide is a recombinant IL-40 polypeptide.

[0353] B19. The method according to any one of embodiments B1 to B17, wherein the IL-40 polypeptide is a human IL-40 polypeptide.

[0354] B20. A method according to embodiment B19, wherein the IL-40 polypeptide is a recombinant human IL-40 polypeptide.

[0355] B21. The method of embodiment B19 or B20, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

[0356] B22. The method of embodiment B19 or B20, wherein the IL-40 polypeptide comprises amino acids 21-265 of SEQ ID NO:1.

[0357] B23. A method according to embodiment B19 or B20, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO: 1.

[0358] B24. A method according to embodiment B19 or B20, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

[0359] B25. A method according to embodiment B19 or B20, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

[0360] B26. The method of any one of embodiments B1 to B17, wherein the IL-40 polypeptide is a mouse IL-40 polypeptide.

[0361] B27. A method according to embodiment B26, wherein the IL-40 polypeptide is a recombinant mouse IL-40 polypeptide.

[0362] B28. A method according to embodiment B26 or B27, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO:3.

[0363] B29. The method of embodiment B26 or B27, wherein the IL-40 polypeptide comprises amino acids 19-252 of SEQ ID NO:3.

[0364] B30. A method according to embodiment B26 or B27, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:3.

[0365] B31. A method according to embodiment B26 or B27, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:3.

[0366] B32. A method according to embodiment B26 or B27, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 3.

[0367] B33. The method of any one of embodiments B1 to B32, wherein the IL-40 polypeptide comprises one or more chemical modifications.

[0368] B34. The method of any one of embodiments B1 to B33, wherein the IL-40 polypeptide comprises a tag.

[0369] B35. The method of any one of embodiments B1 to B34, wherein the IL-40 polypeptide comprises a detectable label.

[0370] B36. A method according to any one of embodiments B1 to B35, wherein the IL-40 polypeptide comprises a fusion polypeptide.

[0371] C1. A method for inducing monocytes to differentiate into macrophages, comprising contacting the monocytes with a first composition comprising an IL-40 polypeptide.

[0372] C2. The method according to embodiment C1, wherein said differentiation of monocytes into macrophages is characterized by changes in cell morphology.

[0373] C3. A method according to embodiment C2, wherein at least about 10% of the cells in the monocyte population show changes in cell morphology.

[0374] C4. A method according to embodiment C1, wherein the differentiation of monocytes into macrophages is characterized by the expression of one or more markers selected from CD14, Ly6C, CD115, CD15s, CD33, CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R.

[0375] C5. A method according to embodiment C1, wherein the differentiation of monocytes into macrophages is characterized by increased expression of one or more markers selected from CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R.

[0376] C6. A method according to embodiment C1, wherein the differentiation of monocytes into macrophages is characterized by reduced expression of one or more markers selected from CD14, Ly6C, CD115, CD15s and CD33.

[0377] C7. A method according to embodiment C1, wherein the differentiation of monocytes into macrophages is characterized by increased production of one or more chemokines selected from CCL10, CCL11, CCL5, CCL8, CCL9, CCL2, CCL3, CCL4, CCL17, CCL22, CCL24, CCL1, CCR2, CCL5, CXCL10, CXCL8, CXCL9 and CXCL16.

[0378] C8. A method according to embodiment C1, wherein the differentiation of monocytes into macrophages is characterized by increased production of one or more cytokines selected from TNF, IL-1β, IL-6, IL-12, IL-23, IL-10, TGF-β, IL-1RA, IL-1, TNF-α and IL-10.

[0379] C9. A method according to embodiment C1, which comprises contacting the monocytes with a second composition comprising a co-stimulatory agent.

[0380] C10. A method according to embodiment C9, wherein the co-stimulatory agent comprises IFN-γ.

[0381] C11. A method according to embodiment C9 or C10, comprising contacting the monocytes with the first composition and the second composition simultaneously.

[0382] C12. The method of embodiment C9 or C10, comprising contacting the monocytes with the second composition before contacting the monocytes with the first composition.

[0383] C13. A method according to any one of embodiments C9 to C12, wherein the differentiation of monocytes into macrophages is characterized by changes in cell morphology.

[0384] C14. A method according to embodiment C13, wherein at least about 30% of the cells in the monocyte population show changes in cell morphology.

[0385] C15. A method according to any one of embodiments C9 to C12, wherein the differentiation of monocytes into macrophages is characterized by the expression of one or more markers selected from CD14, Ly6C, CD115, CD15s, CD33, CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R.

[0386] C16. A method according to any one of embodiments C9 to C12, wherein the differentiation of monocytes into macrophages is characterized by increased expression of one or more markers selected from CD44, CD81, CD49e, CD18, CD11b, CD54 (ICAM-1), CD11c, CD68, CD80, CD86, CD163, IL-1R and CD200R.

[0387] C17. A method according to any one of embodiments C9 to C12, wherein the differentiation of monocytes into macrophages is characterized by reduced expression of one or more markers selected from CD14, Ly6C, CD115, CD15s and CD33.

[0388] C18. A method according to any one of embodiments C9 to C12, wherein the differentiation of monocytes into macrophages is characterized by increased production of one or more chemokines selected from CCL10, CCL11, CCL5, CCL8, CCL9, CCL2, CCL3, CCL4, CCL17, CCL22, CCL24, CCL1, CCR2, CCL5, CXCL10, CXCL8, CXCL9 and CXCL16.

[0389] C19. A method according to any one of embodiments C9 to C12, wherein the differentiation of monocytes into macrophages is characterized by increased production of one or more cytokines selected from TNF, IL-1β, IL-6, IL-12, IL-23, IL-10, TGF-β, IL-1RA, IL-1, TNF-α and IL-10.

[0390] C20. A method according to any one of embodiments C1 to C19, wherein the monocytes are from a subject.

[0391] C21. A method according to any one of embodiments C1 to C19, wherein the monocytes are from a cell line.

[0392] C22. The method of any one of embodiments C1 to C19, wherein the monocytes are isolated monocytes.

[0393] C23. A method according to any one of embodiments C1 to C22, wherein the method is performed ex vivo or in vitro.

[0394] C24. The method of any one of embodiments C1 to C23, wherein the IL-40 polypeptide is a recombinant IL-40 polypeptide.

[0395] C25. The method of any one of embodiments C1 to C23, wherein the IL-40 polypeptide is a human IL-40 polypeptide.

[0396] C26. A method according to embodiment C25, wherein the IL-40 polypeptide is a recombinant human IL-40 polypeptide.

[0397] C27. A method according to embodiment C25 or C26, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or amino acids 21-265 of SEQ ID NO: 1; or a functional fragment thereof; or a modified polypeptide thereof; or a modified functional fragment thereof.

[0398] C28. A method according to any one of embodiments C1 to C23, wherein the IL-40 polypeptide is a mouse IL-40 polypeptide.

[0399] C29. A method according to embodiment C28, wherein the IL-40 polypeptide is a recombinant mouse IL-40 polypeptide.

[0400] C30. A method according to embodiment C28 or C29, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or amino acids 19-252 of SEQ ID NO: 3; or a functional fragment thereof; or a modified polypeptide thereof; or a modified functional fragment thereof.

[0401] D1. A kit comprising:

[0402] a) a first composition comprising an IFN-γ polypeptide;

[0403] b) one or more components for measuring cytokine and / or chemokine production, wherein the cytokines and / or chemokines are selected from one or more of CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, IL-1RA and IL-6; and

[0404] c) Instructions for use.

[0405] D2. A kit according to embodiment D1, which also includes a second composition comprising an IL-40 polypeptide.

[0406] D3. A kit according to embodiment D2, wherein the IL-40 polypeptide is a recombinant IL-40 polypeptide.

[0407] D4. A kit according to embodiment D2, wherein the IL-40 polypeptide is a human IL-40 polypeptide.

[0408] D5. A kit according to embodiment D4, wherein the IL-40 polypeptide is a recombinant human IL-40 polypeptide.

[0409] D6. A kit according to embodiment D4 or D5, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO:1.

[0410] D7. A kit according to embodiment D4 or D5, wherein the IL-40 polypeptide comprises amino acids 21-265 of SEQ ID NO:1.

[0411] D8. A kit according to embodiment D4 or D5, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:1.

[0412] D9. A kit according to embodiment D4 or D5, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:1.

[0413] D10. A kit according to embodiment D4 or D5, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:1.

[0414] D11. A kit according to embodiment D2, wherein the IL-40 polypeptide is a mouse IL-40 polypeptide.

[0415] D12. A kit according to embodiment D10, wherein the IL-40 polypeptide is a recombinant mouse IL-40 polypeptide.

[0416] D13. The kit according to embodiment D11 or D12, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO:3.

[0417] D14. The kit according to embodiment D11 or D12, wherein the IL-40 polypeptide comprises amino acids 19-252 of SEQ ID NO:3.

[0418] D15. The kit according to embodiment D11 or D12, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:3.

[0419] D16. A kit according to embodiment D11 or D12, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:3.

[0420] D17. A kit according to embodiment D11 or D12, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:3.

[0421] D18. A kit according to any one of embodiments D2 to D17, wherein the IL-40 polypeptide comprises one or more chemical modifications.

[0422] D19. A kit according to any one of embodiments D2 to D18, wherein the IL-40 polypeptide comprises a tag.

[0423] D20. A kit according to any one of embodiments D2 to D19, wherein the IL-40 polypeptide comprises a detectable label.

[0424] D21. A kit according to any one of embodiments D2 to D20, wherein the IL-40 polypeptide comprises a fusion polypeptide.

[0425] D22. A kit according to any one of embodiments D1 to D21, wherein each of the one or more components for measuring cytokine and / or chemokine production comprises a binding molecule that immunospecifically binds to one of the cytokines and / or chemokines under binding conditions.

[0426] D23. A kit according to any one of embodiments D1 to D22, which also includes a cell or a cell population.

[0427] ***

[0428] The entire contents of each patent, patent application, publication and document cited herein are hereby incorporated by reference. The citation of the above patents, patent applications, publications and documents neither admits that any of the foregoing is relevant prior art nor constitutes any admission of the contents or dates of these publications or documents. Their citation does not indicate the relevant disclosures retrieved. All statements about the date or contents of documents are based on available information and are not admissions of their accuracy or correctness.

[0429] The foregoing may be modified without departing from the basic aspects of the present technology. Although the present technology has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the present technology.

[0430] The technology described illustratively herein can be appropriately practiced in the absence of any element not specifically disclosed herein. Therefore, for example, in each case herein, any one of the terms "comprising", "consisting essentially of ... " and "consisting of ... " can be replaced by any one of the other two terms. The terms and expressions adopted are used as descriptive and non-restrictive terms, and such terms and expressions used do not exclude any equivalent or part of the features shown and described, and various modifications are possible within the scope of the claimed technology. The term "a kind of" or "one" may refer to one or more elements (for example, "a reagent" may mean one or more reagents) modified by it, unless the context clearly describes an element or more than one element. As used herein, the term "about" refers to a value within 10% of the basic parameter (that is, plus or minus 10%), and the term "about" used at the beginning of a series of values ​​modifies each of the values ​​(that is, "about 1, 2 and 3" refers to about 1, about 2 and about 3). For example, the weight of "about 100 grams" can include a weight between 90 grams and 110 grams. In addition, when a list of values ​​is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate values ​​and fractional values ​​thereof (e.g., 54%, 85.4%). Therefore, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, those skilled in the art may adopt modifications and variations of the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the technology.

[0431] Certain embodiments of the technology are set forth in the following claims.

Claims

1. A method for evaluating the activity of an IL-40 polypeptide, comprising: a) contacting a cell with a first composition and a second composition, wherein the first composition comprises an IL-40 polypeptide and the second composition comprises a co-stimulatory agent, wherein the co-stimulatory agent comprises one or more polypeptides selected from the group consisting of GM-CSF, IFN-α, IFN-γ, IL-1β, IL-4, IL-10, IL-13, TNF-α, TGF-β and M-CSF; b) measuring the production of one or more cytokines, chemokines and / or growth factors selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CCL11, CXCL8, CXCL10, IL-1RA, erythropoietin, PDGF-AA, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL11 and VEGF by the cells, thereby measuring the production of cytokines, chemokines and / or growth factors; as well as c) detecting the activity of the IL-40 polypeptide in the first composition based on the production of the cytokines, chemokines and / or growth factors measured in (b).

2. The method of claim 1, wherein (a) comprises contacting the cell with the first composition and the second composition simultaneously.

3. The method of claim 1, wherein (a) comprises contacting the cell with the second composition before contacting the cell with the first composition.

4. The method of claim 1, wherein production of the one or more cytokines, chemokines and / or growth factors is increased compared to production of the one or more cytokines, chemokines and / or growth factors by cells that have not been exposed to the first composition and the second composition.

5. The method of claim 1, wherein the cell is from a subject.

6. The method of claim 1, wherein the cell is from a cell line.

7. The method of claim 1, wherein the cells are isolated cells.

8. The method of claim 1, wherein the cell is an immune cell.

9. The method of claim 8, wherein the cells are monocytes.

10. The method of claim 8, wherein the cell is a macrophage.

11. The method of claim 1, wherein the cell is a non-immune cell.

12. The method of claim 1, wherein the cell is a stromal cell or a cell derived from the central nervous system.

13. The method of claim 1, wherein the method is performed ex vivo or in vitro.

14. The method of claim 1, wherein the IL-40 polypeptide is a recombinant IL-40 polypeptide.

15. The method of claim 1, wherein the IL-40 polypeptide is a human IL-40 polypeptide.

16. The method of claim 15, wherein the IL-40 polypeptide is a recombinant human IL-40 polypeptide.

17. The method of claim 15 or claim 16, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

18. The method of claim 15 or claim 16, wherein the IL-40 polypeptide comprises amino acids 21-265 of SEQ ID NO:

1.

19. The method of claim 15 or claim 16, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:

1.

20. The method of claim 15 or claim 16, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:

1.

21. The method of claim 15 or claim 16, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:

1.

22. The method of any one of claims 1 to 13, wherein the IL-40 polypeptide is a mouse IL-40 polypeptide.

23. The method of claim 22, wherein the IL-40 polypeptide is a recombinant mouse IL-40 polypeptide.

24. The method of claim 22, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO:

3.

25. The method of claim 22, wherein the IL-40 polypeptide comprises amino acids 19-252 of SEQ ID NO:

3.

26. The method of claim 22, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:

3.

27. The method of claim 22, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:

3.

28. The method of claim 22, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:

3.

29. The method of claim 1, wherein the IL-40 polypeptide comprises one or more chemical modifications.

30. The method of claim 1, wherein the IL-40 polypeptide comprises a tag.

31. The method of claim 1, wherein the IL-40 polypeptide comprises a detectable label.

32. The method of claim 1, wherein the IL-40 polypeptide comprises a fusion polypeptide.

33. A kit comprising: a) a first composition comprising one or more polypeptides selected from the group consisting of IFN-γ, GM-CSF, IFN-α, IL-1β, IL-4, IL-10, IL-13, M-CSF, TGF-β and TNF-α; b) a second composition comprising an IL-40 polypeptide; c) one or more components for measuring the production of cytokines, chemokines and / or growth factors, wherein the cytokines, chemokines and / or growth factors are selected from one or more of CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, IL-1RA, IL-6, erythropoietin, PDGF-AA, VEGF; and d) Instructions for use.

34. The kit of claim 33, wherein the IL-40 polypeptide is a recombinant IL-40 polypeptide.

35. The kit of claim 33, wherein the IL-40 polypeptide is a human IL-40 polypeptide.

36. The kit of claim 35, wherein the IL-40 polypeptide is a recombinant human IL-40 polypeptide.

37. The kit of claim 35 or 36, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

38. The kit of claim 35 or 36, wherein the IL-40 polypeptide comprises amino acids 21-265 of SEQ ID NO:

1.

39. The kit of claim 35 or 36, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:

1.

40. The kit of claim 35 or 36, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:

1.

41. The kit of claim 35 or 36, wherein the IL-40 polypeptide comprises a fragment containing one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:

1.

42. The kit of claim 33, wherein the IL-40 polypeptide is a mouse IL-40 polypeptide.

43. The kit of claim 41, wherein the IL-40 polypeptide is a recombinant mouse IL-40 polypeptide.

44. The kit of claim 42 or 43, wherein the IL-40 polypeptide comprises the amino acid sequence of SEQ ID NO:

3.

45. The kit of claim 42 or 43, wherein the IL-40 polypeptide comprises amino acids 19-252 of SEQ ID NO:

3.

46. ​​The kit of claim 42 or 43, wherein the IL-40 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:

3.

47. The kit of claim 42 or 43, wherein the IL-40 polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:

3.

48. The kit of claim 42 or 43, wherein the IL-40 polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:

3.

49. The kit of any one of claims 33 to 36, wherein the IL-40 polypeptide comprises one or more chemical modifications.

50. The kit of any one of claims 33 to 36, wherein the IL-40 polypeptide comprises a tag.

51. The kit of any one of claims 33 to 36, wherein the IL-40 polypeptide comprises a detectable label.

52. The kit of any one of claims 33 to 36, wherein the IL-40 polypeptide comprises a fusion polypeptide.

53. The kit of any one of claims 33 to 36, wherein each of the one or more components for measuring cytokine, chemokine and / or growth factor production comprises a binding molecule that immunospecifically binds to one of the cytokines, chemokines and / or growth factors under binding conditions.

54. The kit of any one of claims 33 to 36, further comprising a cell or a population of cells.

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