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1. WO2020163469 - MÉTHODES DE DÉTECTION D'UNE ACTIVITÉ DE LA MÉTÉORINE-β

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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METHODS FOR DETECTING METEORIN-b ACTIVITY


This patent application claims the benefit of U.S. provisional patent application no. 62/803,047 filed on February 8, 2019, entitled METHODS FOR DETECTING METEORIN-BETA ACTIVITY, naming Gerardo Arrevillaga BONI et al. as inventors, and designated by attorney docket no. BLD-2002-PV. The entire content of the foregoing application is incorporated herein by reference, including all text, tables, and drawings.

Field

The technology relates in part to methods for detecting the activity of meteorin-b and modified versions thereof.

Background

Cytokines are small secreted proteins involved in immune responses, host defense, inflammation, and immune system development. Cytokines generally exert their effects by binding specific receptors on the membrane of target cells. Examples of cytokines include interleukins,

chemokines, interferons, and members of the tumor necrosis factor superfamily. Certain cytokines are involved in autoimmune diseases, cancer, and other ailments; and may be useful for immunotherapy.

Meteorin-b is generally considered a macrophage-associated cytokine, and may be useful for certain research applications (e.g., studying the pathogenesis of certain diseases; studying immune cell activation and differentiation), diagnostics, and/or certain types of immunotherapy. Provided herein are methods for detecting the activity of meteorin-b (e.g., recombinant meteorin-b, and modified versions thereof).

Summary

Provided herein, in some aspects, are methods for assessing activity of a meteorin-b polypeptide comprising a) contacting a cell with a first composition comprising a co-stimulant and a second

composition comprising a meteorin-b polypeptide; b) measuring production by the cell of one or more cytokines and/or chemokines chosen from CCL2, CCL5, CXCL1 , CXCL8, CXCL9, CXCL10, IL-6 and IL-1 RA, thereby measuring cytokine production; and c) detecting the activity of the meteorin-b polypeptide in the second composition according to the cytokine production measured in (b).

Also provided herein, in some aspects, are methods for identifying whether a cell expresses, or is capable of expressing, a meteorin-b receptor comprising a) contacting a cell with a first composition comprising a stimulant; b) contacting the cell under binding conditions with a second composition comprising a meteorin-b polypeptide; c) detecting the presence, absence, and/or amount of meteorin-b polypeptide bound to the cell; and d) identifying whether the cell expresses, or is capable of expressing, the meteorin-b receptor according to the presence, absence, and/or amount of meteorin-b polypeptide detected in (c).

Also provided herein, in some aspects, are kits comprising a) a first composition comprising one or more co-stimulants chosen from IFN-g, IL-4, IL-10, and TQRb polypeptides; b) one or more components for measuring cytokine and/or chemokine production, where the cytokines and/or chemokines are chosen from one or more of CCL2, CCL5, CXCL1 , CXCL8, CXCL9, CXCL10, IL-6 and IL-1 RA; and c) instructions for use.

Certain embodiments are described further in the following description, examples, claims and drawings.

Brief Description of the Drawings

The drawings illustrate certain embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

Fig. 1 shows the production of CCL2 by THP-1 cells under meteorin-b stimulation (top panel) and IFN-g and meteorin-b co-stimulation (bottom panel).

Fig. 2 shows the production of CXCL10 (top panel), IL1 RA (middle panel), and IL-6 (bottom panel) by THP-1 cells under IFN-y and meteorin-b co-stimulation.

Fig. 3A shows comparative differences between control and meteorin-b treated cells. Relative comparative differences between control and meteorin-b treated cells for different cytokines are indicated in the first column and general activity is indicated in the second column. All samples were co-treated with 1 pg/ml of IFN-y except for sample data shown in the third column (untreated vs. meteorin-b). The fourth column shows control vs. 6XHis peptide (used as an internal control) treatment comparison. The fifth, sixth and seventh columns compare cells co-stimulated with IFN-Y and IFN-g plus 1 pg/ml, 2 pg/ml or 2.5 pg/ml of meteorin-b respectively. Data were obtained using LEGENDplex™ (BioLegend). (-) no differences, (+) less than 50% increase over control,

(++) more than 50% increase over control, (+++) more than 100% increase over control.

Fig. 3B shows comparative differences between cells activated with cytokines (e.g., IFN-g, TQBb, IL-4, and IL-10) alone versus cells activated with cytokines and co-treated with meteorin-b.

Relative comparative differences for several chemokines, listed in the first column, are indicated. Description of the general activity of each chemokine is indicated in the second column. Samples were untreated, treated with 2.5 pg/mL of meteorin-b alone, treated with 1 pg/mL of cytokine alone, or co-treated with 1 pg/mL of cytokine and 2.5 pg/mL of meteorin-b. Data were obtained using LEGENDplex™ (BioLegend). (=) denotes no difference, (--) denotes less than 50% decrease over control, (+++) more than 100% increase over control.

Fig. 4A shows the induction of meteorin-b receptor by IFN-g measured by the capacity of human recombinant meteorin^-6XHis (hrMetm^ -6XHis) to bind meteorin-b receptor in THP-1 cells previously treated with IFN-g (250 ng). An anti-6xHis antibody was used to quantify ligand-receptor interaction. Data were obtained through flow cytometry.

Figs. 4B and 4C show the induction of meteorin-b receptor by IL-10, IL-4, and TΰRb measured by the capacity of human recombinant meteorin^-6xHis (hrMetn^-6xHis) to bind meteorin-b receptor in THP-1 cells previously treated with different cytokines (250 ng). An anti-6xHis antibody was used to quantify ligand-receptor interaction. Data were obtained through flow cytometry.

Fig. 5 shows a schematic of the general strategy to detect meteorin-b receptor in a target cell.

Detailed Description

Meteorin-b may useful for certain research applications, diagnostics, and/or immunotherapy.

Accordingly, bioassays for assessing the activity of meteorin-b, recombinant meteorin-b, meteorin-b variants, meteorin-b fragments, and other modified versions of meteorin-b would be useful for developing practical applications for meteorin-b. Provided herein are methods for assessing the activity of meteorin-b. Also provided herein are methods identifying whether a cell expresses, or is capable of expressing, a meteorin-b receptor.

Meteorin-b

Provided herein are methods for assessing the activity of a meteorin-b polypeptide. Also provided herein are methods identifying whether a cell expresses, or is capable of expressing, a meteorin-b receptor. Meteorin-b, which may be referred to as meteor^, meteorin-beta, meteorin-b, Metrn-b, MbΐGhb, meteorin like, meteorin-glial cell differentiation regulator-like, meteorin-like protein, METRNL, MGC99788, Subfatin, Cometin, interleukin 41 (IL-41), or interleukin 39 (IL-39), is a 311 amino acid (aa) protein that has 58% similarity with Meteorin (in human). Mature human meteorin-b shares 79% and 80% aa sequence identity with mouse and rat meteorin-b, respectively.

Alternate splicing generates an additional isoform that lacks the N-terminal 82 amino acids including the signal peptide. Meteorin-b is expressed in certain adult mouse tissues with the highest expression in muscle tissue, adipose tissue, and activated monocytes. The expression of meteorin-b may be induced in muscle after exercise and in adipose tissue upon cold exposure. Meteorin-b may possess neurotrophic activity as it enhances neurite outgrowth and neuroblast migration in vivo. Meteorin-b also may be indirectly involved in the "browning" of white fat to a more thermogenic "beige fat."

An example human meteorin-b nucleic acid sequence is provided herein as SEQ ID NO: 2

(GENBANK Accession No. NM_001004431.3), and an example human meteorin-b amino acid sequence is provided herein as SEQ ID NO: 1 (GENBANK Accession No. NP_001004431.1). An example mouse meteorin-b nucleic acid sequence is provided herein as SEQ ID NO: 4 (GENBANK Accession No. NM_144797.3), and an example mouse meteorin-b amino acid sequence is provided herein as SEQ ID NO: 3 (GENBANK Accession No. NP_659046.1).

A meteorin-b polypeptide may refer to a precursor meteorin-b polypeptide (includes the signal peptide) or a mature meteorin-b polypeptide (excludes the signal peptide). In some embodiments, a meteorin-b polypeptide is a precursor meteorin-b polypeptide (e.g., a precursor human meteorin-b polypeptide comprising amino acids 1-311 of SEQ ID NO: 1 ; a precursor mouse meteorin-b polypeptide comprising amino acids 1-311 of SEQ ID NO: 3). In some embodiments, a meteorin-b polypeptide is a mature meteorin-b polypeptide (e.g., a mature human meteorin-b polypeptide comprising amino acids 46-311 of SEQ ID NO: 1 ; a mature mouse meteorin-b polypeptide comprising amino acids 46-311 of SEQ ID NO: 3).

In some embodiments, a meteorin-b polypeptide is a recombinant meteorin-b polypeptide. A recombinant meteorin-b polypeptide typically is a meteorin-b polypeptide encoded by DNA (i.e. , meteorin-b nucleic acid sequence) that has been cloned in a vector or system that supports expression of the DNA and translation of messenger RNA. In some embodiments, a meteorin-b polypeptide is a recombinant human meteorin-b polypeptide (rh-meteorin-b). In some

embodiments, a meteorin-b polypeptide is a recombinant mouse meteorin-b polypeptide (rm-meteorin-b).

A meteorin-b polypeptide herein may refer to an unmodified meteorin-b polypeptide. An unmodified polypeptide generally refers to a native or wild-type full-length (precursor or mature) polypeptide having no amino acid substitutions, no insertions, no deletions, no chemical modifications, no amino acid side-chain modifications, no tags, no detectable labels, no fusions, and the like.

A meteorin-b polypeptide herein may refer to a modified meteorin-b 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, and the like and combinations thereof. Modifications may include, for example, addition of one or more fluorophores, glycosylation, prenylation, PEGylation, attachment to a solid surface, biotinylation, antibody conjugation, conjugation to a therapeutic agent, chemical modifications at cysteine (e.g., aminoethylation, iodoacetamides, maleimides, Dha formation, disulfide formation, reaction of Dha with thiols, and desulfurization of disulfides), incorporation of one or more unnatural amino acids, and the like and combinations thereof.

In some embodiments, a meteorin-b polypeptide refers to a meteorin-b variant or mutant. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of a meteorin-b polypeptide. A meteorin-b variant may include any combination of deletion, insertion, and substitution. In some embodiments, a meteorin-b polypeptide comprises one or more amino acid substitutions. These variants have at least one amino acid residue removed from the meteorin-b polypeptide and a different residue inserted in its place. For example, a meteorin-b variant may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions. A meteorin-b 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 meteorin-b activity. Examples of substitutions are listed below:

Example Amino Acid Residue Substitutions

Ala (A) val; leu; ile; val

Arg (R) lys; gin; asn; lys

Asn (N) gin; his; asp, lys; gin; arg

Asp (D) glu; asn

Cys (C) ser; ala

Gin (Q) asn; glu

Glu (E) asp; gin

Gly (G) ala

His (H) asn; gin; lys; arg

Ile (I) leu; val; met; ala; leu; phe; norleucine

Leu (L) norleucine; ile; val; ile; met; ala; phe

Lys (K) arg; gin; asn

Met (M) leu; phe; ile

Phe (F) leu; val; ile; ala; tyr

Pro (P) ala

Ser (S) thr

Thr (T) ser

T rp (W) tyr; phe

Tyr (Y) trp; phe; thr; ser

Val (V) ile; leu; met; phe; ala; norleucine

Substantial modifications in the biological properties of a meteorin-b polypeptide may be accomplished 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 groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions typically entail exchanging a member of one of these classes for another class.

In some embodiments, a meteorin-b polypeptide comprises one or more insertions. In some embodiments, a meteorin-b polypeptide comprises one or more insertions, where 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, a meteorin-b polypeptide comprises one or more deletions. In some embodiments, a meteorin-b polypeptide comprises one or more deletions, where 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.

In some embodiments, a meteorin-b polypeptide comprises a fused polypeptide. A fused polypeptide may be referred to as a fusion protein or chimeric protein. Fused polypeptides typically are created through the joining of two or more genes that code for separate proteins. Translation of this fusion construct may result in a single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant fusion proteins may be created artificially by recombinant DNA technology for use in biological research or therapeutics. Examples of fused polypeptides include meteorin-b fused with a fluorescent protein tag (e.g., green fluorescent protein (GFP)), therapeutic protein (e.g., antibody), or any protein tag described herein. In some embodiments a fused polypeptide comprises a linker (e.g., flexible linker, rigid linker, cleavable linker).

In some embodiments, a meteorin-b polypeptide comprises one or more tags (e.g., one or more amino acid or peptide tags; one or more affinity tags). Tags may facilitate detection, isolation and/or purification of a meteorin-b polypeptide. A tag sometimes specifically binds a molecule or moiety of a solid phase or a detectable label, for example, thereby having utility for isolating, purifying and/or detecting a meteorin-b polypeptide. In some embodiments, a tag comprises one or more of the following elements: Fc (derived from immunoglobulin Fc domain), FLAG (e.g., DYKDDDDKG), V5 (e.g., GKPIPNPLLGLDST), c-MYC (e.g., EQKLISEEDL), HSV (e.g.,

QPELAPEDPED), influenza hemagglutinin, HA (e.g., YPYDVPDYA), VSV-G (e.g.,

YTDIEMNRLGK), bacterial glutathione-S-transferase, maltose binding protein, a streptavidin- or avidin-binding tag (e.g., pcDNA™6 BioEase™ Gateway® Biotinylation System (Invitrogen)), thioredoxin, b-galactosidase, VSV-glycoprotein, a fluorescent protein (e.g., green fluorescent protein or one of its many color variants (e.g., yellow, red, blue)), a polylysine or polyarginine sequence, a polyhistidine sequence (e.g., His6, 6XHis) or other sequence that chelates a metal (e.g., cobalt, zinc, copper), and/or a cysteine-rich sequence that binds to an arsenic-containing molecule. In certain embodiments, a 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 sometimes is CCPGCC. In certain embodiments, the tag comprises a cysteine-rich element and a polyhistidine element (e.g., CCPGCC and His6).

A tag may bind to a binding partner. For example, some tags bind to an antibody (e.g., FLAG) and sometimes specifically bind to a small molecule. For example, a polyhistidine tag specifically chelates a bivalent metal, such as copper, zinc and cobalt; a polylysine or polyarginine tag specifically binds to a zinc finger; a glutathione S-transferase tag binds to glutathione; and a cysteine-rich tag specifically binds to an arsenic-containing molecule. Arsenic-containing molecules include LUMIO™ agents (Invitrogen, California), such as FIAsH™ (EDT2[4',5'-bis(1 ,3,2-dithioarsolan-2-yl)fluorescein-(1 ,2-ethanedithiol)2]) and ReAsH reagents. Such antibodies and small molecules sometimes are linked to a solid phase for isolation of the target protein or target peptide.

In some embodiments, a meteorin-b polypeptide comprises one or more detectable markers or labels. In some embodiments, a meteorin-b polypeptide is conjugated to a detectable marker or label. For example, for research and diagnostic applications, a modified meteorin-b polypeptide may be labeled with a detectable moiety. Numerous labels are available which generally include radioisotopes (e.g., 35S, 14C, 125l, 3H, and 131l), 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™), and enzyme-substrate labels (e.g., described in U.S. Pat. No. 4,275,149, which is incorporated by reference herein, luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456, which is incorporated by reference herein), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like).

In certain instances, a label is indirectly conjugated to a meteorin-b polypeptide. For example, a meteorin-b polypeptide may be conjugated with biotin and any suitable label mentioned above may be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with a meteorin-b polypeptide in this indirect manner. Alternatively, to achieve indirect conjugation of a label with a meteorin-b polypeptide, the meteorin-b polypeptide is conjugated with a small hapten (e.g., digoxin) and one of the types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).

In some embodiments, a meteorin-b polypeptide refers to a fragment of a meteorin-b polypeptide. Generally, a meteorin-b fragment contains fewer amino acids than a full-length mature meteorin-b. For example, a meteorin-b fragment may include a portion of the mature human meteorin-b polypeptide (i.e., a portion of amino acids 46-311 of SEQ ID NO: 1), or a portion of the mature mouse meteorin-b polypeptide (i.e. a portion of amino acids 46-311 of SEQ ID NO: 3). Full-length mature human meteorin-b is 266 amino acids in length. Accordingly, fragments of human meteorin-b may be 265 amino acids in length or shorter. Full-length mature mouse meteorin-b is 266 amino acids in length. Accordingly, fragments of mouse meteorin-b may be 265 amino acids in length or shorter.

In some embodiments, a meteorin-b polypeptide refers to a functional fragment of a meteorin-b polypeptide. Methods for assessing the activity of meteorin-b polypeptides and functional fragments of meteorin-b are provided herein. In some embodiments, a functional fragment of meteorin-b is a fragment that exhibits at least 50% of the activity of a full-length mature meteorin-b. For example, a functional fragment of meteorin-b 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 a full-length mature meteorin-b.

In some embodiments, a meteorin-b polypeptide is immobilized on a solid support or substrate. In some embodiments, a meteorin-b polypeptide is non-diffusively immobilized on a solid support (e.g., the meteorin-b polypeptide does not detach from the solid support). A solid support or substrate can be any physically separable solid to which a meteorin-b polypeptide can be directly or indirectly attached 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. Solid supports also can include, for example, chips, columns, optical fibers, wipes, filters (e.g., flat surface filters), one or more capillaries, glass and modified or functionalized glass (e.g., control led-pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductive materials, quantum dots, coated beads or particles, other

chromatographic materials, magnetic particles; plastics (including acrylics, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethanes, TEFLON™, polyethylene,

polypropylene, polyamide, polyester, polyvinylidenedifluoride (PVDF), and the like),

polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon, silica gel, and modified silicon, Sephadex®, Sepharose®, carbon, metals (e.g., steel, gold, silver, aluminum, silicon and copper), inorganic glasses, conducting polymers (including polymers such as polypyrole and polyindole); micro or nanostructured surfaces such as nucleic acid tiling arrays, nanotube, nanowire, or nanoparticulate decorated surfaces; or porous surfaces or gels such as methacrylates, acrylamides, sugar polymers, cellulose, silicates, or other fibrous or stranded polymers. In some embodiments, the solid support or substrate may be coated using passive or chemically-derivatized coatings with any number of materials, including polymers, such as dextrans, acrylamides, gelatins or agarose. Beads and/or particles may be free or in connection with one another (e.g., sintered). In some embodiments, a solid support or substrate can be a collection of particles. In some embodiments, the particles can comprise silica, and the silica may comprise silica dioxide. In some embodiments the silica can be porous, and in certain

embodiments the silica can be non-porous. In some embodiments, the particles further comprise an agent that confers a paramagnetic property to the particles. In certain embodiments, the agent comprises a metal, and in certain embodiments the agent is a metal oxide, (e.g., iron or iron oxides, where the iron oxide contains a mixture of Fe2+ and Fe3+). A meteorin-b polypeptide may be linked to a solid support by covalent bonds or by non-covalent interactions and may be linked to

a solid support directly or indirectly (e.g., via an intermediary agent such as a spacer molecule or biotin).

Stimulants and co-stimulants

Certain methods provided herein include use of a stimulant or co-stimulant. Use of stimulants and co-stimulants may be included, for example, in methods for assessing the activity of a meteorin-b polypeptide, and/or in methods for identifying whether a cell expresses, or is capable of expressing, a meteorin-b receptor. A stimulant may be used in certain instances (e.g., to stimulate a cell prior to exposure to meteorin-b). A co-stimulant may be used in certain instances (e.g., to co-stimulate a cell during meteorin-b exposure). In some embodiments, a cell or population of cells is contacted with a stimulant/co-stimulant. In some embodiments, a cell or population of cells is simultaneously contacted with a stimulant/co-stimulant and a meteorin-b polypeptide. In some embodiments, a cell or population of cells is contacted with a stimulant/co-stimulant prior to being contacted with a meteorin-b polypeptide.

In certain instances, a stimulant/co-stimulant can strengthen or enhance the effect of meteorin-b on a cell or a population of cells. For example, a stimulant/co-stimulant can enhance production of certain cytokines and/or chemokines in response to meteorin-b stimulation. A stimulant/co stimulant also can upregulate meteorin-b receptor expression. In certain instances, a stimulant/co stimulant can provide a synergistic enhancement when combined with meteorin-b. For example, a stimulant/co-stimulant can synergistically enhance production of certain cytokines and/or chemokines in response to meteorin-b stimulation. An enhancement afforded by a stimulant or co stimulant may be additive, multiplicative, or exponential.

Any suitable stimulant/co-stimulant may be used in conjunction with the methods provided herein.

In some embodiments, a stimulant/co-stimulant comprises a soluble/secreted protein. In some embodiments, a stimulant/co-stimulant comprises a cytokine. In some embodiments, a

stimulant/co-stimulant comprises a chemokine. Non-limiting examples of stimulants/co-stimulants include interferon-y (IFN-g), lipopolysaccharides (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), interleukin 13 (IL-13), macrophage colony-stimulating factor (M-CSF), infection (e.g., fungal infection, helminth infection), immune complexes, interleukin-1 receptor (IL-1 R), interleukin 10 (IL-10), transforming growth factor beta (TGF-b), glucocorticoids, interleukin 6 (IL-6), leukemia inhibitory factor (LIF), adenosine, complement

components, and interleukin 32 (IL-32). In some embodiments, a stimulant/co-stimulant comprises one or more of IFN-g, IL-4, IL-10, and TGF-b.

IFN-Y

Certain methods provided herein include use of interferon-g (IFN-g) as a stimulant or co-stimulant. Interferon-g (also referred to as IFN-g, IFNY, IFN-g, IFNg, IFN-gamma, interferon gamma, immune interferon, type II interferon) is a dimerized soluble cytokine and a type II class of interferon. IFN-g generally is involved in innate and adaptive immunity against certain viral, bacterial, and protozoal infections. IFN-g can function as an activator of macrophages and inducer of Class II major histocompatibility complex (MHC) molecule expression. IFN-g can inhibit viral replication directly, and can provide immunostimulatory and immunomodulatory effects. Aberrant IFN-g expression often is associated with a number of autoinflammatory and autoimmune diseases. IFN-g typically is produced by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response; by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops; and by non-cytotoxic innate lymphoid cells (ILC).

Any suitable IFN-g, or functional fragment, or modified version thereof, may be used in conjunction with the methods described herein (e.g., as a co-stimulant with meteorin-b; as a stimulant prior to meteorin-b exposure). In some embodiments, IFN-g is a human IFN-g. An example human IFN-g nucleic acid sequence is provided herein as SEQ ID NO: 6 (GENBANK Accession No.

NM_000619.3), and an example human IFN-g amino acid sequence is provided herein as SEQ ID NO: 5 (GENBANK Accession No. NP_000610.2). In some embodiments, IFN-g is a mouse IFN-g. An example mouse IFN-g nucleic acid sequence is provided herein as SEQ ID NO: 8 (GENBANK Accession No. NM_008337.4), and an example mouse IFN-g amino acid sequence is provided herein as SEQ ID NO: 7 (GENBANK Accession No. NP_032363.1).

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

comprising amino acids 24-161 of SEQ ID NO: 5; a mature mouse IFN-g polypeptide comprising amino acids 23-155 of SEQ ID NO: 7).

In some embodiments, IFN-g is a recombinant IFN-g polypeptide. A recombinant IFN-g

polypeptide typically is an IFN-g polypeptide encoded by DNA (i.e., IFN-g nucleic acid sequence) that has been cloned in a vector or system that supports expression of the DNA and translation of messenger RNA. In some embodiments, IFN-g is a recombinant human IFN-g polypeptide (rhlFN-Y). In some embodiments, IFN-g is a recombinant mouse IFN-g polypeptide (rmlFN-g). In some embodiments, IFN-g is a commercially available recombinant human IFN-g (e.g., BioLegend cat # 570202). In some embodiments, IFN-g is a commercially available recombinant mouse IFN-g (e.g., BioLegend cat # 575302).

IFN-g may refer to an unmodified IFN-g polypeptide, a modified IFN-g polypeptide, an IFN-g variant, an IFN-g 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-g refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO: 5 or SEQ ID NO: 7. For example, IFN-g 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: 5 or SEQ ID NO: 7.

In some embodiments, IFN-g refers to a fragment of an IFN-g polypeptide. Generally, an IFN-g fragment contains fewer amino acids than a full-length mature IFN-g. For example, an IFN-g fragment may include a portion of the mature human IFN-g 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 the mature mouse IFN-g polypeptide (i.e. a portion of amino acids 23-155 of SEQ ID NO: 7). One example full-length mature human IFN-g is 143 amino acids in length. Accordingly, fragments of human IFN-g may be 142 amino acids in length or shorter. Another example full-length mature human IFN-g is 138 amino acids in length. Accordingly, fragments of human IFN-g may be 137 amino acids in length or shorter. Full-length mature mouse IFN-g is 133 amino acids in length. Accordingly, fragments of mouse IFN-g may be 132 amino acids in length or shorter.

In some embodiments, IFN-g refers to a functional fragment of an IFN-g polypeptide. Any suitable method for assessing the activity of IFN-g and functional fragments of IFN-g may be used to determine whether an IFN-g fragment is a functional fragment. For example, one assay for

assessing IFN-g activity is the induction of an antiviral state in target cells, sometimes referred to as a cytopathic protection effect (CPE) assay. An example CPE assay uses A549 human lung carcinoma cells challenged with encephalomyocarditis virus (EMCV) and the effects are compared to a standard for human IFN-g (e.g., Gxg01-902-535, BEI Resources). In some embodiments, a functional fragment of IFN-g is a fragment that exhibits at least 50% of the activity of an IFN-g standard or a full-length mature IFN-g. For example, a functional fragment of IFN-g 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 IFN-g standard or a full-length mature IFN-g.

IL-4

Certain methods provided herein include use of interleukin 4 (IL-4) as a stimulant or co-stimulant. Interleukin 4 (also referred to as IL-4, B cell growth factor 1 (BCGF-1), B-cell stimulatory factor 1 (BSF-1), interleukin-4, lymphocyte stimulatory factor 1 , MGC79402) is the primary cytokine implicated in the development of Th2-mediated responses, which is associated with allergy and asthma. The Type I receptor comprises IL-4Ra and the common gamma-chain (YC), which is also shared by the cytokines IL-2, -7, -9, -15 and -21 and is present in hematopoietic cells. IL-4 can use the type II complex, comprising IL-4Ra and IL-13Ra1 , which is present in non-hematopoietic cells. This second receptor complex is a functional receptor for IL-13, which shares approximately 25% homology with IL-4. The type I receptor complex can be formed only by IL-4 and is active in Th2 development. In contrast, the type II receptor complex formed by either IL-4 or IL-13 is more active during airway hypersensitivity and mucus secretion and is not found in T cells.

Any suitable IL-4, or functional fragment, or modified version thereof, may be used in conjunction with the methods described herein (e.g., as a co-stimulant with meteorin-b; as a stimulant prior to meteorin-b exposure). In some embodiments, IL-4 is a human IL-4. An example human IL-4 nucleic acid sequence is provided herein as SEQ ID NO: 12 (full mRNA sequence provided as GENBANK Accession No. NM_000589), and an example human IL-4 amino acid sequence is provided herein as SEQ ID NO: 11 (GENBANK Accession No. NP_000580.1). In some

embodiments, IL-4 is a mouse IL-4. An example mouse IL-4 nucleic acid sequence is provided herein as SEQ ID NO: 14 (full mRNA sequence provided as GENBANK Accession No.

NM_021283), and an example mouse IL-4 amino acid sequence is provided herein as SEQ ID NO: 13 (GENBANK Accession No. NP_067258.1).

IL-4 may refer to a precursor IL-4 polypeptide (includes the signal peptide) or a mature IL-4 polypeptide (excludes the 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: 11 ; a precursor mouse IL-4 polypeptide comprising amino acids 1-140 of SEQ ID NO: 13). In some embodiments, an 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: 11 ; a mature mouse IL-4 polypeptide comprising amino acids 21-140 of SEQ ID NO: 13; a mature mouse IL-4 polypeptide comprising amino acids 23-140 of SEQ ID NO: 13).

In some embodiments, IL-4 is a recombinant IL-4 polypeptide. A recombinant IL-4 polypeptide typically is an IL-4 polypeptide encoded by DNA (i.e., IL-4 nucleic acid sequence) that has been cloned in a vector or system that supports expression of the DNA and translation of messenger RNA. In some embodiments, IL-4 is a recombinant human IL-4 polypeptide (rhlL-4). In some embodiments, IL-4 is a recombinant mouse IL-4 polypeptide (rmlL-4). In some embodiments, IL-4 is a commercially available recombinant human IL-4 (e.g., BioLegend cat # 574002). In some embodiments, IL-4 is a commercially available recombinant mouse IL-4 (e.g., BioLegend cat # 574302).

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: 11 or SEQ ID NO: 13. 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: 11 or SEQ ID NO: 13.

In some embodiments, IL-4 refers to a fragment of an IL-4 polypeptide. Generally, 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 the mature human IL-4 polypeptide (i.e., a portion of amino acids 25-153 of SEQ ID NO: 11), or a portion of the mature mouse IL-4 polypeptide (i.e. a portion of amino acids 21-140 of SEQ ID NO: 13). An example full-length mature human IL-4 is 129 amino acids in length. Accordingly, fragments of human IL-4 may be 128 amino acids in length or shorter. An example full-length mature mouse IL-4 is 120 amino acids in length. Accordingly, fragments of mouse IL-4 may be 119 amino acids in length or shorter. Another example full-length mature mouse IL-4 is 118 amino acids in length. Accordingly, fragments of mouse IL-4 may be 117 amino acids in length or shorter.

In some embodiments, IL-4 refers to a functional fragment of an IL-4 polypeptide. Any suitable method for assessing the activity of IL-4 and functional fragments of IL-4 may be used to determine whether an IL-4 fragment is a functional fragment. For example, one assay for assessing IL-4 activity is a cell proliferation assay. An example cell proliferation assay measures ED50 of IL-4 according to dose-dependent stimulation of TF-1 cell proliferation. Another example cell proliferation assay measures ED5o of IL-4 according to dose-dependent stimulation of CTLL-2 cell proliferation. In some embodiments, a functional fragment of IL-4 is a fragment that exhibits at least 50% of the activity of an IL-4 standard (e.g., WHO International Standard for Human IL-4 (NIBSC code: 88/656)) or a 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 a full-length mature IL-4.

IL-10

Certain methods provided herein include use of interleukin 10 (IL-10) as a stimulant or co stimulant. Interleukin 10 (also referred to as IL-10, B-TCGF, CSIF, TGIF) was first described as a cytokine that is produced by T helper 2 (Th2) cell clones. It inhibits interferon (IFN)-y synthesis in Th1 cells, and therefore it was initially called cytokine synthesis inhibiting factor (CSIF).

Macrophages are the main source of IL-10 and its secretion can be stimulated by endotoxin (via Toll-like receptor 4, NF-KB dependent), tumor necrosis factor TNF-a (via TNF receptor p55, NF-KB-dependent), catecholamines, and IL-1. IL-10 controls inflammatory processes by suppressing the expression of proinflammatory cytokines, chemokines, adhesion molecules, as well as antigen-presenting and costimulatory molecules in monocytes/macrophages, neutrophils, and T cells. IL-10 inhibits the production of proinflammatory mediators by monocytes and macrophages such as endotoxin- and IFN-y-induced release of I L- 1 a , IL-6, IL-8, G-CSF, GM-CSF, and TNF-a. In addition, it enhances the production of anti-inflammatory mediators such as IL-1 RA and soluble TNFa receptors. IL-10 inhibits the capacity of monocytes and macrophages to present antigen to T cells. This is realized by down-regulation of constitutive and IFNy-induced cell surface levels of MHC class II, of costimulatory molecules such as CD86 and of some adhesion molecules such as CD58.

Any suitable IL-10, or functional fragment, or modified version thereof, may be used in conjunction with the methods described herein (e.g., as a co-stimulant with meteorin-b; as a stimulant prior to meteorin-b exposure). In some embodiments, IL-10 is a human IL-10. An example human IL-10 nucleic acid sequence is provided herein as SEQ ID NO: 16 (full mRNA sequence provided as GENBANK Accession No. NM_000572), and an example human IL-10 amino acid sequence is provided herein as SEQ ID NO: 15 (GENBANK Accession No. NP_000563.1). In some embodiments, IL-10 is a mouse IL-10. An example mouse IL-10 nucleic acid sequence is provided herein as SEQ ID NO: 18 (full mRNA sequence provided as GENBANK Accession No.

NM_010548), and an example mouse IL-10 amino acid sequence is provided herein as SEQ ID NO: 17 (GENBANK Accession No. NP_034678.1).

IL-10 may refer to a precursor IL-10 polypeptide (includes the signal peptide) or a mature IL-10 polypeptide (excludes the 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: 15; a precursor mouse IL-10 polypeptide comprising amino acids 1-178 of SEQ ID NO: 17). In some embodiments, an 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: 15; a mature mouse IL-10 polypeptide comprising amino acids 19-178 of SEQ ID NO: 17).

In some embodiments, IL-10 is a recombinant IL-10 polypeptide. A recombinant IL-10 polypeptide typically is an IL-10 polypeptide encoded by DNA (i.e. , IL-10 nucleic acid sequence) that has been cloned in a vector or system that supports expression of the DNA and translation of messenger RNA. In some embodiments, IL-10 is a recombinant human IL-10 polypeptide (rhlL-10). In some embodiments, IL-10 is a recombinant mouse IL-10 polypeptide (rmlL-10). In some embodiments, IL-10 is a commercially available recombinant human IL-10 (e.g., BioLegend cat # 715602;

BioLegend cat # 571002). In some embodiments, IL-10 is a commercially available recombinant mouse IL-10 (e.g., BioLegend cat # 575802).

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: 15 or SEQ ID NO: 17. 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: 15 or SEQ ID NO: 17.

In some embodiments, IL-10 refers to a fragment of an IL-10 polypeptide. Generally, 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 the mature human IL-10 polypeptide (i.e. , a portion of amino acids 19-178 of SEQ ID NO: 15), or a portion of the mature mouse IL-10 polypeptide (i.e. a portion of amino acids 19-178 of SEQ ID NO: 17). An example full-length mature human IL-10 is 160 amino acids in length. Accordingly, fragments of human IL-10 may be 159 amino acids in length or shorter. An example full-length mature mouse IL-10 is 160 amino acids in length. Accordingly, fragments of mouse IL-10 may be 159 amino acids in length or shorter.

In some embodiments, IL-10 refers to a functional fragment of an IL-10 polypeptide. Any suitable method for assessing the activity of IL-10 and functional fragments of IL-10 may be used to determine whether an IL-10 fragment is a functional fragment. For example, one assay for assessing IL-10 activity is an IFN-y inhibition assay. An example IFN-y inhibition assay measures the extent to which IL-10 inhibits the induction of INF-y in PHA activated human PBMC. Another assay for assessing IL-10 activity involves dose dependent stimulation MC/9 cell proliferation. In some embodiments, a functional fragment of IL-10 is a fragment that exhibits at least 50% of the activity of an IL-10 standard or a 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 of the activity of an IL-10 standard or a full-length mature IL-10.

TGF-b

Certain methods provided herein include use of transforming growth factor beta (TGF-b) as a stimulant or co-stimulant. Transforming growth factor beta (also referred to as TGF-b, TGF-bI , TGFB, DPD1 , transforming growth factor, Transforming Growth Factor Beta 1 , TGF-Beta-1) is synthesized in cells as a 390-amino acid polypeptide. Furin cleaves the protein at residue 278, yielding an N-terminal cleavage product which corresponds to the latency-associated peptide (LAP), and the 25-kD C-terminal portion of the precursor constitutes the mature TGF-bI . TGF-b activators can release TGF-b from LAP. These activators include proteases that degrade LAP, thrombospondin-1 , reactive oxygen species, and integrins avb6 and avb8. Mouse TGF-b converts naive T cells into regulatory T (Treg) cells that prevent autoimmunity. Although human TGF-bI is widely used for inducing FOXP3+ in vitro, it might not be an essential factor for human Treg differentiation. Th17 murine can be induced from naive CD4+ T cells by the combination of TGF-bI and IL-6 or IL-21. Nevertheless, the regulation of human Th17 differentiation is distinct. TGF-bI seems to have dual effects on human Th17 differentiation in a dose-dependent manner. While TGF-bI is required for the expression of RORyt, in human naive CD4+ T cells from cord blood, TGF-bI can inhibit the function of RORyt at high doses. By using serum-free medium, it has been clarified that the optimum conditions for human Th17 differentiation are TGF-bI , I L- 1 b , and IL-2 in combination with IL-6, IL-21 or IL-23.

Any suitable TGF-b, or functional fragment, or modified version thereof, may be used in

conjunction with the methods described herein (e.g., as a co-stimulant with meteorin-b; as a stimulant prior to meteorin-b exposure). In some embodiments, TGF-b is a human TGF-b. An example human TGF-b nucleic acid sequence is provided herein as SEQ ID NO: 20 (full mRNA sequence provided as GENBANK Accession No. NM_000660.7), and an example human TGF-b amino acid sequence is provided herein as SEQ ID NO: 19 (GENBANK Accession No.

NP_000651.3). Another example human TGF-b nucleic acid sequence is provided as GENBANK Accession No. BC000125.1 , and another example human TGF-b amino acid sequence is provided GENBANK Accession No. P01137. In some embodiments, TGF-b is a mouse TGF-b. An example mouse TGF-b nucleic acid sequence is provided herein as SEQ ID NO: 22 (full mRNA sequence provided as GENBANK Accession No. NM_011577.2), and an example mouse TGF-b amino acid sequence is provided herein as SEQ ID NO: 21 (GENBANK Accession No. NP_035707).

TGF-b may refer to a precursor TGF-b polypeptide (includes the signal peptide) or a mature TGF-b polypeptide (excludes the signal peptide; or excludes the signal peptide and the latency-associated peptide). In some embodiments, TGF-b is a precursor TGF-b polypeptide (e.g., a precursor human TGF-b polypeptide comprising amino acids 1-390 of SEQ ID NO: 19; a precursor mouse TGF-b polypeptide comprising amino acids 1-390 of SEQ ID NO: 21). In some embodiments, a TGF-b polypeptide is a mature TGF-b polypeptide (e.g., a mature human TGF-b polypeptide comprising amino acids 30-390 of SEQ ID NO: 19; a mature human TGF-b polypeptide comprising amino acids 279-390 of SEQ ID NO: 19; a mature mouse TGF-b polypeptide comprising amino acids 29-390 of SEQ ID NO: 21 ; a mature mouse TGF-b polypeptide comprising amino acids 279-390 of SEQ ID NO: 21).

In some embodiments, TGF-b is a recombinant TGF-b polypeptide. A recombinant TGF-b polypeptide typically is a TGF-b polypeptide encoded by DNA (i.e., TGF-b nucleic acid sequence) that has been cloned in a vector or system that supports expression of the DNA and translation of messenger RNA. In some embodiments, TGF-b is a recombinant human TGF-b polypeptide (rhTGF-b). In some embodiments, TGF-b is a recombinant mouse TGF-b polypeptide (rmTGF-b). In some embodiments, TGF-b is a commercially available recombinant human TGF-b (e.g., BioLegend cat # 580704; BioLegend cat # 781802). In some embodiments, TGF-b is a commercially available recombinant mouse TGF-b (e.g., BioLegend cat # 763102).

TGF-b may refer to an unmodified TGF-b polypeptide, a modified TGF-b polypeptide, a TGF-b variant, a TGF-b 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-b refers to a polypeptide comprising an amino acid sequence that is at least about 75% identical to SEQ ID NO: 19 or SEQ ID NO: 21. For example, TGF-b 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: 19 or SEQ ID NO: 21.

In some embodiments, TGF-b refers to a fragment of a TGF-b polypeptide. Generally, a TGF-b fragment contains fewer amino acids than a full-length mature TGF-b. For example, a TGF-b fragment may include a portion of the mature human TGF-b polypeptide (e.g., a portion of amino acids 30-390 of SEQ ID NO: 19; a portion of amino acids 279-390 of SEQ ID NO: 19), or a portion of the mature mouse TGF-b polypeptide (e.g., a portion of amino acids 29-390 of SEQ ID NO: 21 ; a portion of amino acids 279-390 of SEQ ID NO: 21). An example full-length mature human TGF-b is 112 amino acids in length. Accordingly, fragments of human TGF-b may be 111 amino acids in length or shorter. An example full-length mature mouse TGF-b is 112 amino acids in length. Accordingly, fragments of mouse TGF-b may be 111 amino acids in length or shorter.

In some embodiments, TGF-b refers to a functional fragment of a TGF-b polypeptide. Any suitable method for assessing the activity of TGF-b and functional fragments of TGF-b may be used to determine whether a TGF-b fragment is a functional fragment. For example, one assay for assessing TGF-b activity is a cell proliferation inhibition assay. An example cell proliferation inhibition assay measures the extent to which TGF-b inhibits the proliferation of mouse HT-2 cells induced by recombinant mouse IL-4. In some embodiments, a functional fragment of TGF-b is a

fragment that exhibits at least 50% of the activity of a TGF-b standard or a full-length mature TGF-b. For example, a functional fragment of TGF-b 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 a TGF-b standard or a full-length mature TGF-b.

Cells

Certain methods described herein include stimulating a cell or population of cells (e.g., with meteorin-b; with a stimulant; with meteorin-b + a co-stimulant). Cells may be obtained from a subject and/or a cellular source, or may be obtained as an established cell line. A cellular source may include a population of embryonic stem (ES) cells, induced pluripotent stem cells (iPSCs), and the like. Cells may be isolated from an embryo or a stem cell culture derived from an embryo.

Cells may be isolated from an induced pluripotent stem cell (iPSC) culture. Cells may be obtained from a subject in a variety of manners (e.g., harvested from living tissue, such as a biopsy, plucked hair follicles, body fluids like urine or body-cavity fluids, or isolated from circulation). A subject may include any animal, including but not limited to any mammal, such as mouse, rat, canine, feline, bovine, equine, porcine, non-human primate and human. In certain embodiments, a subject is a human. In some embodiments, a subject is an animal or human that has gestated longer than an embryo in a uterine environment and often is a post-natal human or a post-natal animal (e.g., neonatal human, neonatal animal, adult human or adult animal). A subject sometimes is a juvenile animal, juvenile human, adult animal or adult human.

In some embodiments, cells are isolated from a sample from a subject. An isolated cell refers to a cell that has been separated from a component of its original environment (e.g., separated from a host and/or purified from a sample), and thus is altered by human intervention (e.g., "by the hand of man") from its original environment. A sample can include any specimen that is isolated or obtained from a subject or part thereof. Non-limiting examples of specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, or the like), umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), biopsy sample or tissue biopsy, buccal swab, celocentesis sample, washings of female reproductive tract, urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, hard tissues (e.g., liver, spleen, kidney, lung, or ovary), the like or combinations thereof. The term blood encompasses whole blood, blood product or any

fraction of blood, such as serum, plasma, buffy coat, or the like as conventionally defined. Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants. Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated.

In some embodiments, cells comprise normal, healthy cells (e.g., cells that are not diseased). In some embodiments, cells comprise cells that are genetically altered. In some embodiments, cells comprise cells that are not genetically altered. In some embodiments, cells comprise diseased cells. Diseased cells may include cells from a subject carrying disease-causing mutation(s).

Diseased cells may include cells from abnormal tissue, such as from a neoplasia, a hyperplasia, a malignant tumor or a benign tumor. In certain embodiments, diseased cells include cells that are not tumor cells. In certain embodiments, diseased cells may include cells isolated from circulation (e.g., circulating tumor cells (CTCs)) of a subject. In certain embodiments, diseased cells may include cells isolated from bodily samples such as, for example, urine, semen, stool (feces), and the like.

In some embodiments, cells comprise primary cells. Primary cells generally are taken directly from living tissue, such as a biopsy, plucked hair follicles, bodily samples such as a stool sample, body fluids like urine, semen or body-cavity fluids, or isolated from circulation. In certain instances, primary cells have not been passaged. In certain instances, primary cells have been passaged one time. Primary cells may be isolated from differentiated tissue. Typically, primary cells have been freshly isolated, for example, through tissue digestion and plated. Primary cells may or may not be frozen and then thawed at a later time. In addition, the tissue from which the primary cells are isolated may or may not have been frozen of preserved in some other manner immediately prior to processing. Typically, cells are no longer primary cells after the cells have been passaged more than once. Cells passaged once or more and immediately frozen after passaging are also not considered as primary cells when thawed. In certain embodiments, cells are initially primary cells and become non-primary cells after passaging. In some embodiments, cells are maintained or proliferated in cell culture after the cells are isolated from differentiated tissue and prior to use in methods described herein.

In some embodiments, cells comprise non-primary cells, such as cells from an established cell line, transformed cells, thawed cells from a previously frozen collection, and the like. Any suitable cell line may be used in conjunction with the methods described herein. Examples of established cell

lines include, for example, 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 a myeloma), T-47D (breast cancer), U87 (glioblastoma), Vero (African green monkey Chlorocebus kidney epithelial cell line), MC3T3 (embryonic calvarium), GH3 (pituitary tumor), PC12 (pheochromocytoma), CHO (Chinese hamster ovary), MDCK (kidney epithelial), A6 (kidney epithelial), and AB9. In some embodiments, cells comprise THP-1 cells.

In some embodiments, cells comprise immune cells. Immune cells may include, for example, lymphocytes, leukocytes, agranulocytes, monocytes, macrophages, B cells, dendritic cells, granulocytes, neutrophils, innate lymphoid cells (ILC), megakaryocytes, myeloid-derived suppressor cells (MDSC), natural killer cells (NK cells), platelets, red blood cells (RBC), T cells, mast cells, eosinophils, basophils, and thymocytes. In some embodiments, cells comprise, or are derived from, peripheral blood mononuclear cells (PBMCs) which may include, for example, T cells, B cells, natural killer cells, and monocytes.

In some embodiments, cells comprise monocytes. Monocytes are a type of leukocyte, or white blood cell, and can differentiate into macrophages and myeloid lineage dendritic cells. Monocytes are a part of the vertebrate innate immune system and can influence the process of adaptive immunity. There are at least three subclasses of monocytes in human blood, which may be characterized according to certain markers. For example, the classical monocyte is characterized by high level expression of the CD14 cell surface receptor (CD14++ CD16- monocyte); the non-classical monocyte shows low level expression of CD14 and additional co-expression of the CD16 receptor (CD14+CD16++ monocyte); and the intermediate monocyte is characterized by high level expression of CD14 and low level expression of CD16 (CD14++CD16+ monocytes).

In some embodiments, cells comprise macrophages. Macrophages are a type of white blood cell that engulfs and digests foreign proteins and other substances, cellular debris, microbes, and cancer cells. Macrophages may be referred to as phagocytes, histiocytes, Kupffer cells, alveolar macrophages, or microglia, and may be found in nearly all body tissues. Macrophages are involved in nonspecific defense (innate immunity) and can help initiate specific defense

mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes.

Macrophages also have an anti-inflammatory role and can decrease immune reactions through the

release of cytokines. Macrophages that encourage inflammation are often referred to as M1 macrophages, and macrophages that decrease inflammation and encourage tissue repair are often referred to as M2 macrophages. Macrophages are produced by the differentiation of monocytes. Macrophages may be identified by their specific expression of proteins such as CD14, CD40, CD11b, CD64, F4/80, EMR1 , lysozyme M, MAC-1/MAC-3 and CD68 using flow cytometry and/or immunohistochemical staining. Dysfunctional macrophages may cause diseases such as chronic granulomatous disease that results in frequent infections.

In some embodiments, cells comprise non-immune cells. Non-immune cells may include, for example, epithelial cells (e.g., cells lining body cavities), cells derived from the central nervous system (e.g., nerve cells, neurons, neuroglial cells), stromal cells (e.g., connective tissue cells, fibroblasts, pericytes), stem cells (e.g., embryonic stem cells, adult stem cells), muscle cells (e.g., skeletal, cardiac, smooth), cartilage cells (e.g., chondrocytes), bone cells (e.g., osteoblasts, osteoclasts, osteocytes, lining cells), skin cells (e.g., keratinocytes, melanocytes, merkel cells, langerhans cells), endothelial cells (e.g., cells lining blood vessels), fat cells (e.g., white adipocytes, brown adipocytes), and sex cells (spermatozoa, ova).

In some embodiments, cells comprise epithelial cells. An epithelial cell, or epithelium, typically refers to a cell or cells that line hollow organs, as well as those that make up glands and the outer surface of the body. Epithelial cells can comprise squamous epithelial cells, columnar epithelial cells, adenomatous epithelial cells or transitional epithelial cells. Epithelial cells can be arranged in single layers or can be arranged in multiple layers, depending on the organ and location, and may comprise keratinocyte (KE) epithelial cells or non-keratinocyte (NKE) epithelial cells.

Keratinocytes form the squamous epithelium that is found at anatomic sites such as the skin, ocular surface, oral mucosa, esophagus and cervix. Keratinocytes terminally differentiate into flat, highly keratinized, non-viable cells that help protect against the environment and infection by forming a protective barrier. Examples of keratinocyte epithelial cells include, but are not limited to, dermal keratinocyte, ocular epithelial cells, corneal epithelial cells, oral mucosal epithelial cells, esophagus epithelial cells, and cervix epithelial cells.

Non-keratinocyte (NKE) epithelial cells form the epithelium of the body such as found in the breast, prostate, liver, respiratory tract, retina and gastrointestinal tract. NKE cells typically differentiate into functional, viable cells which function, for example, in absorption and/or secretion. These cells typically do not form highly keratinized structures characteristic of squamous epithelial cells.

Examples of NKE cells include, but are not limited to, prostate cells, mammary cells, hepatocytes, liver epithelial cells, biliary epithelial cells, gall bladder cells, pancreatic islet cells, pancreatic beta cells, pancreatic ductal epithelial cells, pulmonary epithelial cells, airway epithelial cells, nasal epithelial cells, kidney cells, bladder cells, urethral epithelial cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, testicular epithelial cells, ovarian epithelial cells, fallopian tube epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymus cells, pituitary cells, glandular cells, amniotic epithelial cells, retinal pigmented epithelial cells, sweat gland epithelial cells, sebaceous epithelial cells and hair follicle cells.

Cytokine and chemokine production

Certain methods described herein include measuring production of one or more cytokines and/or chemokines. Cytokines generally refer to small proteins (generally about 5-20 kDa) that are involved in cell signaling, and a release of cytokines can have an effect on the behavior of cells nearby. Cytokines are often considered as immunomodulating agents 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 may be produced by immune cells (e.g., monocytes, macrophages, B lymphocytes, T lymphocytes, and mast cells), endothelial cells, fibroblasts, and stromal cells.

In some embodiments, a method herein comprises measuring production by a cell of one or more cytokines. In some embodiments, a method herein comprises measuring production by a cell of one or more interleukins. In some embodiments, a method herein comprises measuring production by a cell of one or more IL-6 family cytokines. In some embodiments, a method herein comprises measuring production by a cell of one or more IL-1 family cytokines. A method herein may include measuring production of one or more cytokines provided in Table 1 below. Also provided in Table 1 are corresponding human genes and human receptors, however, cytokines, genes and receptors listed in Table 1 are not limited to human cytokines, genes and receptors. In some embodiments, a method herein comprises measuring production by a cell of a cytokine that binds to one or more receptors provided in Table 1.



Chemokines generally refer to a sub-family of cytokines (signaling proteins secreted by cells). Chemokines can induce directed chemotaxis in nearby responsive cells, and may be referred to as chemotactic cytokines. Chemokines are small (generally about 8-10 kDa) and typically have four cysteine residues in conserved locations for forming their 3-dimensional shape. Certain chemokines are pro-inflammatory and can be induced during an immune response to recruit cells of the immune system to a site of infection, and certain chemokines are homeostatic and are involved in controlling the migration of cells during normal processes of tissue maintenance or development. Chemokines may be classified into subfamilies (i.e., C, CX3C, CC and CXC), and exert their biological effects by interacting with G protein-linked transmembrane receptors

(chemokine receptors), that are found on the surface of target cells.

In some embodiments, a method herein comprises measuring production by a cell of one or more chemokines. In some embodiments, a method herein comprises measuring production by a cell of one or more C-family chemokines. In some embodiments, a method herein comprises measuring production by a cell of one or more CX3C-family chemokines. In some embodiments, a method herein comprises measuring production by a cell of one or more CC-family chemokines. In some embodiments, a method herein comprises measuring production by a cell of one or more CXC-family chemokines. A method herein may include measuring production of one or more chemokines provided in Table 2 below. Also provided in Table 2 are corresponding human genes and human receptors, however, chemokines, genes and receptors herein are not limited to human chemokines, genes and receptors. In some embodiments, a method herein comprises measuring production by a cell of a chemokine that binds to one or more receptors provided in Table 2.



In some embodiments, a method herein comprises measuring production by a cell of one or more cytokines and/or chemokines chosen from CCL2, CXCL10, IL-6 and IL-1 RA. In some

embodiments, a method herein comprises measuring production by a cell of one or more cytokines and/or chemokines chosen from CCL2, CCL5, CXCL1 , CXCL8, CXCL9, CXCL10, IL-6 and IL-1 RA.

In some embodiments, a method herein comprises measuring production by a cell of CCL2. In some embodiments, a method herein comprises measuring production by a cell of CXCL10. In some embodiments, a method herein comprises measuring production by a cell of IL-6. In some embodiments, a method herein comprises measuring production by a cell of IL-1 RA. In some embodiments, a method herein comprises measuring production by a cell of CCL5. In some embodiments, a method herein comprises measuring production by a cell of CXCL1. In some embodiments, a method herein comprises measuring production by a cell of CXCL8. In some embodiments, a method herein comprises measuring production by a cell of CXCL9.

Cytokine and/or chemokine production may be measured using any suitable method, apparatus or machine for measuring protein secretion and/or DNA expression (e.g., mRNA). For example, cytokine and/or chemokine production may be measured by immunoassay (e.g., enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, Western blot, 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, a luminometer, a fluorometer, a spectrophotometer, a suitable gene-chip or microarray analysis, mass spectrometry, chromatography, cytofluorimetric analysis, fluorescence microscopy, a suitable fluorescence or digital imaging method, confocal laser scanning microscopy, laser scanning cytometry, affinity chromatography, manual batch mode separation, electric field suspension, a suitable nucleic acid sequencing method and/or nucleic acid sequencing apparatus, the like and combinations thereof. Example immunoassays include LEGEND MAX™ ELISA kits with pre-coated plates (BioLegend), Macrophage/Microglia

LEGENDplex™ panels (BioLegend), and Chemokine Inflammatory LEGENDplex™ panels

(BioLegend).

Methods for assessing meteorin-b activity

Provided herein are methods for assessing meteorin-b activity. Methods for assessing meteorin-b activity generally are performed ex vivo (i.e. , outside of an organism) or in vitro (i.e. , performed or taking place in a test tube, culture dish, or other vessel outside of an organism). The terms ex vivo and in vitro may be used interchangeably, and typically are used in conjunction with methods performed in an artificial environment or artificial system. Methods herein generally include contacting a cell with meteorin-b or meteorin-b and a co-stimulant, and such contacting is typically performed ex vivo/in vitro (e.g., in cell culture).

In some embodiments, a method comprises contacting a cell with meteorin-b, measuring production of one or more cytokines and/or chemokines described herein, and detecting the activity of meteorin-b according to the cytokine production. In some embodiments, detecting the activity of meteorin-b comprises comparing the cytokine/chemokine production under test conditions (meteorin-b stimulation) to cytokine/chemokine production under control conditions (no meteorin-b stimulation). In some embodiments, detecting the activity of meteorin-b comprises comparing cytokine/chemokine production under test conditions to cytokine/chemokine production in a standard curve (e.g., cytokine/chemokine production measured for a plurality of different amounts of active meteorin-b). An active meteorin-b may include a meteorin-b standard (e.g., an unmodified full-length mature meteorin-b; a full-length mature meteorin-b comprising activity enhancing modifications). In some embodiments, detecting the activity of meteorin-b comprises comparing cytokine/chemokine production under test conditions to cytokine/chemokine production measured for a control, thereby providing a comparison. A control may include, for example, cytokine/chemokine production measured in the absence of meteorin-b. Typically, for a control, or for generating a standard curve, cytokine production is measured for the same cell (e.g., same cell-type, same cellular source, and/or same cell population) used for assessing meteorin-b activity (test conditions). Typically, for a control, or for generating a standard curve, cytokine/chemokine production can be a measured level of a cytokine/chemokine, or measured levels of

cytokines/chemokines in a combination of cytokines, chemokines, or cytokines and chemokines. Often, the level of the same cytokine/chemokine, or the levels of the same cytokines, chemokines, or cytokines and chemokines in the same cytokine/chemokine combination, is/are measured for test conditions and for control conditions or conditions for a standard curve.

In some embodiments, a method comprises contacting a cell with meteorin-b and a co-stimulant (e.g., IFN-g, IL-4, IL-10, TGF-b), measuring production of one or more cytokines and/or

chemokines described herein, and detecting the activity of meteorin-b according to the cytokine production. In some embodiments, detecting the activity of meteorin-b comprises comparing the cytokine/chemokine production under test conditions (meteorin-b + co-stimulant) to

cytokine/chemokine production under control conditions (no meteorin-b, no co-stimulant, or no meteorin-b + co-stimulant). In some embodiments, detecting the activity of meteorin-b comprises comparing cytokine/chemokine production under test conditions to cytokine/chemokine production in a standard curve (e.g., cytokine/chemokine production measured for a plurality of different amounts of active meteorin-b or active meteorin-b + co-stimulant). An active meteorin-b may

include a meteorin-b standard (e.g., an unmodified full-length mature meteorin-b; a full-length mature meteorin-b comprising activity-enhancing modifications). In some embodiments, detecting the activity of meteorin-b comprises comparing cytokine/chemokine production under test conditions to cytokine/chemokine production measured for a control, thereby providing a comparison. A control may include, for example, cytokine/chemokine production measured in the absence of meteorin-b; cytokine/chemokine production measured in the absence of a co-stimulant; cytokine/chemokine production measured in the absence of meteorin-b and a co-stimulant.

Typically, for a control, or for generating a standard curve, cytokine production is measured for the same cell (e.g., same cell-type, same cellular source, and/or same cell population) used for assessing meteorin-b activity (test conditions). Typically, for a control, or for generating a standard curve, cytokine/chemokine production can be a measured level of a cytokine/chemokine, or measured levels of cytokines/chemokines in a combination of cytokines, chemokines, or cytokines and chemokines. Often, the level of the same cytokine/chemokine, or the levels of the same cytokines, chemokines, or cytokines and chemokines in the same cytokine/chemokine

combination, is/are measured for test conditions and for control conditions or conditions for a standard curve.

In some embodiments, the production of one or more cytokines and/or chemokines under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is increased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, production of one or more cytokines and/or chemokines under test conditions is increased by at least about 10% compared to the production under control conditions. For example, production of one or more cytokines and/or chemokines under test conditions may be increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more compared to the production under control conditions. In some embodiments, production of one or more cytokines and/or chemokines under test conditions is increased by at least about 2-fold compared to the production under control conditions. For example, production of one or more cytokines and/or chemokines under test conditions may 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 the production under control conditions.

In some embodiments, the production of CCL2 under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is increased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, the production of CCL2 under test conditions (e.g., meteorin-b + IFN-g, or meteorin-b + IL-4) is increased compared to the production under control conditions (e.g., IFN-g or IL-4, respectively, without meteorin-b).

In some embodiments, the production of CCL5 under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is increased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, the production of CCL5 under test conditions (e.g., meteorin-b + IFN-g, meteorin-b + IL-4, or meteorin-b + IL-10) is increased compared to the production under control conditions (e.g., IFN-g, IL-4, or IL-10, respectively, without meteorin-b).

In some embodiments, the production of CXCL9 under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is increased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, the production of CXCL9 under test conditions (e.g., meteorin-b + IFN-g) is increased compared to the production under control conditions (e.g., IFN-g without meteorin-b).

In some embodiments, the production of CXCL10 under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is increased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, the production of CXCL10 under test conditions (e.g., meteorin-b + IFN-g) is increased compared to the production under control conditions (e.g., IFN-g without meteorin-b).

In some embodiments, the production of IL-6 under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is increased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, the production of IL-6 under test conditions (e.g., meteorin-b + IFN-g) is increased compared to the production under control conditions (e.g., IFN-g without meteorin-b).

In some embodiments, the production of IL-1 RA under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is increased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some

embodiments, the production of IL-1 RA under test conditions (e.g., meteorin-b + IFN-g) is increased compared to the production under control conditions (e.g., IFN-g without meteorin-b).

In some embodiments, the production of one or more cytokines and/or chemokines under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is decreased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, production of one or more cytokines and/or chemokines under test conditions is decreased by at least about 10% compared to the production under control conditions. For example, production of one or more cytokines and/or chemokines under test conditions may be decreased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more compared to the production under control conditions. In some embodiments, production of one or more cytokines and/or chemokines under test conditions is decreased by at least about 2-fold compared to the production under control conditions. For example, production of one or more cytokines and/or chemokines under test conditions may be decreased 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 the production under control conditions.

In some embodiments, the production of CXCL8 under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is decreased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, the production of CXCL8 under test conditions (e.g., meteorin-b + IL-10) is decreased compared to the production under control conditions (e.g., IL-10 without meteorin-b).

In some embodiments, the production of CXCL1 under test conditions (i.e., under meteorin-b stimulation or under meteorin-b + co-stimulant stimulation) is decreased compared to the production under control conditions (i.e., absence of meteorin-b or meteorin-b + co-stimulant). In some embodiments, the production of CXCL1 under test conditions (e.g., meteorin-b + IFN-g) is decreased compared to the production under control conditions (e.g., IFN-g without meteorin-b).

Meteorin-b receptor expression

In some embodiments, a method herein comprises identifying whether a cell expresses, or is capable of expressing, a meteorin-b receptor. Such method may include contacting a cell or a

population of cells (e.g., cells described herein) with a stimulant (e.g., a stimulant described herein (e.g., IFN-g, IL-4, IL-10, TGF-b)). Typically, a cell or population of cells is contacted with a stimulant for a duration of time prior to exposure to meteorin-b. A duration of time for contact with a stimulant may comprise about 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours, 48 hours or more. In some embodiments, a cell is contacted with a stimulant for about 18 hours. Conditions for contacting a cell with a stimulant may include a temperature between about 35°C and 40°C. In some embodiments, a cell is contacted with a stimulant at 37°C.

In some embodiments, a method may include contacting a cell or a population of cells under binding conditions with meteorin-b. Binding conditions typically include conditions (temperature, media components, pH) favorable to ligand-receptor binding (e.g., meteorin-b binding to a receptor). Ligand-receptor binding typically is non-covalent and occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces. The rate of binding may be referred to as ligand-receptor binding affinity. Generally, high-affinity ligand binding results from greater intermolecular force between the ligand and its receptor, while low-affinity ligand binding involves less intermolecular force between the ligand and its receptor. High-affinity binding typically results in a higher degree of occupancy for the ligand at its receptor binding site than for low-affinity binding.

Typically, a cell or population of cells is contacted with meteorin-b after contacting the cell with a stimulant for a duration of time. A duration of time for contact with meteorin-b may comprise about 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes or more. In some embodiments, a cell is contacted with meteorin-b for about 90 minutes. Conditions for contacting a cell with meteorin-b may include a temperature between about 1 °C and 20°C.

Conditions for contacting a cells with meteorin-b may include a temperature between about 1 °C and 19°C, or about 1°C and 18°C. In some embodiments, a cell is contacted with a stimulant at 4°C. Without being limited by theory, conditions for contacting a cell with meteorin-b may be performed at low temperatures to prevent activation and potential internalization of a meteorin-b receptor.

In some embodiments, a method may include detecting the presence, absence, and/or amount of meteorin-b polypeptide bound to a cell. Any suitable protein detection method may be used to detect the presence, absence, and/or amount of meteorin-b polypeptide bound to a cell, such as a

protein detection method described herein (e.g., immunoassay, flow cytometry). In some embodiments, meteorin-b comprises a tag, such as a tag described herein, and a method comprises contacting tagged meteorin-b with an agent capable of binding to the tag. In some embodiments, the agent is an antibody, an antibody comprising a detectable label, or a fragment thereof. In some embodiments, meteorin-b comprises a member of a binding pair, and a method comprises contacting meteorin-b with a second member of the binding pair. Any suitable binding pair may be used, and examples of binding pairs are described herein. In some embodiments, meteorin-b, or an agent that binds meteorin-b, comprises a detectable label, such as a detectable label described herein, and a method comprises detecting the detectable label (e.g., by flow cytometry).

In some embodiments, a cell assessed for expression of a meteorin-b is from a subject having a disease, disorder, syndrome, condition, infection or illness, or suspected of having a disease, disorder, syndrome, condition, infection or illness. Cells from a subject having a disease, disorder, syndrome, condition, infection or illness may be referred to as affected cells. In some

embodiments, the disease, disorder, syndrome, condition, infection or illness is characterized by altered expression of meteorin-b and/or a meteorin-b receptor. In some embodiments, the disease, disorder, syndrome, condition, infection or illness is characterized by increased expression of meteorin-b and/or a meteorin-b receptor. In some embodiments, the disease, disorder, syndrome, condition, infection or illness is characterized by decreased expression of meteorin-b and/or a meteorin-b receptor. Altered, increased or decreased expression of meteorin-b and/or a meteorin-b receptor may be relative to a cell or cells from a subject not having the disease, disorder, syndrome, condition, infection or illness. The subject not having the disease, disorder, syndrome, condition, infection or illness may be a different subject; or may be the same subject prior to developing the disease, disorder, syndrome, condition, infection or illness, or the same subject after recovery from the disease, disorder, syndrome, condition, infection or illness. Cells from a subject not having the disease, disorder, syndrome, condition, infection or illness may be referred to as a control cells.

In some embodiments, expression of meteorin-b and/or a meteorin-b receptor in affected cells is increased by at least about 10% compared to expression in control cells. For example, expression of meteorin-b and/or a meteorin-b receptor in affected cells may be increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more compared to expression in control cells. In some embodiments, expression of meteorin-b and/or a meteorin-b receptor in affected cells is increased by at least about 2-fold compared to expression in control cells. For example, expression of meteorin-b and/or a meteorin-b receptor in affected cells may 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 expression in control cells.

Kits

Provided in certain embodiments are kits. The kits may include any components and compositions described herein (e.g., IFN-g, IL-4, IL-10, and/or TGF-b; one or more components for measuring cytokine and/or chemokine production; a cell or population of cells) useful for performing any of the methods described herein, in any suitable combination. In some embodiments, a kit further includes meteorin-b (e.g., for use as a standard/control and/or for the user to generate a standard curve). Meteorin-b for testing may be provided by the user/purchaser of the kit. Kits may further include any reagents, buffers, or other components useful for carrying out any of the methods described herein. For example, a kit may include one or more binding molecules that

immunospecifically bind to one or more cytokines and/or chemokines under binding conditions.

Components of a kit may be present in separate containers, or multiple components may be present in a single container. Suitable containers include a single tube (e.g., vial), a cell culture plate, one or more wells of a plate (e.g., a 6-well plate, a 12-well plate, a 24-well plate, a 96-well plate, a 384-well plate, and the like), and the like.

Kits may also comprise instructions for performing one or more methods described herein and/or a description of one or more components described herein. Instructions and/or descriptions may be in printed form and may be included in a kit insert. In some embodiments, instructions and/or descriptions are provided as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD, CD-ROM, diskette, and the like. A kit also may include a written description of an internet location that provides such instructions or descriptions.

The examples set forth below illustrate certain embodiments and do not limit the technology.

Example 1: Production of cytokines and chemokines by THP-1 cells under IFN-g and meteorin-b stimulation

In this Example, the production of certain cytokines and chemokines by THP-1 cells under meteorin-b stimulation and IFN-y + meteorin-b co-stimulation was measured. A 6XHis-tagged meteorin-b (R & D Systems, cat # 9339-MN-050) and human recombinant IFN-g (BioLegend, cat # 752802) were used for the assays described below. The meteorin-b product included the following components: N-terminus- human meteorin-b (Gln46-Asp311 ; accession # Q641Q3); HP; linker peptide (GGGSGGGSGGGS (SEQ ID NO: 9)); and 6XHis (HHHHHH (SEQ ID NO: 10)) - C-terminus. The meteorin-b activity may be measured by its ability to induce STAT3 phosphorylation in E16E18 embryonic rat cortical neurons.

The production of CCL2 by THP-1 cells under IFN-g and meteorin-b co-stimulation was measured. THP-1 cells (5X104) were incubated on 96-well culture plates with RPMI 1% FBS and p/s 1X at 37°C (200 pi), with or without 1 pg/ml of rhlFN-g (BioLegend, cat # 570202) and with or without different concentrations (1 pg/ml, 2 pg/ml or 2.5 pg/ml) of meteorin-b (R & D Systems, cat # 9339-MN-050) or its control peptide (6XHis 2.5 pg/ml; BioLegend) for 24 hrs. For cytokine

measurements, samples were analyzed using human CCL2 ELISA (BioLegend), and the results are shown in Fig. 1.

The production of CXCL10, IL1 RA, and IL-6 by THP-1 cells under IFN-g and meteorin-b co stimulation was measured. THP-1 cells (5X104) were incubated on 96-well culture plates with RPMI 1 % FBS and p/s 1X at 37°C (200 mI), with or without 1 pg/ml of rhlFN-g (BioLegend, cat # 570202) and with or without different concentrations (1 pg/ml, 2 pg/ml or 2.5 pg/ml) of meteorin-b (R & D Systems, cat # 9339-MN-050) or its control peptide (6XHis 2.5 pg/ml; BioLegend) for 24 hrs. For cytokine measurements, samples were analyzed using LEGENDplex™ (BioLegend), and the results are shown in Fig. 2 (CXCL10 (top panel), IL1 RA (middle panel), and IL-6 (bottom panel)).

The production of other cytokines (i.e., ARGINASE, IL12p70, TNFa, IL-4, IL-10, IL-1b, CCL17 (also referred to as TARC), IL12p40, and IL-23) by THP-1 cells under IFN-y and meteorin-b co stimulation was measured, and the comparative results are provided in Fig. 3A.

Similarly, the production of chemokines by THP-1 cells under meteorin-b plus IFN-g, TΰRb, IL-4, or IL-10 co-stimulation was measured, and comparative results are provided in Fig. 3B.

Example 2: Identification of meteorin-b receptor expression in THP-1 cells

In this Example, identification of meteorin-b receptor expression in THP-1 cells is shown (see Fig.

5 for a schematic of the general detection strategy). Specifically, THP-1 cells (3X105) were incubated on 24-well culture plates with RPMI 1% FBS and p/s 1X at 37°C (400 pi), with or without 250 ng of rhlFN-g (BioLegend, cat # 570202), IL-10 (BioLegend, cat #715602), IL-4 (BioLegend, cat #574002), or TQBb (BioLegend, cat #580704) overnight (18 hrs). Then cells were transferred and washed with 2 ml of staining buffer (ST) in 2 ml Eppendorf tubes. Cells were pre-incubated with 5 mI of Fc receptor blocking solution (Human TruStain FcX™ (BioLegend)) per 100 mI of cell suspension for 10 minutes at room temperature (RT). Cells were then incubated with meteorin-b for 90 minutes at 4°C. After cells were washed 1X with 2 ml of PBS and vortexed, cells were incubated in a PBS/Zombie NIR™ (BioLegend) solution at 1 :300 for 10 minutes at RT in the dark. Cells were washed once with 2 ml of ST. Next anti 6XHis antibody coupled to phycoerythrin (BioLegend, Inc. cat # J095G46) was added to 100 ml of suspended cells for 30 minutes at 4°C in the dark. Finally, cells were washed 2X with ST and read by flow cytometry (Figs. 4A, 4B, 4C).

Example 3: Examples of amino acid and nucleic acid sequences








Example 4: Examples of embodiments

The examples set forth below illustrate certain embodiments and do not limit the technology.

A1. A method for assessing activity of a meteorin-b polypeptide comprising:

a) contacting a cell with a first composition comprising a co-stimulant and a second composition comprising a meteorin-b polypeptide;

b) measuring production by the cell of one or more cytokines and/or chemokines chosen from CCL2, CCL5, CXCL1 , CXCL8, CXCL9, CXCL10, IL-6 and IL-1 RA, thereby measuring cytokine production; and

c) detecting the activity of the meteorin-b polypeptide in the second composition according to the cytokine production measured in (b).

A2. The method of embodiment A1 , wherein the production of the one or more cytokines and/or chemokines is increased compared to the production by a cell not contacted with the first composition and the second composition.

A3. The method of embodiment A1 , wherein the production of the one or more cytokines and/or chemokines is increased compared to the production by a cell contacted with the first composition and not contacted with the second composition.

A3.1 The method of embodiment A1 , wherein the production of the one or more cytokines and/or chemokines is decreased compared to the production by a cell contacted with the first composition and not contacted with the second composition.

A4. The method of embodiment A3, wherein the production of the one or more cytokines and/or chemokines is increased by at least about 10% compared to the production by a cell contacted with the first composition and not contacted with the second composition.

A4.1 The method of embodiment A3.1 , wherein the production of the one or more cytokines and/or chemokines is decreased by at least about 10% compared to the production by a cell contacted with the first composition and not contacted with the second composition.

A5. The method of any one of embodiments A1 to A4.1 , wherein the cell is simultaneously contacted with the first composition and the second composition.

A6. The method of any one of embodiments A1 to A4.1 , comprising contacting the cell with the first composition prior to contacting the cell with the second composition.

A7. The method of any one of embodiments A1 to A6, wherein the co-stimulant comprises IFN-y.

A7.1 The method of any one of embodiments A1 to A6, wherein the co-stimulant comprises one or more polypeptides chosen from IFN-g, IL-4, IL-10, and TQRb polypeptides.

A8. The method of any one of embodiments A1 to A7.1 , wherein the cell is from a subject.

A9. The method of any one of embodiments A1 to A7.1 , wherein the cell is from a cell line.

A10. The method of any one of embodiments A1 to A9, wherein the cell is an isolated cell.

A11. The method of any one of embodiments A1 to A10, wherein the cell is an immune cell.

A12. The method of embodiment A6, wherein the cell is a monocyte.

A13. The method of embodiment A6, wherein the cell is a macrophage.

A14. The method of any one of embodiments A1 to A10, wherein the cell is a non-immune cell.

A15. The method of embodiment A14, wherein the cell is a stromal cell or a cell derived from the central nervous system.

A16. The method of any one of embodiments A1 to A15, wherein the cell is contacted with the first composition and the second composition ex vivo or in vitro.

A17. The method of any one of embodiments A1 to A16, wherein the meteorin-b polypeptide is a recombinant meteorin-b polypeptide.

A18. The method of any one of embodiments A1 to A16, wherein the meteorin-b polypeptide is a human meteorin-b polypeptide.

A19. The method of embodiment A18, wherein the meteorin-b polypeptide is a recombinant human meteorin-b polypeptide.

A20. The method of embodiment A18 or A19, wherein the meteorin-b polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

A21. The method of embodiment A18 or A19, wherein the meteorin-b polypeptide comprises amino acids 46-311 of SEQ ID NO: 1.

A22. The method of embodiment A18 or A19, wherein the meteorin-b polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO: 1.

A23. The method of embodiment A18 or A19, wherein the meteorin-b polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

A24. The method of embodiment A18 or A19, wherein the meteorin-b polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

A25. The method of any one of embodiments A1 to A16, wherein the meteorin-b polypeptide is a mouse meteorin-b polypeptide.

A26. The method of embodiment A25, wherein the meteorin-b polypeptide is a recombinant mouse meteorin-b polypeptide.

A27. The method of embodiment A25 or A26, wherein the meteorin-b polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

A28. The method of embodiment A25 or A26, wherein the meteorin-b polypeptide comprises amino acids 46-311 of SEQ ID NO: 3.

A29. The method of embodiment A25 or A26, wherein the meteorin-b polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO: 3.

A30. The method of embodiment A25 or A26, wherein the meteorin-b polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 3.

A31. The method of embodiment A25 or A26, wherein the meteorin-b polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 3.

A32. The method of any one of embodiments A1 to A31 , wherein the meteorin-b polypeptide comprises one or more chemical modifications.

A33. The method of any one of embodiments A1 to A32, wherein the meteorin-b polypeptide comprises a tag.

A34. The method of any one of embodiments A1 to A33, wherein the meteorin-b polypeptide comprises a detectable label.

A35. The method of any one of embodiments A1 to A34, wherein the meteorin-b polypeptide comprises a fused polypeptide.

B1. A kit, comprising:

a) a first composition comprising an IFN-g polypeptide;

b) one or more components for measuring cytokine and/or chemokine production, wherein the cytokines and/or chemokines are chosen from one or more of CCL2, CXCL10, IL-6 and IL-1 RA; and

c) instructions for use.

B1.1 A kit, comprising:

a) a first composition comprising one or more co-stimulants chosen from IFN-g, IL-4, IL-10, and TQRb polypeptides;

b) one or more components for measuring cytokine and/or chemokine production, wherein the cytokines and/or chemokines are chosen from one or more of CCL2, CCL5, CXCL1 , CXCL8, CXCL9, CXCL10, IL-6 and IL-1 RA; and

c) instructions for use.

B2. The kit of embodiment B1 or B1.1 , further comprising a second composition comprising a meteorin-b polypeptide.

B3. The kit of embodiment B2, wherein the meteorin-b polypeptide is a recombinant meteorin-b polypeptide.

B4. The kit of embodiment B2, wherein the meteorin-b polypeptide is a human meteorin-b polypeptide.

B5. The kit of embodiment B4, wherein the meteorin-b polypeptide is a recombinant human meteorin-b polypeptide.

B6. The kit of embodiment B4 or B5, wherein the meteorin-b polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

B7. The kit of embodiment B4 or B5, wherein the meteorin-b polypeptide comprises amino acids 46-311 of SEQ ID NO: 1.

B8. The kit of embodiment B4 or B5, wherein the meteorin-b polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO: 1.

B9. The kit of embodiment B4 or B5, wherein the meteorin-b polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

B10. The kit of embodiment B4 or B5, wherein the meteorin-b polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

B11. The kit of embodiment B2, wherein the meteorin-b polypeptide is a mouse meteorin-b polypeptide.

B12. The kit of embodiment B10, wherein the meteorin-b polypeptide is a recombinant mouse meteorin-b polypeptide.

B13. The kit of embodiment B11 or B12, wherein the meteorin-b polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

B14. The kit of embodiment B11 or B12, wherein the meteorin-b polypeptide comprises amino acids 46-311 of SEQ ID NO: 3.

B15. The kit of embodiment B11 or B12, wherein the meteorin-b polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO: 3.

B16. The kit of embodiment B11 or B12, wherein the meteorin-b polypeptide comprises one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 3.

B17. The kit of embodiment B11 or B12, wherein the meteorin-b polypeptide comprises a fragment comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 3.

B18. The kit of any one of embodiments B2 to B17, wherein the meteorin-b polypeptide comprises one or more chemical modifications.

B19. The kit of any one of embodiments B2 to B18, wherein the meteorin-b polypeptide comprises a tag.

B20. The kit of any one of embodiments B2 to B19, wherein the meteorin-b polypeptide comprises a detectable label.

B21. The kit of any one of embodiments B2 to B20, wherein the meteorin-b polypeptide comprises a fused polypeptide.

B22. The kit of any one of embodiments B1 to B21 , wherein each of the one or more components for measuring cytokine production comprises a binding molecule that immunospecifically binds to one of the cytokines under binding conditions.

B23. The kit of any one of embodiments B1 to B22, further comprising a cell or a population of cells.

C1. A method for identifying whether a cell expresses, or is capable of expressing, a meteorin-b receptor comprising:

a) contacting a cell with a first composition comprising a stimulant;

b) contacting the cell under binding conditions with a second composition comprising a meteorin-b polypeptide;

c) detecting the presence, absence, and/or amount of meteorin-b polypeptide bound to the cell; and

d) identifying whether the cell expresses, or is capable of expressing, the meteorin-b receptor according to the presence, absence, and/or amount of meteorin-b polypeptide detected in (c).

C2. The method of embodiment C1 , wherein the stimulant comprises a soluble protein.

C2.1 The method of embodiment C1 or C2, wherein the stimulant comprises a cytokine or a chemokine.

C2.2 The method of any one of embodiments C1 to C2.1 , wherein the stimulant comprises IFN-y.

C2.3 The method of any one of embodiments C1 to C2.1 , wherein the stimulant comprises one or more polypeptides chosen from IFN-g, IL-4, IL-10, and TQRb.

C3. The method of any one of embodiments C1 to C2.3, wherein the cell is from a subject.

C4. The method of embodiment C3, wherein the subject has a disease, disorder, syndrome, condition, infection or illness, or is suspected of having a disease, disorder, syndrome, condition, infection or illness.

C5. The method of embodiment C4, wherein the disease, disorder, syndrome, condition, infection or illness is characterized by altered expression of meteorin-b and/or a meteorin-b receptor.

C5.1 The method of embodiment C4, wherein the disease, disorder, syndrome, condition, infection or illness is characterized by increased expression of meteorin-b and/or a meteorin-b receptor.

C5.2 The method of embodiment C4, wherein the disease, disorder, syndrome, condition, infection or illness is characterized by decreased expression of meteorin-b and/or a meteorin-b receptor.

C6. The method of any one of embodiments C1 to C2.2, wherein the cell is from a cell line.

C7. The method of any one of embodiments C1 to C6, wherein the cell is an isolated cell.

C8. The method of any one of embodiments C1 to C7, wherein the cell is an immune cell.

C9. The method of embodiment C8, wherein the cell is a monocyte.

C10. The method of embodiment C8, wherein the cell is a macrophage.

C11. The method of any one of embodiments C1 to C7, wherein the cell is a non-immune cell.

C12. The method of embodiment C11 , wherein the cell is a stromal cell or a cell derived from the central nervous system.

C13. The method of any one of embodiments C1 to C12, wherein the method is performed ex vivo or in vitro.

C14. The method of any one of embodiments C1 to C13, wherein the meteorin-b polypeptide is a recombinant meteorin-b polypeptide.

C15. The method of any one of embodiments C1 to C13, wherein the meteorin-b polypeptide is a human meteorin-b polypeptide.

C16. The method of embodiment C15, wherein the meteorin-b polypeptide is a recombinant human meteorin-b polypeptide.

C17. The method of any one of embodiments C1 to C13, wherein the meteorin-b polypeptide is a mouse meteorin-b polypeptide.

C18. The method of embodiment C17, wherein the meteorin-b polypeptide is a recombinant mouse meteorin-b polypeptide.

C19. The method of any one of embodiments C1 to C18, wherein the meteorin-b polypeptide comprises a tag.

C20. The method of embodiment C19, wherein (c) comprises contacting the meteorin-b polypeptide with an agent capable of binding to the tag.

C21. The method of embodiment C20, wherein the agent is an antibody.

C22. The method of any one of embodiments C1 to C18, wherein the meteorin-b polypeptide comprises a first member of a binding pair.

C23. The method of embodiment C22, wherein (c) comprises contacting the meteorin-b polypeptide with a second member of the binding pair.

C24. The method of any one of embodiments C1 to C18, wherein the meteorin-b polypeptide comprises a detectable label.

C25. The method of embodiment C24, wherein (c) comprises detecting the detectable label.

The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.

Modifications may be made to the foregoing without departing from the basic aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill 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 technology.

The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms“comprising,”“consisting essentially of,” and“consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. The term“a” or“an” can refer to one of or a plurality of the elements it modifies (e.g.,“a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term“about” as used herein refers to a value within 10% of the underlying parameter (i.e. , plus or minus 10%), and use of the term“about” at the beginning of a string of values modifies each of the values (i.e.,“about 1 , 2 and 3” refers to about 1 , about 2 and about 3). For example, a weight of “about 100 grams” can include weights between 90 grams and 110 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes

all intermediate and fractional values thereof (e.g., 54%, 85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative

embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this technology.

Certain embodiments of the technology are set forth in the claim(s) that follow(s).