Compositions and Methods for Modulating Circulating Factors

Abstract

The present disclosure provides compositions such as polypeptides, polynucleotides, gene editing systems, small molecules, vectors, or host cells that include and / or modulate the expression or activity of immunoregulatory-related proteins in various embodiments. The present disclosure also provides, in various embodiments, methods of treating aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer, infectious diseases, immune diseases, hormones, growth factors, and / or indications treated with protein replacement, or combinations thereof, using agents that include and / or modulate the expression or activity of immunoregulatory-related proteins, as well as methods of identifying such agents.

Description

Technical Field

[0001] Incorporation by reference of materials This application claims the benefit of U.S. Provisional Application No. 63 / 345,771, filed May 25, 2022. The entire teachings of the above application are incorporated herein by reference.

[0002] Incorporation by reference of materials into XML This application incorporates by reference the Sequence Listing contained in the following Extensible Markup Language (XML) file, filed concurrently herewith. a) File name: 57081056002.xml; created on May 25, 2023, size 89,306,685 bytes.

Background Art

[0003] Endocrine circulating factors are released from endocrine organs and are found in the circulation. Endocrine circulating factors regulate the body's homeostasis and metabolism. Dysregulation of these factors can be affected by genetic factors and diseases such as cancer or hormonal imbalances. Therefore, endocrine circulating factors can be markers of disease states or biological / disease pre-phenotypes. Since dysregulation of endocrine circulating factors can affect the body's homeostasis, such dysregulation can cause a wide range of symptoms and can affect growth, development, metabolism, sexual function, and mood. Therefore, there is a significant need to identify additional novel endocrine circulating factors as biomarkers and therapeutic targets.

Summary of the Invention

Means for Solving the Problems

[0004] The disclosure provided herein is based in part on the identification of non-canonical (e.g., proteins encoded by non-canonical open reading frames (ORFs)) circulating factors (e.g., endocrine circulating factors).

[0005] In one aspect, the present disclosure relates to an agent that includes and / or modulates (e.g., increases or decreases) the expression and / or activity of a target protein identified herein (e.g., the target proteins listed in the Sequence Listing, Table A) or a variant of the foregoing. In some embodiments, the agent includes a target protein identified herein (e.g., the target proteins listed in the Sequence Listing, Table A) or a variant of the foregoing. In certain embodiments, the agent modulates (e.g., increases or decreases) the expression and / or activity of a target protein identified herein (e.g., the target proteins listed in the Sequence Listing, Table A or a variant of the foregoing). In some embodiments, the agent comprises, consists essentially of, or consists of a polypeptide, polynucleotide, gene editing system, small molecule, or cell (e.g., cell therapy). The agent can be an inhibitor or activator of a target protein identified herein. In some embodiments, the agent modulates the expression of a target protein identified herein. In some embodiments, the agent modulates the activity of a target protein identified herein.

[0006] In another aspect, the present disclosure provides a pharmaceutical composition comprising a target protein identified herein and a pharmaceutically acceptable carrier.

[0007] In another aspect, the present disclosure provides a pharmaceutical composition comprising an agent that modulates the expression or activity of a target protein identified herein and a pharmaceutically acceptable carrier.

[0008] In other aspects, the present disclosure relates to a polynucleotide encoding a polypeptide described herein, an expression vector comprising a polynucleotide encoding a polypeptide described herein, and a host cell comprising a polynucleotide encoding a polypeptide described herein.

[0009] In another aspect, the present disclosure provides a method for detecting a disease or condition in a subject or determining the likelihood of developing a disease or condition in a subject, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject, wherein the level of expression or activity of the target protein in the sample indicates the likelihood of developing a disease or condition in the subject, and the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infectious diseases, immune diseases (e.g., inflammation and / or autoimmune diseases), indications treated with hormones, growth factors and / or protein replacement (e.g., enzyme replacement, antibody replacement), or combinations thereof.

[0010] In another aspect, the present disclosure provides a method for preparing a sample useful for determining the likelihood of developing a disease or condition in a subject, the method comprising: a) obtaining or having obtained a sample from the subject; b) adding a protease inhibitor, a control peptide, a standard peptide, or a combination thereof to the sample to prepare a sample useful for detecting the likelihood of developing cancer; and c) quantifying the expression or activity of a target protein in the sample prepared in step b). comprising wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infectious diseases, immune diseases (e.g., inflammation and / or autoimmune diseases), indications treated with hormones, growth factors and / or protein replacement, or combinations thereof.

[0011] In some embodiments, the method further comprises treating a subject predicted to be at risk of developing a disease or condition with an effective amount of an agent that modulates the expression or activity of the target protein identified herein and / or administering to the subject a pharmaceutical composition comprising the agent.

[0012] In another aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof (e.g., a human subject having cancer), the method comprising administering to the subject an effective amount of an agent that modulates and / or comprises the expression or activity of a target protein identified herein, or a pharmaceutical composition comprising the agent.

[0013] In another aspect, the present disclosure provides a method of selecting a subject suitable for treatment of a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject, and selecting a subject suitable for treatment of the disease or condition according to the level of the expression or activity of the target protein in the sample, wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infectious diseases, immune diseases (e.g., inflammation and / or autoimmune diseases), indications treatable with hormones, growth factors and / or protein supplements, or combinations thereof.

[0014] In another aspect, the present disclosure provides a method of modulating the expression or activity of a target protein identified in the Sequence Listing, Table A or a variant of the foregoing in a cell (e.g., a cancer cell such as a cancer cell in a subject), the method comprising contacting the cell (e.g., in vitro, ex vivo, or in vivo) with an agent that modulates and / or comprises the expression or activity of the target protein identified herein, or a pharmaceutical composition comprising the agent.

[0015] In another aspect, the present disclosure provides a method of identifying an agent that modulates the expression or activity of a target protein identified herein, a) contacting the target protein with the agent; and b) determining whether the agent modulates the expression or activity of the target protein comprising, wherein a difference in the expression or activity of the target protein contacted with the agent, as compared to a reference, with respect to the expression or activity of the target protein, indicates that the agent modulates the expression or activity of the target protein.

[0016] The foregoing will be apparent from the following more particular description of the exemplary embodiments, as illustrated in the accompanying drawings, wherein like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

Brief Description of the Drawings

[0017]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0018] The description of the exemplary embodiments continues below.

[0019] Target protein In one aspect, the present disclosure provides a target protein identified herein. As used herein, the expressions "target protein identified herein" and "target protein of the present disclosure" refer to the polypeptide disclosed in the sequence listing (for example, a target protein comprising an amino acid sequence selected from any of SEQ ID NO: 3585, SEQ ID NO: 24296, SEQ ID NO: 74164, SEQ ID NO: 7353, SEQ ID NO: 26888, SEQ ID NO: 36277, SEQ ID NO: 24296, SEQ ID NO: 32262, SEQ ID NO: 49310, SEQ ID NO: 42382, SEQ ID NO: 38427, SEQ ID NO: 246513, SEQ ID NO: 75388, and SEQ ID NOs: 75451-75473), its variants (for example, a target protein comprising an amino acid sequence selected from SEQ ID NO: 36277_T31A; SEQ ID NO: 7353_P25L, SEQ ID NO: 74164_I36T, SEQ ID NO: 26888_L8V, SEQ ID NO: 24296 rs221797 V-to-A, V-to-G or V-to-D), and both the peptides disclosed in Table A herein. The target protein can be produced recombinantly (for example, via DNA or mRNA) or synthetically.

[0020] In various embodiments, the target protein is an extracellular (secreted) protein.

[0021] In various embodiments, the target protein is the protein of the sequence listing or Table A. In some embodiments, the target protein is a protein comprising the amino acid sequence described in the sequence listing or Table A. In some embodiments, the target protein consists of the amino acid sequence described in the sequence listing or Table A. In some embodiments, the target protein comprises an amino acid sequence having one amino acid substitution relative to the amino acid sequence described in the sequence listing or Table A, wherein the substitution is a substitution of the methionine (Met) residue of the N-terminal residue in the amino acid sequence of the sequence listing or Table A. In some embodiments, the target protein consists of an amino acid sequence having one amino acid substitution relative to the amino acid sequence described in the sequence listing or Table A, wherein the substitution is a substitution of the methionine (Met) residue of the N-terminal residue in the amino acid sequence of the sequence listing or Table A. In some embodiments, the target protein comprises the amino acid sequence described in the sequence listing or Table A and further comprises a methionine (Met) residue at its N-terminus. In some embodiments, the target protein consists of the amino acid sequence described in the sequence listing or Table A and the methionine (Met) residue at its N-terminus.

[0022]

Table A-1

[0023]

Table A-2

[0024]

Table A-3

[0025]

Table A-4

[0026]

Table A-5

[0027] The specific target proteins of the sequence listing and Table A are differentially expressed (e.g., upregulated or downregulated) in diseases and / or conditions selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors) and infections, immune diseases (e.g., inflammation and / or autoimmune diseases), indications treated with hormones, growth factors and / or protein supplements, or combinations thereof, as compared to a reference state (e.g., a normal state), such that modulation of the level and / or activity of the target protein acts to treat, ameliorate and / or prevent the onset of the disease or condition.

[0028] As used herein, the term "differentially expressed" refers to at least one recognizable difference in protein expression. It can be a quantitatively measurable, semi-quantitatively estimable or qualitatively detectable difference in protein expression. Thus, a protein that is differentially expressed, i.e., a "DEP", can have a higher expression level in a reference state (e.g., a normal state) than in a disease state in which the DEP has a lower expression level or is not expressed at all. Conversely, a DEP can have a higher expression level in a disease state than in a reference state (e.g., a normal state) in which the DEP has a lower expression level or is not expressed at all. Further, expression can be considered differential if the DEP changes recognizably (e.g., mutates) between the two states being compared. Recognizable changes can include amino acid substitutions, insertions and / or deletions, including N-terminal and C-terminal cleavage, as well as modifications (e.g., post-translational modifications).

[0029] As used herein, the term "reference" refers to a standard used for comparison purposes. One of ordinary skill in the art can select an appropriate reference for a particular comparison purpose. Thus, for example, a reference for a disease state may be a normal healthy state; a reference for a mutant protein may be a non-mutant protein; a reference for disease treatment may be no treatment or a standard treatment. In some embodiments, particularly those including methods for identifying an agent that modulates the expression and / or activity of a target protein, the reference is the activity and / or expression of the target protein in the absence of the agent. In some embodiments, the reference is based on a predetermined level, such as functional expression or an empirical assay. In some embodiments, the reference is obtained from one cell, sample or subject (e.g., a healthy subject, a cell or sample from a subject without a particular disease; a healthy subject, a subject without a particular disease). In some embodiments, the reference is obtained from two or more cells, samples or subjects (e.g., a population thereof) (e.g., a healthy subject, a cell or sample from a subject without a particular disease; a healthy subject, a subject without a particular disease), such as two, three, four, five, ten, twenty, thirty, fifty, one hundred or more, or a statistically significant number of cells, samples or healthy subjects. A reference obtained from two or more cells, samples or subjects can be expressed as a statistic (e.g., an average or a median).

[0030] In some embodiments, the protein is used as a marker of immunity and / or disease state.

[0031] In certain embodiments, the target protein has a higher expression level in endocrine organs (e.g., hypothalamus, pituitary gland, thyroid gland, parathyroid gland, adrenal gland, pineal gland, pancreas, ovary and / or testis) and / or secretory cells (e.g., alpha cells, beta cells and / or delta cells). In some embodiments, the target protein has an expression level that is at least about 0.5-fold higher, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher (e.g., 50-fold higher, 100-fold higher) in endocrine organs and / or secretory cells than the target protein expression level in a reference organ and / or cell.

[0032] In certain embodiments, the target protein has a lower expression level in endocrine organs and / or secretory cells. In some embodiments, the target protein has an expression level in endocrine organs and / or secretory cells that is at least about 0.5-fold lower, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold lower (e.g., 50-fold lower, 100-fold lower) than the target protein expression level in a reference organ and / or cell.

[0033] In some embodiments, the target protein has an expression level that is at least about 0.5-fold higher, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher (e.g., 50-fold higher, 100-fold higher) in a disease (e.g., determined from a sample from a cell or tissue of a subject having the disease) than the target protein expression level in a reference (e.g., a sample derived from a cell or tissue of a subject without the disease).

[0034] In some embodiments, the target protein has an expression level that is at least about 0.5-fold lower, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) in a disease (e.g., determined from a sample comprising or obtained from the cells or tissue of a subject having the disease) than the target protein expression level in a reference (e.g., a sample derived from the cells or tissue of a subject without the disease). In some embodiments, the target protein is not expressed or is expressed at undetectable levels in a disease (e.g., when determined from a sample comprising or obtained from the cells or tissue of a subject having the disease).

[0035] In some embodiments, the target protein has a transcript level that is at least about 0.5-fold higher, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher (e.g., 50-fold higher, 100-fold higher) in a disease (e.g., determined from a sample from the cells or tissue of a subject having the disease) than the target protein transcript level in a reference (e.g., a sample derived from the cells or tissue of a subject without the disease). In certain embodiments, the increase in the transcription level of the target protein contributes to (e.g., results in) the disease or condition described herein.

[0036] In some embodiments, the target protein has a transcript level that is at least about 0.5-fold lower, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) in a disease (e.g., determined from a sample comprising or obtained from the cells or tissue of a subject having the disease) than the target protein transcript level in a reference (e.g., a sample from cells or tissue of a subject without the disease). In some embodiments, the transcription of the target protein is not expressed or is expressed at undetectable levels in a disease (e.g., when determined from a sample comprising or obtained from the cells or tissue of a subject having the disease). In certain embodiments, the decrease in the transcription level of the target protein contributes to (e.g., results in) the disease or condition described herein.

[0037] In certain embodiments, the gene encoding the target protein comprises at least one mutation (e.g., fusion of amino acid repeats, deletion, insertion, point mutation, and / or expansion) in the disease described herein.

[0038] Non-limiting examples of (biological) samples include blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., biopsy or micro-biopsy), pancreatic juice, chorionic villus samples, and cells isolated from a subject.

[0039] In some embodiments, the target protein is translated from non-coding RNA. In some embodiments, the non-coding RNA is a long intergenic non-coding RNA (lincRNA). In certain embodiments, the non-coding RNA is a long non-coding RNA (lncRNA). In some embodiments, the non-coding RNA is a microRNA (miRNA or miR).

[0040] In some embodiments, the target protein is translated from a non-exon element in unprocessed precursor mRNA (pre-mRNA). In some embodiments, the non-exon element is an intron in the pre-mRNA. In some embodiments, the non-exon element is the 5' untranslated region (5'-UTR) in the pre-mRNA. In some embodiments, the non-exon element is the 3' untranslated region (3'-UTR) in the pre-mRNA.

[0041] In some embodiments, the target protein has a length of 2,000 amino acids or less, such as 1000 amino acids or less, 750 amino acids or less, 500 amino acids or less, 250 amino acids or less, 150 amino acids or less, or 100 amino acids or less. In some embodiments, the target protein has a length of 7 amino acids or more, such as 8, 9, 10, 15, 18, 25, 50, 75, or 100 amino acids or more. In certain embodiments, the target protein has a length of about 50 to about 200 amino acids, such as about 100 to about 150 amino acids. In certain embodiments, the target protein has a length of 7 amino acids or more. In more specific embodiments, the target protein has a length of about 18 amino acids.

[0042] In some embodiments, the target protein is involved in acyl-CoA hydrolysis, acylglyceride metabolism, alanine, aspartic acid and glutamic acid metabolism, amino sugar and nucleotide sugar metabolism, aminoacyl-tRNA biosynthesis, androgen metabolism, arachidonic acid metabolism, arginine and proline metabolism, ascorbic acid and aldaric acid metabolism, beta-oxidation of branched-chain fatty acids (mitochondria), beta-oxidation of di-unsaturated fatty acids (n-6) (mitochondria), beta-oxidation of di-unsaturated fatty acids (n-6) (peroxisome), beta-oxidation of even-chain fatty acids (mitochondria), beta-oxidation of even-chain fatty acids (peroxisome), beta-oxidation of odd-chain fatty acids (mitochondria), beta-oxidation of phytanic acid (peroxisome), beta-oxidation of poly-unsaturated fatty acids (mitochondria), beta-oxidation of unsaturated fatty acids (n-7) (mitochondria), beta-oxidation of unsaturated fatty acids (n-7) (peroxisome), beta-oxidation of unsaturated fatty acids (n-9) (mitochondria), beta-oxidation of unsaturated fatty acids (n-9) (peroxisome), beta-alanine metabolism, bile acid biosynthesis, bile acid recycling, biopterin metabolism, biotin metabolism, blood group biosynthesis, butanoic acid metabolism, C5-branched dibasic acid metabolism, carnitine shuttle (cytoplasm, endoplasmic reticulum, mitochondria, and / or peroxisome), cholesterol biosynthesis 1 (Bloch pathway), cholesterol biosynthesis 2, cholesterol biosynthesis 3 (Kandustch-Russell pathway), cholesterol metabolism, chondroitin / heparan sulfate biosynthesis, chondroitin sulfate degradation, CoA synthesis, cysteine and methionine metabolism, drug metabolism, eicosanoid metabolism, estrogen metabolism, ether lipid metabolism, fatty acid activation (cytoplasm and / or endoplasmic reticulum), fatty acid biosynthesis (even-chain and / or odd-chain), fatty acid biosynthesis (unsaturated), fatty acid unsaturation (even-chain and / or odd-chain), fatty acid elongation (even-chain and / or odd-chain), fatty acid oxidation, folate metabolism, formation and hydrolysis of cholesterol esters, fructose and mannose metabolism, galactose metabolism, glucocorticoid biosynthesis, glutathione metabolism, glycerolipid metabolism, glycerophospholipid metabolism, glycine, serine and threonine metabolism, glycolysis / gluconeogenesis, sphingolipid biosynthesis - ganglio series, globo series sphingoglycolipid biosynthesis, lacto-neolacto series sphingoglycolipid biosynthesis, sphingoglycolipid metabolismIt is an upstream signaling molecule selected from glycosylphosphatidylinositol (GPI)-anchor biosynthesis, heme degradation, heme synthesis, heparan sulfate degradation, histidine metabolism, inositol phosphate metabolism, isolation, keratan sulfate biosynthesis, keratan sulfate degradation, leukotriene metabolism, linoleic acid metabolism, lipoic acid metabolism, lysine metabolism, metabolism of other amino acids, N-glycan metabolism, nicotinic acid and nicotinamide metabolism, nucleotide metabolism, O-glycan metabolism, omega-3 fatty acid metabolism, omega-6 fatty acid metabolism, oxidative phosphorylation, pantothenic acid and CoA biosynthesis, pentose and glucuronate interconversions, pentose phosphate pathway, phenylalanine metabolism; tyrosine and tryptophan biosynthesis, phosphatidylinositol phosphate metabolism, pool reactions, porphyrin metabolism, propanoate metabolism, prostaglandin biosynthesis, protein assembly, proteolysis, protein modification, purine metabolism, pyrimidine metabolism, pyruvate metabolism, retinol metabolism, riboflavin metabolism, ROS detoxification, serotonin and melatonin biosynthesis, sphingolipid metabolism, starch and sucrose metabolism, steroid metabolism, sulfur metabolism, terpenoid backbone biosynthesis, thiamine metabolism, transport reactions, tricarboxylic acid cycle and glyoxylate / dicarboxylate metabolism, tryptophan metabolism, tyrosine metabolism, ubiquinone synthesis, urea cycle, valine; leucine; and isoleucine metabolism, vitamin A metabolism, vitamin B2 metabolism, vitamin B6 metabolism, vitamin B12 metabolism, vitamin C metabolism, vitamin D metabolism, vitamin E metabolism, xenobiotic metabolism, or a combination thereof.,

[0043] Certain target proteins disclosed herein (e.g., SEQ ID NO: 49310; SEQ ID NO: 42382) have been identified as modulators of G protein-coupled receptors (GPCRs) (see, e.g., Example 12). In some embodiments, the target proteins of the present disclosure are modulators of one or more GPCRs. In some embodiments, the target protein is an agonist of one or more GPCRs. In some embodiments, the target protein is an antagonist of one or more GPCRs. In some embodiments, the target protein is a direct modulator of one or more GPCRs, e.g., a ligand of one or more GPCRs. In some embodiments, the target protein is an indirect modulator of one or more GPCRs.

[0044] The expression and / or activity of various GPCRs are associated with various diseases / disorders, conditions, and indications, including those shown in Table B (e.g., see Kenakin, T., Biased Receptor Signaling in Drug Discovery, Pharmacol Rev 71:267-315, April 2019; Harmar, A.J., et al., IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels, Nucleic Acids Research, 2009, Vol. 37; and Davenport AP, Scully CCG, de Graaf C, Brown AJH, and Maguire JJ. Advances in therapeutic peptides targeting G protein-coupled receptors. Nat Rev Drug Discov. 2020 Jun. 19(6):389-413; the entire contents of each are hereby incorporated by reference in their entirety). Accordingly, in some embodiments, the target proteins disclosed herein that are modulators of GPCRs are useful for treating and / or diagnosing one or more diseases / disorders, conditions, and / or indications known to be associated with GPCR expression and / or activity, such as cancer or pre-cancerous conditions, or any of the diseases / disorders, conditions, and indications listed in Table B.

[0045]

Table B-1

[0046]

Table B-2

[0047]

Table B-3

[0048]

Table B-4

[0049]

Table B-5

[0050] Agents that regulate the target protein In the present specification, agents that regulate the expression of the target proteins disclosed herein, such as the target proteins in the Sequence Listing, Table A, or variants or fragments of the foregoing (e.g., biologically active fragments of the target proteins) are provided. The expression of the target protein or its variant or fragment can be regulated by a wide range of processes that directly or indirectly result in an increase or decrease in the level of the target protein. Non-limiting examples include changes in the copy number of the gene encoding the target protein, transcription initiation, elongation or termination, RNA processing, RNA stability (e.g., mRNA stability), RNA degradation, translation initiation, post-translational modification of the protein, protein stability, proteolysis (e.g., cleavage such as protease cleavage), or combinations thereof.

[0051] In some embodiments, the agent regulates (e.g., increases or decreases) the expression of the gene or gene transcript encoding the target protein. In some embodiments, the agent regulates the expression or activity of the target protein. In some embodiments, the agent decreases (e.g., inhibits, reduces or neutralizes) the activity of the target protein. In some embodiments, the agent increases (e.g., activates) the activity of the target protein. In some aspects, the agent decreases (e.g., inhibits or down-regulates) the expression of the target protein. In other embodiments, the agent increases (e.g., activates or up-regulates) the expression of the target protein.

[0052] As used herein, the term "raising" or "raise" refers to an adjustment that results in a higher level of expression, activity, function, or a combination or metric thereof (e.g., cancer cell death or DNA methylation at a target site) of a target protein as compared to a reference (e.g., the level before or in the absence of modulation by an agent). In some embodiments, the agent raises the expression or activity or metric of the target protein by at least about 5%, e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% as compared to the reference.

[0053] As used herein, the term "lowering" or "lower" refers to an adjustment that results in a lower level of expression, activity, function, or a combination or metric thereof (e.g., cancer cell death or DNA methylation at a target site) of a target protein as compared to a reference (e.g., the level before or in the absence of modulation by an agent). In some embodiments, the agent lowers the expression or activity or metric of the target protein by at least about 5%, e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% as compared to the reference.

[0054] Non-limiting examples of metrics include energy production or conversion in the liver (e.g., regulation of ATP synthesis, β-oxidation, oxidation of metabolites derived from glycolysis, oxidation of metabolites derived from amino acids), mitochondrial transcription, mitochondrial ribosome assembly, mitochondrial translation, mitochondrial thermogenesis, hormonal signaling (e.g., mitochondrial estrogen receptor (mtER) signaling), redox maintenance (e.g., NADH and / or FADH 2) Cell cycle regulation, cell migration, cell morphology, apoptosis, necrosis, membrane potential, ion (e.g., calcium or zinc) storage, ion (e.g., calcium or zinc) homeostasis, metabolite synthesis (e.g., heme biosynthesis or steroid biosynthesis), nutrient sensing, endoplasmic reticulum stress response pathway, signal transduction processes (e.g., calcium signal transduction) are included.

[0055] In some embodiments, the level of expression, activity, function or combination thereof, or metric of the target protein is measured, for example, after initiating a treatment regimen, after contacting the agent (e.g., with cells) or administering the agent (e.g., to a subject) for at least about 1 day, for example, at least about 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months or 6 months.

[0056] In some embodiments, the agent comprises, consists essentially of, or consists of a polypeptide, polynucleotide, gene editing system, small molecule, or cell (e.g., cell therapy).

[0057] In some embodiments, the target protein activates immune cells and the agent modulates (e.g., increases or decreases) the level of expression, activity, function or combination thereof of the target protein. In some embodiments, the target protein inhibits immune cells (e.g., inhibits activation of immune cells, induces immune cell death (e.g., apoptosis), or a combination thereof), and the agent modulates (e.g., increases or decreases) the level of expression, activity, function or combination thereof of the target protein.

[0058] In certain embodiments, the agent modulates (e.g., increases or decreases) the expression, activity, function, or a combination thereof of a target protein in cancer cells (e.g., metastatic cancer cells), cells in the tumor microenvironment (e.g., stromal cells), target cells of an inflammatory response (e.g., epithelial cells, endothelial cells, stem cells, or non-immune cells), immune cells (e.g., effector T cells, helper T cells, Th1 cells, Th2 cells, Th17 cells, B cells, natural killer (NK) cells, innate lymphoid cells (e.g., ILC1 cells, ILC2 cells, ILC3 cells), macrophages (e.g., M1 macrophages, M2 macrophages), monocytes, and / or antigen-presenting cells (e.g., dendritic cells), or combinations thereof. In certain embodiments, the agent modulates the expression, activity, function, or a combination thereof of a target protein in a tumor, tumor microenvironment, metastatic site, lymph node, spleen, secondary lymphoid organ, tertiary lymphoid organ, barrier tissue, skin, intestine, airway, wound, other immune tissue, non-immune tissue, or combinations thereof.

[0059] In some embodiments, the agent modulates (e.g., increases or decreases) inflammation, decreases the level of autoantibodies, increases organ function, decreases the rate or number of relapses or recurrences, decreases the viral load, controls infection, or performs a combination of the above.

[0060] In some embodiments, the agent induces downregulation of the target protein (e.g., increases target protein degradation); prevents multimerization (e.g., dimerization) of the target protein; sequesters the target protein (e.g., secreted target protein); modulates (e.g., agonizes, antagonizes, or disrupts) a known function of the target protein; decreases the binding between the target protein and a binding partner (e.g., via steric hindrance); modulates (e.g., increases or decreases) downstream cell signaling; induces antibody-dependent cell killing, phagocytosis, and / or opsonization of cells expressing the target protein; or combinations thereof. In certain embodiments, the agent lacks agonist activity against the target protein. In certain embodiments, the agent has agonist activity against the target protein. In some embodiments, the agent lacks antagonist activity against the target protein. In some embodiments, the agent has antagonist activity against the target protein. In certain embodiments, the agent binds to at least one residue of the target protein involved in binding to the binding partner. In some embodiments, the agent binds to one or more binding sites and / or domains of the target protein involved in binding to the binding partner of the target protein.

[0061] Non-limiting examples of binding partners include androgen receptor, calcitriol receptor, corticotropin-releasing hormone receptor 1, corticotropin-releasing hormone receptor 2, estrogen receptor, follicle-stimulating hormone receptor, glucagon receptor, gonadotropin receptor, gonadotropin-releasing hormone receptor, growth hormone receptor, insulin receptor, luteinizing hormone, progesterone receptor, retinoid receptor, somatostatin receptor, thyroid hormone receptor, and thyrotropin receptor.

[0062] In some embodiments, the agent induces downregulation of a binding partner of the target protein; sequesters a binding partner of the target protein (e.g., a secreted binding partner); prevents multimerization (e.g., dimerization) of a binding partner of the target protein; sequesters a binding partner of the target protein (e.g., a secreted binding partner); modulates (e.g., agonizes, antagonizes, or disrupts) a known function of a binding partner of the target protein; decreases the binding between the target protein and the binding partner (e.g., via steric hindrance); modulates (e.g., increases or decreases) downstream cell signaling; induces antibody-dependent cell death, phagocytosis, and / or opsonization of cells expressing a binding partner of the target protein; or combinations of the above. In certain embodiments, the agent lacks agonist activity against a binding partner of the target protein. In certain embodiments, the agent has agonist activity against a binding partner of the target protein. In some embodiments, the agent lacks antagonist activity against a binding partner of the target protein. In some embodiments, the agent has antagonist activity against a binding partner of the target protein. In certain embodiments, the agent further binds to at least one residue of a binding partner of the target protein involved in the binding between the target protein and the binding partner. In more specific embodiments, the agent further binds to one or more binding sites and / or domains of a binding partner of the target protein involved in the binding between the target protein and the binding partner.

[0063] In some embodiments, the agent modulates (e.g., activates or inhibits) immune signaling, cytokine signaling, inflammatory signaling, or combinations of the foregoing.

[0064] In some embodiments, the agent enhances signals involved in T cell activation and / or survival. In certain embodiments, the agent activates stimulatory checkpoint molecules. Non-limiting examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, GITR, inducible T cell co-stimulator (ICOS). In certain embodiments, the agent is an agonist for CD28.

[0065] In some embodiments, the agent reduces signals involved in T cell anergy and / or exhaustion. In certain embodiments, the agent inhibits inhibitory checkpoint molecules. Non-limiting examples of inhibitory checkpoint molecules include PD-1, PD-L1, PD-L2, TIM-3, LAG-3, CTLA-4, A2AR, CD276, B7-H4, BTLA, IDO, KIR, NOX2, VISTA, SIGLEC 7 and SIGLEC 9. In certain aspects, the agent is an inhibitor for PD-1 (e.g., a blocking antibody).

[0066] In certain embodiments, the agent modulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of a variant of the target protein disclosed herein. In some embodiments, the variant comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the target protein disclosed herein. For example, the sequence identity to the variant can be at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the sequence identity is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the sequence identity is about 70-99%, 75-99%, 75-95%, 80-99%, 80-98%, 80-95%, 80-90%, 85-98%, 85-97%, 85-90%, 90-97%, 90-96%, 90-85%, 90-80%, or 95-99%. In some embodiments, the variant comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to the amino acid sequence of the target protein disclosed herein.

[0067] As used herein, the term "sequence identity" refers to the extent to which two nucleotide sequences or two amino acid sequences have the same residue at the same position when the sequences are aligned to achieve the maximum level of identity expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as the reference sequence and is compared to the test sequence. The sequence identity between the reference sequence and the test sequence is expressed as the percentage of positions over the full length of the reference sequence at which the reference sequence and the test sequence share the same nucleotide or amino acid when aligned to achieve the maximum level of identity. As an example, if, upon alignment to achieve the maximum level of identity, the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the full length of the reference sequence, the two sequences are considered to have 70% sequence identity.

[0068] The alignment of the comparison arrays to achieve the maximum level of identity can be readily implemented by one of ordinary skill in the art using an appropriate alignment method or algorithm. In some cases, the alignment may include gaps introduced to provide the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (generally, see Ausubel et al., Current Protocols in Molecular Biology).

[0069] When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, coordinates are then specified as necessary, and the sequence algorithm program parameters are specified. Next, the sequence comparison algorithm calculates the percent sequence identity of the test sequence to the reference sequence based on the specified program parameters. A commonly used tool for determining percent sequence identity is the Protein Basic Local Alignment Search Tool (BLASTP) available from the National Center for Biotechnology Information of the National Library of Medicine of the National Institutes of Health, USA. (Altschul et al., 1990).

[0070] In some embodiments, the amino acid sequence of a variant of the target polypeptide disclosed herein comprises at least one amino acid substitution relative to the amino acid sequence of the target protein. In some embodiments, the number of amino acid substitutions in the variant relative to the amino acid sequence of the target protein disclosed herein is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, the number of amino acid substitutions is at least about 5, 6, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60. In some embodiments, the number of amino acid substitutions is at most about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 6, or 5. In some embodiments, the number of amino acid substitutions is about 1 - 60, 1 - 55, 2 - 55, 2 - 50, 3 - 50, 3 - 45, 4 - 45, 4 - 40, 5 - 40, 5 - 35, 6 - 35, 6 - 30, 7 - 30, 7 - 25, 8 - 25, 8 - 20, 9 - 20, 9 - 15, 10 - 15, 5 - 60, 10 - 60, 10 - 55, 15 - 55, 15 - 50, 20 - 50, 20 - 45, 25 - 45, 25 - 40, or 30 - 40. In some embodiments, the number of amino acid substitutions is about 10 - 35, 10 - 33, 11 - 33, 11 - 31, 12 - 31, 12 - 29, 13 - 29, 13 - 27, 14 - 27, or 14 - 25.

[0071] The amino acid substitutions in the variant can be substitutions with standard or non - standard amino acids. Non - standard amino acids include, but are not limited to, D - amino acids such as the D - version of standard L - amino acids.

[0072] In some embodiments, the amino acid substitutions are conservative substitutions. The term "conservative amino acid substitution" or "conservative substitution" refers to an amino acid substitution having a value of 0 or more in BLOSUM62.

[0073] In some embodiments, the amino acid substitutions are highly conservative substitutions. The term "highly conservative amino acid substitution" or "highly conservative substitution" refers to an amino acid substitution having a value of at least 1 (e.g., at least 2) in BLOSUM62.

[0074] In some embodiments, variants of the target protein of the present disclosure contain from about 5 to 60 amino acid substitutions relative to the amino acid sequence of the target protein disclosed herein. In some embodiments, the amino acid substitutions include at least one conservative substitution. In some embodiments, the amino acid substitutions include at least one highly conservative substitution.

[0075] A. Polypeptide agent The terms “polypeptide,” “peptide,” or “protein” mean a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A protein, peptide, or polypeptide can include any suitable L- and / or D-amino acids, such as common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine), 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). Amino, carboxyl, and / or other functional groups on a peptide may be free (e.g., unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups, are known in the art and are disclosed, for example, in Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. The functional groups of a protein, peptide, or polypeptide can also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label such as a fluorophore or hapten) using methods known in the art. A protein, peptide, or polypeptide can optionally include one or more modifications (e.g., amino acid linker, acylation, acetylation, amidation, methylation, terminal modification factor (e.g., cyclization modification), N-methyl-α-amino group substitution). Further, a protein, peptide, or polypeptide can be an analog of a known and / or natural peptide, such as a peptide analog having conservative amino acid residue substitutions.

[0076] In some embodiments, the agent comprises a polypeptide. In some embodiments, the polypeptide is an isolated polypeptide (e.g., isolated or extracted from a biological sample or source). In some embodiments, the polypeptide is a recombinant polypeptide. In some embodiments, the polypeptide is an inhibitor (e.g., a direct inhibitor or an indirect inhibitor) of the expression and / or activity of a target protein disclosed herein. In some embodiments, the polypeptide is an activator (e.g., a direct activator or an indirect activator) of the expression and / or activity of a target protein disclosed herein. In some embodiments, the polypeptide decreases the expression or activity of a target protein disclosed herein. In other embodiments, the polypeptide increases the expression or activity of a target protein disclosed herein. In some embodiments, the polypeptide is a target protein disclosed herein or a part thereof (e.g., its biologically active part, e.g., a biologically active fragment of the target protein).

[0077] In some embodiments, the polypeptide is an immunoglobulin molecule, such as an antibody (e.g., a whole antibody, an intact antibody) or an antigen-binding fragment of an antibody. In some embodiments, the antibody or its antigen-binding fragment binds to a target protein. In some embodiments, the antibody or its antigen-binding fragment binds to a protein that can regulate the expression or activity of a target protein.

[0078] In some embodiments, the polypeptide is an antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" refers to a full-length antibody comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or multimers thereof (e.g., IgM). Each heavy chain comprises a heavy-chain variable region (V Hand a heavy chain constant region (including domains CH1, hinge, CH2 and CH3). Each light chain comprises a variable light chain region (V L ) and a light chain constant region (CL). V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that are interspersed within framework regions (FRs). V H and V L each contain three CDRs and four FR segments and are arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibody can be of any species, such as rodent (e.g., mouse, rat, guinea pig) antibodies, human antibodies, or the antibody can be a humanized or chimeric antibody.

[0079] In some embodiments, the antibody comprises an IgA (e.g., IgA1 or IgA2) heavy chain constant region, an IgD heavy chain constant region, an IgE heavy chain constant region, an IgG (e.g., IgG1, IgG2 (e.g., IgG2a, IgG2b or IgG2c), IgG3 or IgG4) heavy chain constant region or an IgM heavy chain constant region. In some embodiments, the antibody comprises an IgG heavy chain constant region. In some embodiments, the antibody comprises a κ light chain constant region. In some embodiments, the antibody comprises a λ light chain constant region.

[0080] In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is human or chimeric. In some embodiments, the antibody is primatized (e.g., humanized). In some embodiments, the antibody is multispecific, e.g., bispecific, trispecific or tetravalent. In some embodiments, the antibody is a heteroconjugate antibody.

[0081] In some embodiments, the polypeptide agent is an antigen-binding fragment of an immunoglobulin molecule (e.g., an antibody). The term "antigen-binding fragment" refers to a portion of an immunoglobulin molecule (e.g., an antibody) that retains the antigen-binding properties of the parent full-length antibody. Non-limiting examples of antigen-binding fragments include the V H region, the V L region, Fab fragment, F(ab’) 2 fragment, Fd fragment, Fv fragment, and domain antibodies (dAbs) consisting of one V H domain or one V L domain. VH and VL domains can be linked to each other via a synthetic linker to form various types of single-chain antibody designs where the V H / V L domains pair within the molecule or, when the V H and V L domains are expressed by separate chains, pair intermolecularly, forming a monovalent antigen-binding site such as a single-chain Fv (scFv) or a diabody. In some embodiments, the polypeptides disclosed herein are antigen-binding fragments selected from Fab, Fab’, F(ab’) 2 , Fd, Fv, disulfide-bonded Fv (sdFv, e.g., diabody, triabody or tetrabody), scFv, SMIP or rlgG. In some embodiments, the polypeptide is an scFv. Antigen-binding fragments can be generated by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.

[0082] Polypeptide agents (e.g., monoclonal antibodies) can be monovalent, bivalent or multivalent. Monoclonal antibodies can be monospecific or multispecific (e.g., bispecific). Monospecific antibodies bind to one antigen epitope. Multispecific antibodies such as bispecific or trispecific antibodies are included within the term monoclonal antibody.

[0083] "Multispecificity" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes within an antigen, such as three, four, or five different antigens or epitopes. "Bispecificity" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen.

[0084] "Isolated antibody" refers to an antibody or antigen-binding fragment thereof that is substantially free of other antibodies having different antigen specificities (e.g., an isolated anti-target protein antibody is substantially free of antibodies that specifically bind to antigens other than the target protein). In the case of a bispecific antibody, the bispecific antibody specifically binds to the two antigens of interest and is substantially free of antibodies that specifically bind to antigens other than the two antigens of interest. In some embodiments, the polypeptide agent (e.g., a monoclonal antibody) is at least 80% pure, e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0085] In some embodiments, the polypeptide is an antagonist antibody that binds to a target protein (e.g., a target protein whose expression or activity is elevated in a cancer state compared to a reference state). In some embodiments, the antibodies described herein are antagonist antibodies that bind to a protein that can regulate the expression or activity of a target protein. As used herein, the term "antagonist antibody" refers to an antibody that, when bound to an antigen (e.g., a target protein or a protein that can regulate the expression or activity of a target protein), reduces (e.g., inhibits) the function of the antigen. In some embodiments, the antigen is a receptor and the antagonist antibody binds to the ligand-binding domain of the receptor. In some embodiments, the antigen is a transmembrane protein and the antagonist antibody binds to the extracellular region of the transmembrane protein. In some embodiments, the antigen is an enzyme or a signaling molecule and the antagonist antibody reduces the activity of the enzyme or attenuates the signaling pathway mediated by the signaling molecule. In some embodiments, the antagonist antibody reduces the antigen function by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 99%.

[0086] In some embodiments, the polypeptide is an agonist antibody that binds to a target protein (e.g., a target protein whose expression or activity is reduced in a cancerous state compared to a reference state). In some embodiments, the antibody is an agonist antibody that binds to a protein that can regulate the expression or activity of the target protein. As used herein, the term "agonist antibody" refers to an antibody that, when binding to an antigen (e.g., a target protein or a protein that can regulate the expression or activity of the target protein), increases the function of the antigen. In some embodiments, the antigen is a receptor and the agonist antibody binds to the ligand-binding domain of the receptor. In some embodiments, the antigen is a transmembrane protein and the agonist antibody binds to the extracellular region of the transmembrane protein. In some embodiments, the antigen is an enzyme or a signaling molecule and the agonist antibody increases the activity of the enzyme or activates a signaling pathway mediated by the signaling molecule. In some embodiments, the agonist antibody increases the antigen function by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1,000%.

[0087] In some embodiments, the agonist antibody does not exhibit at least one of the following functional properties: reducing (e.g., inhibiting) the activity of the antigen; inducing antibody-dependent cell death of cells expressing the antigen (e.g., by natural killer (NK) cells, monocytes, macrophages, neutrophils, dendritic cells, or eosinophils); inducing phagocytosis of cells expressing the antigen (e.g., by macrophages); inducing opsonization of cells expressing the antigen; and inducing downregulation of the antigen on the cell surface (e.g., by cross-linking or clustering the antigen to induce internalization and degradation).

[0088] Appropriate techniques, assays, and reagents for making and using therapeutic antibodies against antigens are known in the art. See, for example, methods for making recombinant antibodies, including antibody engineering, use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis; antibody testing and characterization; antibody pharmacokinetics and pharmacodynamics; antibody purification and storage; and screening and labeling techniques in Therapeutic Monoclonal Antibodies: From Bench to Clinic (Zhiqiang An eds., 1st ed. 2009); Antibodies: A Laboratory Manual (Edward A. Greenfield eds., 2d ed. 2013); Ferrara et al., Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker, PLoS ONE 7(11):e49535 (2012).

[0089] In some embodiments, the polypeptide is an antibody mimetic that binds to a target protein disclosed herein. The term "antibody mimetic" refers to a polypeptide that can mimic the ability of an antibody to bind an antigen but has a structure that is structurally different from the native antibody structure. Non-limiting examples of antibody mimetics include adnectin, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, nanobody, nanoCLAMP, and Versabody.

[0090] In some embodiments (e.g., when the expression or activity of the target protein is reduced in the disease state as compared to the reference state), the agent is a polypeptide (e.g., an isolated polypeptide) comprising an amino acid sequence that is at least 70% identical to at least a portion of the target protein (e.g., a biologically active portion or fragment). For example, the percent identity can be at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the full-length target protein or a biologically active portion or fragment thereof. In some embodiments, the polypeptide comprises the amino acid sequence of the full-length target protein. In some embodiments, the polypeptide comprising the amino acid sequence of the full-length target protein is a recombinant polypeptide. In some embodiments, the polypeptide comprising the amino acid sequence of the full-length target protein is a synthetic polypeptide.

[0091] In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence having at least one amino acid substitution relative to the target protein. For example, the number of amino acid substitutions can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the amino acid substitution is a highly conservative substitution.

[0092] In some embodiments, a polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence that is at least 70% identical to at least a portion of a protein that can regulate the expression or activity of a target protein. For example, the percent identity can be at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the polypeptide comprises the amino acid sequence of a protein that can regulate the expression or activity of a target protein.

[0093] In some embodiments, a polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence having at least one amino acid substitution relative to a protein that can regulate the expression or activity of a target protein. For example, the number of amino acid substitutions can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about 1 - 20, 1 - 19, 2 - 19, 2 - 18, 2 - 17, 3 - 17, 3 - 16, 4 - 16, 4 - 15, 5 - 15, 5 - 14, 6 - 14, 6 - 13, 7 - 13, 7 - 12, 8 - 12, 8 - 11 or 9 - 11. In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the amino acid substitutions are highly conservative substitutions.

[0094] In some embodiments, the polypeptide is a cell-penetrating peptide. In certain embodiments, the polypeptide is linked to a cell-penetrating peptide. Suitable cell-penetrating peptide sequences can be protein-derived, designed, or chimeric (modified). See, for example, Regberg, et al., Applications of cell-penetrating peptides for tumor targeting and future cancer therapies, Pharmaceuticals 5(9):991-1007 (2012). Non-limiting examples of cell-penetrating peptides include TAT(48-60), Penetratin, pVEC, MPG8, Transportan, Transportan10, PepFect3, PepFect 6, PepFect 14, polyarginine, stearyl-polyarginine, Pep-1, Pep-3, CADY, YTA2, YTA4, SynB1, SynB3, Maurocalcine, and PTD4.

[0095] In some embodiments, the polypeptide is a circulating factor (e.g., a cytokine).

[0096] In some embodiments, the polypeptide (e.g., an isolated polypeptide) and the biological properties of the target protein (e.g., biological activity or half-life) are similar. Non-limiting examples of biological activity include, inter alia, enzyme activity or properties (e.g., selectivity, steady state or kinetics), binding activity (e.g., nucleic acid (DNA, RNA) binding protein binding) or properties (e.g., specificity, affinity or kinetics), cell signaling activity, immunological activity, and structural activity (e.g., cell adhesion). Non-limiting examples of enzyme activity include transferase activity (e.g., transferring a functional group from one molecule to another), oxidoreductase activity (e.g., catalyzing an oxidation-reduction reaction), hydrolase activity (e.g., cleaving a chemical bond by hydrolysis), lyase activity (e.g., generating a double bond), ligase activity (e.g., joining two molecules via a covalent bond) and isomerase activity (e.g., catalyzing an intramolecular structural change from one isomer to another).

[0097] In some embodiments, the polypeptide (e.g., an isolated polypeptide) is a recombinant protein. In other embodiments, the polypeptide (e.g., an isolated polypeptide) is a synthetic protein. Methods for producing therapeutic polypeptides are known in the art. See, for example, Therapeutic Proteins: Methods and Protocols (Mark C. Smales & David C James eds., 2005); Pharmaceutical Biotechnology: Fundamentals and Applications (Daan J.A. Crommelin, Robert D. Sindelar & Bernd Meibohm eds., 2013). The polypeptide can be recombinantly expressed using, for example, mammalian cells, insect cells, yeast or bacteria under the control of an appropriate promoter.

[0098] In some embodiments, the polypeptides described herein (e.g., a target protein or a portion thereof, a polypeptide agent that modulates a target protein) are modified, for example, by cleavage (e.g., protease cleavage) or post-translational modification. In certain embodiments, the modification affects the activity of the polypeptide, for example, by activating an inactive polypeptide or by changing (e.g., increasing, decreasing) the activity level of the polypeptide. In certain embodiments, the polypeptides described herein are provided as prodrugs that can be converted, for example, in vivo (e.g., by proteolytic cleavage, post-translational modification) into active polypeptides. In some embodiments, the polypeptide includes post-translational modification or other chemical modification. Non-limiting examples of post-translational modifications include acetylation, amidation, formylation, glycosylation, hydroxylation, methylation, myristoylation, phosphorylation, deamidation, prenylation (e.g., farnesylation, geranylation, etc.), ubiquitination, ribosylation, and sulfation. Phosphorylation can occur on amino acids such as tyrosine, serine, threonine, or histidine.

[0099] In some embodiments, the polypeptide is coupled (e.g., via a covalent bond such as a peptide bond, or a non-covalent bond) to a heterologous peptide or protein such as a conjugate or a fusion protein. In some embodiments, the polypeptide includes a tag (e.g., a detectable label such as a fluorophore or an enzyme, or a purification tag such as an epitope tag).

[0100] In some embodiments, the polypeptide comprises one or more neoantigens selected from the sequence listing, Table A, or variants of the foregoing. As used herein, the term "neoantigen" refers to a tumor antigen arising from a target protein described herein. In some aspects, the neoantigen is a cancer-specific neoantigen. There are various ways to produce neoantigens. For example, a neoantigen can be produced in vitro as a polypeptide before being formulated into a neoplasm vaccine or immunogenic pharmaceutical composition. In some embodiments, the immunogenic pharmaceutical composition comprises an effective amount of one or more neoantigens or a pharmaceutically acceptable salt thereof. In some embodiments, the immunogenic pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, adjuvant, or additive.

[0101] Alternatively, a neoantigen can be produced in vivo by introducing a polynucleotide or expression vector (e.g., a viral expression vector) encoding the neoantigen into cells or tissues (e.g., of a subject in need thereof). In certain embodiments, the polypeptide comprises at least two neoantigens. In some embodiments, the polypeptide comprises a T cell enhancer amino acid sequence. In some embodiments, the T cell enhancer is selected from the group consisting of an invariant chain, a leader sequence of tissue-type plasminogen activator, a PEST sequence, a cyclin destruction box, a ubiquitination signal, and a SUMOylation signal.

[0102] B. Polynucleotide Agent In some embodiments, the agent comprises a polynucleotide or an analog or derivative thereof. In some embodiments, the polynucleotide or an analog or derivative thereof is an inhibitor of a target protein. In some embodiments, the polynucleotide or an analog or derivative thereof is an activator of a target protein. In some embodiments, the polynucleotide or an analog or derivative thereof decreases (e.g., reduces or neutralizes) the expression or activity of a target protein. In other embodiments, the polynucleotide or an analog or derivative thereof increases the expression or activity of a target protein.

[0103] A polynucleotide can have a sequence containing naturally occurring ribonucleotide or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or combinations thereof. Thus, a polynucleotide can include, for example, nucleotides containing naturally occurring bases (e.g., A, G, C, or T) and nucleotides containing modified bases (e.g., 7-deazaguanosine, inosine, or methylated nucleotides such as 5-methyl dCTP and 5-hydroxymethylcytosine). In some embodiments, the polynucleotide includes at least one modified nucleotide. Non-limiting examples of modified nucleotides include 2'-fluoro, 2'-O-methyl, 2'-deoxy, locked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'-thiouridine, and 2'-deoxyuridine. In some embodiments, the modification is to increase nuclease resistance, increase serum stability, decrease immunogenicity, or a combination thereof.

[0104] In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the polynucleotide is an RNA molecule. In some embodiments, the polynucleotide is a vector (e.g., an expression vector, a plasmid).

[0105] In some embodiments, the polynucleotide includes a polynucleotide analog or derivative. In some embodiments, the analog or derivative is a peptide nucleic acid (PNA). In some embodiments, the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative includes one or more phosphorothioate linkages. In some embodiments, the agent includes deoxyribonucleoguanidine (DNG) nucleotides. In some embodiments, the agent includes ribonucleoguanidine (RNG) nucleotides.

[0106] In some embodiments, the polynucleotide regulates the expression and / or activity of a nucleic acid encoding a target protein disclosed herein (e.g., a target protein in the Sequence Listing or Table A) or a part thereof (e.g., a biologically active part or a fragment thereof).

[0107] In some embodiments, the polynucleotide comprises a nucleotide sequence that is complementary (e.g., fully complementary or partially complementary) to at least a part of a gene or gene transcript encoding a target protein disclosed herein, such that the polynucleotide sequence can hybridize or anneal to the gene or gene transcript (e.g., under physiological conditions). In other embodiments, the polynucleotide comprises a nucleotide sequence that is complementary to at least a part of a gene or gene transcript encoding a protein that can regulate the expression or activity of a target protein disclosed herein.

[0108] In some embodiments, the polynucleotide encodes a target protein disclosed herein, or a variant thereof (e.g., a biologically active variant thereof), or a part thereof (e.g., a biologically active part or a fragment thereof).

[0109] In some embodiments, the nucleic acid encoding the target protein, or a variant thereof, or a part thereof (e.g., a fragment) is a gene sequence or a part thereof. In some embodiments, the encoding nucleic acid is an unprocessed RNA transcript (e.g., pre-mRNA) or a part thereof (e.g., 5'-UTR, 3'-UTR, intron). In some embodiments, the encoding nucleic acid is an mRNA molecule or a part thereof. In some embodiments, the encoding nucleic acid is present in a non-coding RNA (e.g., long intergenic non-coding RNA (lincRNA), long non-coding RNA (lncRNA), or miRNA).

[0110] The encoding nucleic acid may contain a standard open reading frame (ORF) or a non-standard ORF. In certain embodiments, the encoding nucleic acid contains a non-standard ORF.

[0111] The polynucleotide can be single-stranded (ss) or double-stranded (ds). In some embodiments, the polynucleotide is double-stranded (ds). In some embodiments, the length of the ds polynucleotide is about 15 to 50 base pairs, such as about 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 18 to 50, 18 to 45, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 50, 35 to 45, 35 to 40 or 40 to 50 base pairs. In some embodiments, the length of the polynucleotide is about 19 to 23 base pairs. In some embodiments, the length of the polynucleotide is about 21 base pairs.

[0112] In some embodiments, the polynucleotide is single-stranded (ss). In some embodiments, the length of the ss polynucleotide is about 15 to 50 nucleotides, such as about 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 18 to 50, 18 to 45, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 50, 35 to 45, 35 to 40 or 40 to 50 nucleotides.

[0113] In some embodiments, the polynucleotide inhibits the maturation of a newly generated nuclear RNA transcript into mRNA for transcription. In some embodiments, the polynucleotide comprises a nucleotide sequence complementary to a sequence at the intron-exon boundary.

[0114] In some embodiments, the polynucleotide (e.g., an antisense oligonucleotide) can hybridize to the mRNA encoding the target protein (e.g., under physiological conditions). In some embodiments, the length of the polynucleotide is at least about 10 nucleotides, such as at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or about 10 - 30, 15 - 30, 15 - 25, 20 - 25 nucleotides. In some embodiments, the polynucleotide is at least 75% identical to the antisense sequence of the same target transcript, such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical.

[0115] In some embodiments, the polynucleotide further comprises an overhang sequence (e.g., unpaired overhang nucleotides that are not directly involved in the formation of the double helix structure by the core sequence). In some embodiments, the polynucleotide comprises a 3' overhang, a 5' overhang, or both. In some embodiments, the overhang is about 1 - 5 nucleotides. In some embodiments, the overhang comprises modified ribonucleotides or deoxynucleotides, such as phosphorothioates, phosphorothioate or deoxynucleotide inversion (linked 3' to 3') nucleotides.

[0116] Non-limiting examples of polynucleotide agents suitable for use in the compositions, kits and methods described herein include small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antagomir, antisense DNA, antisense RNA, morpholino nucleic acid (MNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), aptamer and guide RNA (gRNA).

[0117] In some embodiments, the polynucleotide inhibits gene expression (e.g., via the biological process of RNA interference (RNAi)). Polynucleotides suitable for RNA interference can be readily designed and made by those skilled in the art using techniques, assays, and reagents known in the art, including computational tools. See, for example, Pei et al. 2006, Reynolds et al. 2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005, Chalk et al. 2004, Amarzguioui et al. 2004.

[0118] In some embodiments, the polynucleotide is a miRNA. In some embodiments, the miRNA is about 22 nucleotides in length. The miRNA binds to a target site on the mRNA molecule and silences the mRNA, for example, by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA.

[0119] In some embodiments, the polynucleotide is a siRNA. In some embodiments, the siRNA comprises a nucleotide sequence identical to about 15 - 25 contiguous mRNA sequences encoding the target protein. In some embodiments, the siRNA is a double-stranded RNA molecule having about 19 - 25 base pairs. In some embodiments, the siRNA begins with the dinucleotide AA. In some embodiments, the siRNA has a GC content of about 30 - 70%, such as about 30 - 65%, 30 - 60%, 30 - 55%, 30 - 50%, 40 - 70%, 40 - 65%, 40 - 60%, 40 - 55%, 45 - 70%, 45 - 65%, 45 - 60%, or 45% - 55%.

[0120] In some embodiments, the polynucleotide is shRNA. shRNA is an RNA molecule containing a hairpin turn that reduces the expression of a target gene via RNAi. shRNA can be delivered to cells in the form of a plasmid, such as a viral vector or a bacterial vector, for example, by transfection, electroporation or transduction.

[0121] siRNAs and shRNAs are similar to intermediates in the processing pathway of endogenous microRNA (miRNA) genes (see, e.g., Bartel, Cell 116:281-97 (2004)). In some embodiments, siRNAs function as miRNAs; in other embodiments, miRNAs function as siRNAs (see, e.g., Zeng et al., Mol Cell 9:1327-33 (2002); Doench et al., and Genes Dev 17:438-42 (2003)). MicroRNAs such as siRNAs use RISC to downregulate target genes, but unlike siRNAs, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein output through translational repression or poly(A) removal and mRNA decay (see, e.g., Wu et al., Proc Natl Acad Sci USA 103:4034-39 (2006)). Known miRNA binding sites are within the mRNA 3’UTR; miRNAs are thought to target sites with near perfect complementarity to nucleotides 2-8 from the 5’ end of the miRNA (see, e.g., Rajewsky, Nat Genet 38 Suppl:S8-13 (2006) and Lim et al., Nature 433:769-73 (2005)). This region is known as the seed region. Since siRNAs and miRNAs are interchangeable, exogenous siRNAs downregulate mRNAs that have seed complementarity to the siRNA (see, e.g., Birmingham et al., Nat Methods 3:199-204 (2006)). Multiple target sites within the 3’UTR result in stronger downregulation (see, e.g., Doench et al., Genes Dev 17:438-42 (2003)).

[0122] In some embodiments, the polynucleotide is a messenger RNA (mRNA) or circular RNA (circRNA) encoding a target protein or a variant thereof (e.g., a variant that is at least about 70% identical to the wild-type protein, e.g., at least about 75%, 80%, 85%, 90%, 95%, 98% or 99% identical) disclosed herein. In some embodiments, the mRNA is codon-optimized (e.g., to improve the efficiency of protein synthesis and limit mRNA destabilization by rare codons; see, e.g., Presnyak et al., Cell. 160(6):1111-24(2015) and Thess et al., Mol Ther. 23(9):1456-64(2015)).

[0123] In some embodiments, the polynucleotide comprising RNA is chemically synthesized. In some embodiments, the polynucleotide comprising RNA is recombinantly expressed. In some embodiments, the RNA is transcribed in vitro. The production and use of RNA therapeutics are known in the art. See, e.g., RNA Therapeutics: Function, Design, and Delivery (Mouldy Sioud eds., 2010) and Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Medicine 9:60(2017).

[0124] In some embodiments, the mRNA is produced by in vitro transcription. In some embodiments, the mRNA is modified to optimize its activity. In some embodiments, the mRNA comprises modified bases, a 5' cap, a 5' cap analog, an anti-reverse cap analog (ARCA), or a combination thereof.

[0125] In some embodiments, the mRNA comprises a poly(A) tail. In some embodiments, the poly(A) tail is about 100 to 200 nucleotides. In some embodiments, the poly(A) tail improves the expression and / or stability of the mRNA (see, e.g., Kaczmarek et al., Genome Medicine 9:60 (2017)).

[0126] In some embodiments, the mRNA comprises a 5' cap. In some embodiments, the mRNA comprises a 5' cap analog. In some embodiments, the 5' cap analog is a 1,2-dithiophosphate-modified cap (see, e.g., Strenkowska et al., Nucleic Acids Res. 44:9578-90 (2016)).

[0127] In some embodiments, the mRNA comprises a modified 3' untranslated region (UTR), 5' UTR, or both. In some embodiments, the modified UTR comprises sequences involved in the recruitment of RNA-binding proteins (RBPs) and miRNAs (e.g., to enhance the level of protein production) (see, e.g., Kaczmarek et al., Genome Medicine 9:60 (2017)). In some embodiments, the 3' UTR, 5' UTR, or both are modified to encode regulatory elements. In some embodiments, the regulatory elements comprise a K-turn motif, miRNA binding site, or a combination thereof for controlling RNA expression in a cell-specific manner (see, e.g., Wroblewska et al., Nat Biotechnol. 33:839-41 (2015)).

[0128] In some embodiments, the mRNA comprises an RNA base modification. In some embodiments, the mRNA comprises pseudouridine. In some embodiments, the mRNA comprises N1-methyl-pseudouridine (e.g., to mask immune-stimulatory activity and enhance translation initiation) (see, e.g., Andries et al., J Control Release 217:337-44 (2015) and Svitkin et al., Nucleic Acids Res. 45:6023-36 (2017)).

[0129] In some embodiments, the RNA (e.g., mRNA) is circular RNA.

[0130] Compositions and methods for producing mRNA are described, for example, in WO 2016 / 011306, WO 2016 / 014846, WO 2016 / 022914, WO 2016 / 077123, WO 2016 / 164762, WO 2016 / 201377, WO 2017 / 049275, US Patent No. 9,937,233, US Patent No. 8,710,200, US Patent No. 10,022,425, US Patent No. 9,878,056, US Patent No. 9,572,897, WO 2010 / 084371, US Patent No. 9,353,153, WO 2015 / 034925, and WO 2019 / 236673. See, for example, Jemielity et al., RNA 9(9):1108-22(2003); Mockey et al., Biochem Biophys Res Commun. 340:1062-88(2006); Strenkowska et al., Nucleic Acids Res. 44:9578-90(2016); Presnyak et al., Cell 160:1111-24(2015) and Kaczmarek et al., Genome Medicine 9:60(2017)). In some embodiments, the mRNA is prepared in a lipid nanoparticle (LNP) formulation (see, for example, for in vivo delivery, US Patent No. 9,764,036, US Patent No. 9,682,139, Kauffman et al., Nano Lett. 15:7300-6(2015) and Fenton et al., Adv Mater. 28:2939-43(2016)).

[0131] In some embodiments, the polynucleotide is an aptamer. In certain embodiments, the aptamer binds to a target protein disclosed herein. In certain embodiments, the aptamer binds to a binding partner of a target protein disclosed herein.

[0132] In some embodiments, the polynucleotide is linked (e.g., by a covalent bond) to the delivery polymer. In some embodiments, the linkage between the polynucleotide and the delivery polymer is reversible. In some embodiments, the polynucleotide is linked to the delivery polymer via a physiologically labile linker. In some embodiments, the physiologically labile linker is a disulfide bond.

[0133] In some embodiments, the polynucleotide is conjugated to the polymer in the presence of excess polymer. In some embodiments, the excess polymer is removed prior to administration (e.g., to a cell or subject).

[0134] One of ordinary skill in the art can readily make appropriate polynucleotide agents for use in the compositions, kits, and methods described herein using the locus information of the protein sequences, such as chromosomal location, start nucleotide position, and end nucleotide position, and polymorphism identification, contained in the sequence listing and Table A incorporated herein.

[0135] C. Agent Comprising a Gene Editing System In some embodiments, the agent comprises a gene editing system. In some embodiments, the gene editing system results in a nucleotide deletion, nucleotide substitution, nucleotide addition, or a combination thereof in the gene encoding the target protein.

[0136] In some embodiments, the gene editing system is a CRISPR / Cas system, a transposon-based gene editing system, or a transcription activator-like effector nuclease (TALEN) system. In some embodiments, the gene editing system is a CRISPR / Cas system. In some embodiments, the gene editing system is a class II CRISPR / Cas system.

[0137] In some embodiments, the gene editing system (e.g., the CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates the expression of the target protein (e.g., via gene knockout). In some embodiments, the gene editing system (e.g., the CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates the expression of a protein capable of regulating the expression or activity of the target protein (e.g., via gene knockout). In some embodiments, the gene editing system (e.g., the CRISPR / Cas system) increases the expression of the target protein (e.g., via gene knock-in or gene replacement). In some embodiments, the gene editing system (e.g., the CRISPR / Cas system) increases the expression of a protein capable of regulating the expression or activity of the target protein (e.g., via gene knock-in or gene replacement).

[0138] In some embodiments, the CRISPR system specifically catalyzes the cleavage of the gene encoding the target protein, thereby inactivating the gene. Repair of nucleic acid strand breaks via non-homologous end joining (NHEJ) often results in changes in the DNA sequence at the cleavage site, resulting in small insertions or deletions (Indels). In some embodiments, NHEJ is used to knockout the gene encoding the target protein. In some embodiments, homologous recombination repair (HDR) is used to simultaneously inactivate the gene encoding the target protein and insert a heterologous sequence at the inactivated locus. Cells in which knockout and / or knock-in events have occurred can be identified and / or selected by methods well known in the art.

[0139] In some embodiments, the gene editing system comprises a single Cas endonuclease or a polynucleotide encoding a single Cas endonuclease. In some embodiments, the single Cas endonuclease is Cas9, Cpf1, C2C1 or C2C3. In some embodiments, the single Cas endonuclease is Cas9 (e.g., of Streptococcus Pyogenes). In some embodiments, the single Cas endonuclease is Cpf1. In some embodiments, Cpf1 is AsCpf1 (derived from the Acidaminococcus species) or LbCpf1 (derived from the Lachnospiraceae species). The selection of the nuclease and gRNA is typically determined according to whether nucleotide deletions, substitutions or additions to the target sequence are desired.

[0140] In some embodiments, the type II Cas endonuclease is Cas9 (e.g., of Streptococcus pyogenes). In some embodiments, the modified Cas9 is nickase Cas9, dead Cas9 (dCas9) or eSpCas9. In some embodiments, the nickase Cas9 is Cas9 D10A. In some embodiments, dCas9 is D10A or H840A. In some embodiments, the gene editing system comprises dual nickase Cas9 (see, e.g., Ran et al., Cell 154:1380-89 (2013) for achieving more accurate genome editing). Wild-type Cas9 generates a double-strand break (DSB) at a specific DNA sequence targeted by the gRNA. Nickase Cas9 generates only single-strand breaks. dCas9 is catalytically inactive. In some embodiments, dCas9 is fused to a nuclease (e.g., FokI for generating DSBs at target sequences homologous to two gRNAs). Various CRISPR / Cas9 plasmids are publicly available from the Addgene repository (Addgene, Cambridge, MA: addgene.org / crispr / ).

[0141] In some embodiments, the gene editing system comprises: a) a wild-type or modified type II Cas endonuclease, or a polynucleotide encoding a wild-type or modified type II Cas endonuclease; b) a CRISPR RNA (“crRNA”); and c) a trans-activating crRNA (“tracrRNA”). Including.

[0142] In some embodiments, the crRNA comprises at least one “guide RNA” (sgRNA), such as at least 2, 3, or 4 gRNAs. In some embodiments, the gRNA comprises a sequence identical to a portion of the gene sequence of the target protein. In some embodiments, the gRNA comprises a sequence identical to a portion of the gene sequence of a protein capable of regulating the expression or activity of the target protein. In some embodiments, the gRNA is at least about 16 nucleotides, such as at least about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; or about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; or about 16-24, 17-24, 17-23, 18-23, 18-22, 19-22, or 19-21, or 19, 20, or 21 nucleotides. In some embodiments, the sgRNA is chemically modified.

[0143] The design of gRNA sequences for gene editing is known in the art. See, for example, Cong et al., Science, 339:819-23 (2013) and Ran et al., Nature Protocols 8:2281-308 (2013). Cas9 requires a gRNA sequence of at least about 16 or 17 nucleotides to cleave DNA, and Cpf1 requires a gRNA sequence of at least about 16 nucleotides to cleave DNA. In practice, the gRNA sequence has a length of about 17-24 nucleotides (e.g., about 19, 20, or 21 nucleotides) and is complementary to the target gene. Custom gRNA generators and algorithms are commercially available. Chemically modified sgRNAs have also been demonstrated to be effective for genome editing (see, for example, Hendel et al., Nature Biotechnol., 985-91 (2015)).

[0144] In some embodiments, the crRNA further comprises a sequence capable of binding to the tracrRNA. When bound, the partially double-stranded structure is cleaved by RNase III, and the resulting crRNA / tracrRNA hybrid instructs the Cas9 endonuclease to recognize and cleave the target DNA sequence.

[0145] In some embodiments, the target DNA sequence is proximal to a "protospacer adjacent motif" ("PAM") specific to the Cas endonuclease. The PAM sequence appears throughout the given genome. CRISPR endonucleases of various prokaryotic species have specific PAM sequence requirements. Non-limiting examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endonucleases, such as the Cas9 endonuclease, associate with a G-rich PAM site, such as 5'-NGG, and perform blunt-end cleavage of the target DNA at a position 3 nucleotides upstream (5') of the PAM site.

[0146] In some embodiments, the gene editing system is: a) a wild-type or modified type II Cas endonuclease, or a polynucleotide encoding a wild-type or modified type II Cas endonuclease; and b) a crRNA comprising.

[0147] Cpf1-related CRISPR arrays are processed into mature crRNAs without the need for tracrRNA. The Cpf1 endonuclease associates with T-rich PAM sites, such as 5'-TTN. Cpf1 can also recognize the 5'-CTA PAM motif. Cpf1 introduces offset or staggered double-strand breaks with 5'-overhangs of 4 or 5 nucleotides, for example, by cleaving target DNA having an offset or staggered cut of 5 nucleotides located 18 nucleotides downstream (3') of the PAM site on the coding strand and 23 nucleotides downstream of the PAM site on the complementary strand. The 5-nucleotide overhangs resulting from such offset cuts allow for more accurate genome editing by DNA insertion via homologous recombination rather than by insertion into blunt-ended cut DNA. See, for example, Zetsche et al., Cell 163:759-71 (2015).

[0148] In some embodiments, the gene editing system activates or suppresses transcription of the target gene. In some embodiments, the gene editing system comprises: a) a chimeric protein comprising dCas9 and one or more effector domains; and b) one or more sgRNAs and.

[0149] In some embodiments, the chimeric protein suppresses expression of the target protein (CRISPRi). In some embodiments, the chimeric protein activates expression of the target protein (CRISPRa). In some embodiments, the chimeric protein methylates the DNA sequence recognized by the sgRNA. In some embodiments, the chimeric protein demethylates the DNA sequence recognized by the sgRNA.

[0150] The effector domain comprises the biologically active portion of an effector protein (e.g., a transcriptional activator or a transcriptional repressor). In some embodiments, the gene editing system comprises one effector domain. In some embodiments, the gene editing system comprises at least two effector domains, e.g., 2, 3, or 4 effector domains. In some embodiments, the effector domain comprises KRAB. In some embodiments, the effector domain comprises VP64. In some embodiments, the effector domain comprises VP64, p65, and Rta. In some embodiments, dCas9 is D10A. In some aspects, dCas9 is H840A.

[0151] Since dCas9 is catalytically inactive, dCas9 does not cleave target DNA and interferes with transcription by steric hindrance. dCas9 chimeric proteins (e.g., dCas9-VPR) are guided by one or more gRNAs to sequences upstream of the transcription start site (TSS) of the target gene and regulate the transcription of the target gene. For example, Gilbert et al., CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes, Cell 154, 442-51 (2013); Cheng et al., Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system, Cell Res. 23:1163-71 (2013); Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation, Cell 159:647-61 (2014); Tanenbaum et al., A protein-tagging system for signal amplification in gene expression and fluorescence imaging, Cell 159:635-46 (2014); Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Nature 517:583-88 (2015); Chavez et al., Highly efficient Cas9-mediated transcriptional programming, Nat. Methods. 12:326-28 (2015); Zalatan et al., Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Cell 160:339-50 (2015); Horlbeck et al.See Chavez et al., Comparison of Cas9 activators in multiple species, Nat Methods. 7:563-67 (2016); eLife. 5:e19760 (2016); Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.

[0152] CRISPR technology for editing eukaryotic genes is disclosed in US Patent Application Publication No. 2016 / 0138008A1, US Patent Application Publication No. 2015 / 0344912A1, US Patent No. 8,697,359, US Patent No. 8,771,945, US Patent No. 8,945,839, US Patent No. 8,999,641, US Patent No. 8,993,233, US Patent No. 8,895,308, US Patent No. 8,865,406, US Patent No. 8,889,418, US Patent No. 8,871,445, US Patent No. 8,889,356, US Patent No. 8,932,814, US Patent No. 8,795,965, and US Patent No. 8,906,616. The Cpf1 endonuclease and corresponding guide RNA and PAM site are disclosed in US Patent Application Publication No. 2016 / 0208243A1. CRISPR technology for causing mtDNA dysfunction in the mitochondrial genome is disclosed in Jo et al., BioMed Res. Int. 2015:305716 (2015). Co-delivery of Cas9 and sgRNA with nanoparticles is disclosed in Mout et al., ACS Nano 11(3):2452-58 (2017).

[0153] In some embodiments, the agent comprises a transposon-based gene editing system. An example of a transposon-based gene editing system suitable for use in the disclosure provided herein is the Gene Writer system described in International Publication No. WO 2020 / 047124, published on March 5, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0154] In some embodiments, the agent comprises a transcription activator-like effector nuclease (TALEN) system. TALEN-based systems include proteins comprising a TAL effector DNA-binding domain and an enzyme domain. They are created by fusing the TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). The above-described FokI restriction enzyme is an exemplary enzyme domain suitable for use in TALEN-based gene regulatory systems.

[0155] TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when infecting plants. The DNA-binding domain contains a repeated, highly conserved 33-34 amino acid sequence with different 12th and 13th amino acids. These two positions, called repeat variable diresidues (RVDs), are highly variable and strongly correlate with specific nucleotide recognition. Thus, the TAL effector domain can be engineered to bind to a specific target DNA sequence by selecting a combination of repeat segments containing appropriate RVDs. The nucleic acid specificities for combinations of RVDs are as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (however, in some embodiments, NN can also bind to adenine with lower specificity).

[0156] In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to the target DNA sequence of the target protein. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to the target DNA sequence defined by a set of genomic coordinates.

[0157] In some embodiments, the gene regulatory system comprises two or more TAL effector fusion proteins each comprising a TAL effector domain, and at least one of the TAL effector domains binds to a target DNA sequence of the target protein. In some embodiments, at least one of the two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to the target DNA sequence defined by a set of genomic coordinates.

[0158] Methods and compositions for assembling TAL-effector repeats are known in the art. See, for example, Cermak et al, Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting, Nucleic Acids Res 39(12):e82(2011). Plasmids for construction of TAL-effector repeats are commercially available, for example, from Addgene.

[0159] In some embodiments, the agent comprises a zinc finger nuclease (ZFN) system. The ZFN domain can be generated using commercially available plasmids, such as plasmid pairs from Sigma Aldrich (St. Louis, MO), e.g., CSTZFN-1KT COMPOZR® Custom Zinc Finger Nuclease (ZFN) R-3257609. The plasmids can be prepared using commercially available systems according to the manufacturer's protocol (e.g., NEB Monarch Miniprep (Catalog No. T1010), New England Biolabs, Ipswich, MA).

[0160] In some embodiments, the agent comprises a vector designed to deliver conventional gene therapy (e.g., gene knockout or knock-in by homologous recombination). Non-limiting examples of such vectors include retroviruses (e.g., lentivirus 5), adenoviruses, adeno-associated viruses, herpes simplex viruses, nanoparticles, and DNA transposons.

[0161] D. Small molecule agents In some embodiments, the agent comprises a small molecule. In some embodiments, the small molecule binds to a target protein. In some embodiments, the small molecule binds to a protein that can regulate the expression or activity of the target protein. In some embodiments, the small molecule is an inhibitor of the target protein (e.g., a direct inhibitor, an indirect inhibitor). In some embodiments, the small molecule is an activator of the target protein (e.g., a direct activator, and an indirect activator).

[0162] Examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic, or inorganic compounds. The atoms in a small molecule are typically linked to each other via covalent and / or ionic bonds. In certain embodiments, the small molecule is an organic small molecule. The arrangement of atoms in an organic small molecule may represent a chain (e.g., a carbon-carbon chain or a carbon-heteroatom chain), or a ring containing carbon atoms, such as benzene or a polycyclic system, or a combination of carbon and heteroatoms, i.e., a heterocycle such as pyrimidine or quinazoline. Small molecules can have a wide range of molecular weights, but generally include molecules having a molecular weight of less than about 5,000 Daltons. For example, such small molecules can be less than about 1,000 Daltons, preferably less than about 750 Daltons, or more preferably less than about 500 Daltons. Small molecules can be found in nature (e.g., identified, isolated, purified) and / or produced synthetically (e.g., by traditional organic synthesis, biocatalyzed synthesis, or a combination thereof). See, for example, Ganesan, Drug Discov. Today 7(1):47-55 (January 2002); Lou, Drug Discov. Today, 6(24):1288-1294 (December 2001). Examples of naturally occurring small molecules include, but are not limited to, hormones, neurotransmitters, nucleotides, amino acids, sugars, lipids, and derivatives thereof.

[0163] In certain embodiments, the agent comprises a proteolysis targeting chimera (PROTAC).

[0164] Small molecules suitable for use in the compositions, kits, and methods of the present disclosure can be identified by one of ordinary skill in the art using any of the screening methods disclosed herein.

[0165] E. Therapeutic Cells and Cell-Based Therapies In some embodiments, the agent comprises therapeutic cells. In certain embodiments, the therapeutic cells express and / or are engineered to express a target protein described herein (e.g., a target protein in the Sequence Listing, Table A, or a variant of the foregoing), a polypeptide (e.g., an antibody, antigen-binding fragment, or polypeptide comprising an amino acid sequence that is at least 70% identical to at least a portion of the target protein), a polynucleotide (e.g., recombinant DNA, RNA, such as mRNA or siRNA), and / or a gene editing system (e.g., the CRISPR / Cas system).

[0166] In some embodiments, the polypeptides (e.g., antibodies or antigen-binding fragments) disclosed herein are incorporated into cell-based therapies. In some embodiments, the polypeptide is an engineered T cell receptor. In some embodiments, the polypeptide is a chimeric antigen receptor (CAR) (e.g., expressed on T (CAR-T) cells, natural killer (CAR-NK) cells, or macrophage (CAR-M) cells). In some embodiments, the CAR comprises a transmembrane domain and an antigen recognition portion that binds to a target protein. In certain embodiments, the polypeptide is expressed by therapeutic cells (e.g., CAR-T, CAR-NK, or CAR-M cells). In certain embodiments, the polypeptide is a cytokine receptor expressed on the membrane of CAR-T, CAR-NK, or CAR-M cells. In more specific embodiments, the polypeptide is a cytokine secreted from CAR-T, CAR-NK, or CAR-M cells.

[0167] Therapeutic cells suitable for use in the compositions, kits, and methods of the present disclosure can be generated, identified, and / or enriched by methods known to those of ordinary skill in the art. Non-limiting examples of such methods include purifying, expanding, and / or differentiating cells from a subject (e.g., a human) into specific cell products; engineering somatic cells for gene therapy; immortalizing cells; ex vivo gene modification of cells (e.g., using viral vector and / or lipid nanoparticle delivery technologies); in vivo gene modification of cells (e.g., using viral vector and / or lipid nanoparticle delivery technologies); genome editing; cell plasticity technologies; gene modification; and flow cytometry. In some embodiments, the therapeutic cells are autologous or syngeneic. In other embodiments, the therapeutic cells are allogeneic.

[0168] Expression Vectors and Hosts In another aspect, the present disclosure provides an expression vector comprising the polynucleotide described herein.

[0169] The term "expression vector" refers to a replicable nucleic acid capable of expressing one or more proteins when the expression vector is transformed into a suitable expression host cell.

[0170] In some embodiments, the expression vector comprises an expression control polynucleotide sequence operably linked to a polynucleotide, a polynucleotide sequence encoding a selectable marker, or both. In some embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In some embodiments, the expression control polynucleotide sequence comprises an inducible promoter sequence. The term "promoter" refers to the region of DNA to which RNA polymerase binds to initiate transcription of a gene. The term "operably linked" means that a nucleic acid is positioned in a recombinant polynucleotide, such as a vector, in such a way that it enables expression of the nucleic acid under the control of the element (e.g., a promoter) to which it is linked. The term "selectable marker element" is an element that confers a trait suitable for artificial selection. A selectable marker element can be a negative or positive selectable marker. Non-limiting examples of expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Molecular Cloning: A Laboratory Manual (Michael R. Green & Joseph Sambrook eds., 4th ed. 2012).

[0171] In another aspect, the present disclosure provides an expression host cell comprising any one or more of the polynucleotides or expression vectors described herein.

[0172] The term "expression host cell" refers to a cell useful for receiving, maintaining, replicating, and / or amplifying a vector.

[0173] Non-limiting examples of expression host cells include mammals, such as hybridoma cells, baby hamster kidney fibroblasts (BHK cells), Chinese hamster ovary (CHO) cells, COS cells, HeLa cells, and human embryonic kidney (HEK), yeast cells, such as Pichia pastoris cells, or bacterial cells, such as DH5α. For example, for the process of host cell culture for producing protein therapeutics, see Mammalian Cell Cultures for Biologics Manufacturing (Weichang Zhou & Anne Kantardjieff eds., 2014), and for the purification of protein therapeutics, see Protein Biotechnology: Isolation, Characterization, and Stabilization (Felix Franks eds., 2013) and Protein Purification Protocols (Paul Cutler eds., 2010); and for the formulation of therapeutic proteins, see Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic (Brian K Meyer eds., 2012).

[0174] The polynucleotides or expression vectors described herein can be introduced into appropriate or desired host cells using techniques known in the art, including transformation, electroporation, and transduction. The introduced nucleic acid can be extrachromosomal within the host cell or integrated into the genome of the host cell.

[0175] Pharmaceutical composition In another aspect, the present disclosure provides a pharmaceutical composition comprising an agent disclosed herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a composition having a pharmacological activity or other direct effect in the alleviation, treatment, or prevention of cancer, or a finished dosage form or formulation thereof.

[0176] In some embodiments, the composition (e.g., pharmaceutical composition) includes a pharmaceutically acceptable carrier, excipient, stabilizer, diluent, or tonic (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents, or tonics include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, or dextrin); chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-protein complexes); nonionic surfactants (e.g., Tween®), PLURONICS® and polyethylene glycol (PEG).

[0177] In some embodiments, the agent of the pharmaceutical composition (e.g., polypeptide, polynucleotide, or small molecule) is modified, e.g., conjugated to a heterologous moiety. The term "conjugated" refers to being linked via a covalent or non-covalent interaction. Conjugation can be carried out using any suitable linker; non-limiting examples include peptide linkers, compound linkers, and chemical crosslinking agents.

[0178] In some embodiments, the heterologous moiety is a marker (e.g., a fluorescent marker or a radioactive marker), a molecule that stabilizes an agent, a molecule that targets an agent (e.g., to a specific cell or tissue, such as to facilitate or prevent passage across the blood-brain barrier), or a combination thereof.

[0179] In some embodiments, the heterologous moiety is polyethylene glycol (PEG), hexadecanoic acid, hydrogel, nanoparticles, multimerization domain, and carrier peptide. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are polymer nanoparticles. In some embodiments, the polymer is an amphiphilic polymer. In other embodiments, the polymer is a hydrophobic or hydrophilic polymer. Non-limiting examples of polymers include poly(lactic acid)-poly(ethylene glycol), poly(lactic-co-glycolic acid)-poly(ethylene glycol), poly(lactic-co-glycolic acid) (PLGA), poly(lactic-co-glycolic acid)-d-α-tocopheryl polyethylene glycol succinate, poly(lactic-co-glycolic acid)-ethylene oxide fumarate, poly(glycolic acid)-poly(ethylene glycol), polycaprolactone-poly(ethylene glycol), or any salt thereof. In some embodiments, the polymer nanoparticles comprise poly(lactic-co-glycolic acid) (PLGA).

[0180] In some embodiments, the composition (e.g., a pharmaceutical composition) is formulated for an appropriate dosing schedule and route. Non-limiting examples of the route of administration include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical. In some embodiments, the composition (e.g., a pharmaceutical composition) is stored in the form of an aqueous solution or a dry formulation (e.g., lyophilized). In some embodiments, the composition is formulated to be administered by infusion (e.g., intravenous infusion).

[0181] In some embodiments, the composition is formulated to be administered as a combination therapy with one or more additional therapeutic agents (e.g., with a second therapeutic agent). As used herein, "combination therapy" or "administered in combination" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. Non-limiting examples of additional agents or treatments include biologic agents (e.g., antibodies, peptides), steroid hormones, protein replacement therapy, substrate therapy, enzyme therapy, cell therapy, gene therapy, small molecules, and agents that affect metabolic activity.

[0182] The treatment regimen defines the dosage and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, two or more agents are administered sequentially as part of a prescription regimen. In other embodiments, the delivery of two or more agents is simultaneous or co-temporaneous. In some embodiments, two or more agents are co-formulated. In some embodiments, by administering two or more agents or treatments in combination, a reduction in other parameters associated with the symptom or disorder is achieved that is greater than that observed when one agent or treatment is delivered alone or in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Each of the two or more therapeutic agents can be administered by any suitable route including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The two or more therapeutic agents can be administered by the same route or different routes.

[0183] In some embodiments, the agent or pharmaceutical composition of the disclosure is delivered by a viral vector, e.g., by contacting cells with the viral vector, and is administered locally to a tumor (e.g., by injection) or systemically to a subject (e.g., a human patient) (e.g., intravenously or orally).

[0184] The viral genome provides a rich source of vectors that can be used for the efficient delivery of exogenous genes into mammalian cells. The viral genome is a particularly useful vector for gene delivery because the polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Non-limiting examples of viral vectors include retroviruses (e.g., retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses, such as picornaviruses and alphaviruses, and double-stranded DNA viruses, such as adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-deficient herpesviruses) and poxviruses (e.g., vaccinia, modified vaccinia virus Ankara (MVA), fowlpox and canarypox). Further non-limiting examples include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus and hepatitis virus.Non-limiting examples of retroviruses include avian leukosis sarcoma, avian C-type virus, mammalian C-type, B-type virus, D-type virus, oncovirus, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (see, e.g., Coffin JM. Retroviridae: The viruses and their replication. In: Fields BN, Knipe DM, Howley PM et al, eds. Fundamental Virology. 3rd ed. Philadelphia: Lippincott-Raven Publishers, 1996:763-843). Further non-limiting examples include murine leukemia virus, murine sarcoma virus, murine mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, chimpanzee endogenous virus, talapoin monkey leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Further non-limiting examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the teachings of which are incorporated herein by reference.

[0185] In some embodiments, the agents or pharmaceutical compositions of the present disclosure are formulated to be delivered by a membrane-based carrier in vivo, in vitro, ex vivo, or in situ. In some embodiments, the membrane-based carrier is a cell-based carrier (e.g., a mammal such as a human cell). In some embodiments, the membrane-based carrier is a vesicle-based carrier. In some embodiments, the membrane-based carrier comprises one or more vectors (e.g., plasmids, viruses, virus-like particles, or virions) described herein.

[0186] In some embodiments, the agents or pharmaceutical compositions of the present disclosure are formulated to be delivered by one or more liposomes. Liposomes are spherical vesicular structures composed of a single or multiple lipid bilayers surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible and non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB) (see, e.g., Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)).

[0187] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, the teachings of which regarding the preparation of multilamellar vesicle lipids are incorporated herein by reference). Vesicle formation can be spontaneous when lipid membranes are mixed with an aqueous solution, but can also be facilitated by applying force in the form of agitation using a homogenizer, sonicator, or extrusion device (see, e.g., Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)). Extruded lipids can be prepared by extruding them through small-sized filters as described in Templeton et al., Nature Biotech, 15:647-52 (1997), the teachings of which regarding the preparation of extruded lipids are incorporated herein by reference).

[0188] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by lipid nanoparticles (LNPs). In one embodiment, the LNP preparation comprising the agent or pharmaceutical composition of the present disclosure has one or more of the following characteristics: (a) the LNP preparation comprises a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid, and (b) the LNP preparation has an average particle size of 80 nm to 160 nm.

[0189] Nanostructured lipid carriers (NLCs) are solid lipid nanoparticles (SLNs) that retain the characteristics of modified SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are important components for drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier combining liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs are composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, the two components enhance drug encapsulation efficiency, facilitate surface modification, and prevent the leakage of water-soluble drugs. See, for example, Li et al., Nanomaterials 7(6):122 (2017).

[0190] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by a carbohydrate carrier (e.g., anhydride-modified phytoglycogen or glycogen-like material). Non-limiting examples of carbohydrate carriers include octenyl succinic acid phytoglycogen, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin.

[0191] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by a protein carrier (e.g., a protein covalently linked to a cyclic polynucleotide). Non-limiting examples of protein carriers include human serum albumin (HAS), low density lipoprotein (LDL), high density lipoprotein (HDL), and globulin.

[0192] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by a cationic carrier (e.g., a cationic lipopolymer or transfection reagent). Non-limiting examples of cationic carriers include Lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimer, chitosan, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(’\N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HC1), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC).

[0193] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by exosomes, adipocytes, and / or red blood cells. See, for example, Ha et al., Acta Pharm Sin B. 6(4):287-96 (2016).

[0194] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more fusosomes. The fusosomes are engineered to confer target cell specificity for fusion and payload delivery, thereby enabling the generation of a delivery vehicle with programmable cell specificity. See, for example, WO 2020014209 pamphlet, the teachings of which regarding the design, preparation, and use of fusosomes are incorporated herein by reference.

[0195] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by ex vivo differentiated red blood cells. See, for example, WO 2015073587 pamphlet; WO 2017123646 pamphlet; WO 2017123644 pamphlet; WO 2018102740 pamphlet; WO 2016183482 pamphlet; WO 2015153102 pamphlet; WO 2018151829 pamphlet; WO 2018009838 pamphlet; Shi et al., PNAS, 111(28):10131-36 (2014); US Patent No. 9,644,180; Huang et al., Nature Communications 8:423 (2017).

[0196] In some embodiments, the agents or pharmaceutical compositions of the present disclosure are formulated to be delivered by one or more microsomes, virus-like particles (VLPs), or plant nanovesicles and plant messenger packs (PMPs). See, for example, WO 2011 / 097480, WO 2013 / 070324, WO 2017 / 004526, and WO 2020 / 041784.

[0197] In some embodiments, the agents or pharmaceutical compositions of the present disclosure are formulated to be delivered by one or more anellosomes. The preparation and use of anellosomes for the delivery of therapeutic products are described in U.S. Patent No. 11,166,996, the teachings of which regarding the design, preparation, and use of anellosomes are incorporated herein by reference.

[0198] Method for modulating a target protein In another aspect, the present disclosure provides a method for modulating the expression or activity of a target protein identified in the Sequence Listing, Table A, or a variant of the foregoing in a cell (target cell, cell of a target tissue), the method comprising contacting the cell (e.g., in vitro, ex vivo, or in vivo) with an agent that comprises and / or modulates the expression or activity of the target protein identified herein, or a pharmaceutical composition comprising the agent.

[0199] The target cell can be any cell type. In some embodiments, the target cell is a hepatocyte (e.g., hepatocyte (HC), hepatic stellate cell (HSC), Kupffer cell (KC), and / or liver sinusoidal endothelial cell (LSEC)); pancreatic cell (e.g., alpha cell, beta cell, delta cell, and / or PP cell); thyroid cell; glandular cell; or a combination thereof. In certain embodiments, the target cell is a hepatocyte (HC), Kupffer cell (KC), pancreatic beta cell, muscle cell, heart cell, brain cell, kidney cell, adipocyte, or a combination thereof.

[0200] In some embodiments, the target cells are related to and / or involved in inflammation. In certain embodiments, the target cells are epithelial cells, endothelial cells, stem cells, non-immune cells, or combinations thereof.

[0201] In some embodiments, the target cells are related to and / or involved in fibrosis, aging, senescence, or combinations thereof. In certain embodiments, the target cells are epithelial cells, endothelial cells, non-immune cells, or combinations thereof.

[0202] In some embodiments, the target cells are immune cells. In certain embodiments, the target cells are effector T cells, helper T cells, Th1 cells, Th2 cells, Th17 cells, B cells, natural killer (NK) cells, innate lymphoid cells (e.g., ILC1 cells, ILC2 cells, ILC3 cells), macrophages (e.g., M1 macrophages, M2 macrophages), monocytes, and / or antigen-presenting cells (e.g., dendritic cells), or combinations thereof.

[0203] In some embodiments, the target proteins of the present disclosure are used to mediate depletion of cell populations (e.g., populations of cancer cells such as tumor cells; populations of immune cells). In some embodiments, the target proteins of the present disclosure facilitate cell targeting (e.g., delivering a therapeutic agent in a cell-type specific manner), for example as a binder of surface markers.

[0204] The target tissue can be any tissue of the body.

[0205] The target tissue can be any gland of the body (e.g., adrenal gland, pituitary gland, parathyroid gland, and / or pineal gland) or reproductive tissue (e.g., ovaries, testes). In certain embodiments, the target tissue includes the liver, pancreas, thyroid, ovaries, testes, muscle, heart, brain, kidney, adipose tissue, or combinations thereof. In some embodiments, the target tissue includes the adrenal gland, pituitary gland, parathyroid gland, pineal gland, or combinations of the foregoing.

[0206] In some embodiments, the target tissue is immune tissue. In some embodiments, the target cell is non-immune tissue. In some embodiments, the target tissue includes lymph nodes, spleen, secondary lymphoid organs, tertiary lymphoid organs, barrier tissues, skin, intestine, airway, wound, immune tissue, non-immune tissue, or combinations thereof.

[0207] In certain embodiments, the effective amount is sufficient to reduce the expression of the target protein in the target cell and / or target tissue. In some embodiments, the reduction is at least about 10%, for example, at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10-99%, for example, about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0208] In certain embodiments, the effective amount is sufficient to increase the expression of the target protein in the target cell and / or target tissue. In some embodiments, the increase is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is about 10-99%, such as about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%. In some embodiments, the increase is about 1-100 fold, such as about 1-75, 1-50, 1-25, 1-20, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3 or 1-2 fold.

[0209] In some embodiments, the effective amount is sufficient to modulate nuclear factor kappa B (NF-κB) signaling, growth factor signaling, cell death (e.g., apoptosis), cell cycle (e.g., mitosis), cell migration, inflammation, or combinations thereof. In some embodiments, the effective amount is sufficient to modulate a signaling pathway involving the Janus kinase (JAK) signaling family (e.g., JAK1, JAK2, JAK3 and TYK2), members of the signal transducer and activator of transcription (STAT) protein family (e.g., STAT1, STAT3), members of the protein kinase B family (e.g., RAC-α, RAC-β or RAC-γ serine / threonine-protein kinase), members of the interferon regulatory factor (IRF) family, mitogen-activated protein kinase (MAPK) or combinations thereof.

[0210] Methods of diagnosis and treatment In another aspect, the present disclosure provides a method for detecting a disease or condition in a subject or predicting the likelihood (or risk level) of developing a disease or condition in a subject, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject, wherein the level of expression or activity of the target protein in the sample indicates the likelihood of developing a disease or condition in the subject, and the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), and infectious diseases, immune diseases (e.g., inflammation and / or autoimmune diseases), indications treated with hormones, growth factors, and / or protein supplements, or combinations thereof.

[0211] In another aspect, the present disclosure provides a method for classifying a subject based on the predicted likelihood of developing a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject; predicting the likelihood of developing a disease or condition based on the expression or activity of the target protein in the sample; and classifying the subject based on the predicted likelihood, wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), and infectious diseases, immune diseases (e.g., inflammation and / or autoimmune diseases), indications treated with hormones, growth factors, and / or protein supplements, or combinations thereof.

[0212] In another aspect, the present disclosure provides a method for stratifying a set of subjects having a disease or condition, the method comprising quantifying the expression and / or activity of a target protein in a sample derived from an individual subject within the set; and stratifying the set of subjects for treatment according to the level of expression and / or activity of the target protein in the sample of each individual subject, wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), and infectious diseases, immune diseases (e.g., inflammation and / or autoimmune diseases), indications treated with hormones, growth factors, and / or protein supplements, or combinations thereof. In certain embodiments, the disease or condition is or involves inflammation.

[0213] In some embodiments, a higher expression or activity level of a target protein in a sample from a subject, as compared to an appropriate control (e.g., a reference standard), indicates a disease or condition or a likelihood of developing a disease or condition. In some embodiments, a lower expression or activity level of a target protein in a sample from a subject, as compared to an appropriate control (e.g., a reference standard), indicates a disease or condition or a likelihood of developing a disease or condition.

[0214] In some embodiments, the method further comprises administering to a subject determined or predicted to have a likelihood of developing a disease or condition (or at risk of developing) an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein.

[0215] In some embodiments, the method further comprises administering to a subject determined or predicted to have a likelihood of developing a disease or condition an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein.

[0216] In another aspect, the disclosure is a method of preparing a sample useful for detecting a likelihood of developing a disease or condition in a subject, comprising: a) obtaining or having obtained a sample from the subject; b) adding a protease inhibitor, a control peptide, a standard peptide, or a combination thereof to the sample to prepare a sample useful for detecting a likelihood of developing cancer; c) quantifying the expression or activity of a target protein in the sample prepared in step b). comprising wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic disease, cardiovascular disease, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), immune diseases (e.g., inflammation and / or autoimmune diseases), indications treated with hormones, growth factors, and / or protein replacement, or combinations thereof, and provides a method.

[0217] In another aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein.

[0218] In another aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein, wherein the subject has an altered level of expression and / or activity of a target protein disclosed herein.

[0219] As used herein, the terms "treatment" or "treating" refer to the medical management of a subject with the goal of improving, alleviating, stabilizing (i.e., not worsening), preventing or curing a disease, medical condition, or disorder. "Treatment" includes active treatment (treatment directed at improving a disease, medical condition, or disorder), causal treatment (treatment directed at the cause of a related disease, medical condition, or disorder), palliative treatment (treatment designed to relieve symptoms), prophylactic treatment (treatment directed at minimizing or inhibiting, in part or completely, the onset of a related disease, medical condition, or disorder); and adjuvant treatment (treatment utilized to supplement another therapy). Treatment includes a detectable or undetectable decrease in the degree of a disease or condition; prevention of the spread of a disease or condition; delay or deceleration of the progression of a disease or condition; improvement or alleviation of a disease or condition; and remission (partial or complete). "Improving" or "alleviating" a disease or condition means that the degree and / or undesirable clinical symptoms of the disease, disorder, or condition are reduced and / or the time course of progression is decelerated or lengthened as compared to the degree or time course in the absence of treatment. "Treatment" also includes extending survival as compared to expected survival in the absence of treatment. Persons in need of treatment include those who already have the condition or disorder, those who are prone to having the condition or disorder, or those in whom the condition or disorder should be prevented.

[0220] In some embodiments, the subject is an animal. In other embodiments, the subject is a bird, such as a hen, rooster, turkey or parrot. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. Non-limiting examples of non-human mammals include cows (e.g., dairy cows or beef cattle), sheep, goats, pigs, horses, dogs, cats, mice, rats, and the like. In some embodiments, the subject is a human. In some embodiments, the human is a neonate. In some embodiments, the human is a pediatric patient. In some embodiments, the human is a young person. In some embodiments, the human is an adult. In some embodiments, the human is under 18 years old. In some embodiments, the human is at least 18 years old. In some embodiments, the human is between 18 and 25 years old. In some embodiments, the human is at least 25 years old, e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 years old.

[0221] As used herein, the terms "effective amount", "therapeutically effective amount" or "sufficient amount" refer to an amount sufficient to effect a treatment (e.g., to produce a beneficial or desired result), including effects at the cellular, tissue or clinical level, when administered to a subject (e.g., a mammal such as a human cancer patient). Thus, the term depends on the context in which it is applied. For example, in the context of treating cancer, it is an amount of the agent sufficient to achieve a response as compared to the response obtained without administration of the agent. The amount of a given composition described herein corresponding to such an amount will vary depending on various factors such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, subject (e.g., age, sex, weight, etc.) or identity of the host being treated, but can nonetheless be routinely determined by one of ordinary skill in the art. In some embodiments, a "therapeutically effective amount" of the compositions of the present disclosure is an amount that produces a beneficial or desired result in a subject (e.g., as compared to a control). The therapeutically effective amount of the compositions of the present disclosure can be readily determined by one of ordinary skill in the art by routine methods known in the art. The dosing regimen can be adjusted to provide an optimal therapeutic response.

[0222] The therapeutic agents described herein can be administered via various routes of administration, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intraarterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion, and inhalation (e.g., intratracheal, intranasal, or oral inhalation, intranasal instillation), depending on the compound and the particular disease or condition being treated. Administration can be local or systemic, as indicated. The preferred mode of administration can vary depending on the particular compound selected.

[0223] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more additional therapeutic agents (e.g., a second therapeutic agent).

[0224] Administration of two or more therapeutic agents includes co-administering the therapeutic agents substantially simultaneously, such as in a combination pharmaceutical. Alternatively, such administration includes co-administering each therapeutic agent in multiple or separate containers (e.g., capsules, powders, and liquids). Such administration also includes using the therapeutic agents in a sequential manner, either substantially simultaneously or at different time points. When two or more therapeutic agents are administered, the therapeutic agents can be administered via the same route of administration or via different routes of administration.

[0225] In another aspect, the present disclosure provides a method of modulating the expression or activity of a target protein identified in the Sequence Listing, Table A, or a variant of the foregoing in a cell, the method comprising contacting the cell with an agent disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cell is within a subject.

[0226] In another aspect, the present disclosure provides a method of identifying an agent that modulates the expression and / or activity of a target protein (e.g., the target protein of Sequence Listing, Table A, or a variant of the foregoing), the method comprising: a) contacting a sample comprising the target protein (e.g., a biological sample such as a cell or tissue) with an agent (e.g., a candidate agent being tested for its ability to modulate the expression and / or activity of the target); and b) determining whether the agent modulates the expression or activity of the target protein comprising There is provided a method, which shows that the difference in the expression or activity of a target protein contacted with an agent, as compared to a reference, indicates that the agent modulates the expression or activity of the target protein.

[0227] In some embodiments, a difference in the expression or activity of a protein contacted with an agent of at least about 10% as compared to a reference indicates that the agent modulates the expression or activity of the protein. In some embodiments, the difference is at least about 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% or more.

[0228] In some embodiments, a decrease in the expression or activity of a target protein contacted with an agent as compared to a reference indicates that the agent inhibits the expression or activity of the target protein. In some embodiments, an increase in the expression or activity of a protein as compared to a reference indicates that the agent activates the expression or activity of the protein.

[0229] Indications Aging The methods described herein are applicable to the treatment of aging, aging-related conditions, and / or aging-related disorders or diseases. In some embodiments, the methods described herein include the treatment of overall health, body temperature, weight, height, abdominal circumference, reproductive ability, body fat, heart rate, blood pressure, pulse rate, blood oxygen level, respiratory rate, respiratory pattern, blood glucose level, blood pH, cardiac output, cardiac rhythm, and / or the concentration of specific substances in the blood. In some embodiments, the treatment can be evaluated by measurement of complete blood count, red blood cells, white blood cells, platelets, hemoglobin, hematocrit, mean corpuscular volume, basic metabolic panel, blood glucose, calcium, electrolyte tests, renal function, blood enzyme tests, troponin, creatine kinase, lipoprotein panel, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, coagulation panel, and / or bone marrow examination.

[0230] Senescence In some embodiments, the methods disclosed herein are for the treatment of aging. In still further embodiments, the methods disclosed herein relate to the treatment of aging-related diseases or disorders. In some embodiments, the disease or disorder is selected from diabetes, metabolic syndrome, and obesity. In other embodiments, the disease or disorder is related to photosensitivity or photoaging. In still further embodiments, the disease or disorder is selected from arthritis, Alzheimer's disease, asthma, blindness, cancer, chronic bronchitis, chronic kidney disease, chronic obstructive pulmonary disease, coronary heart disease, deep vein thrombosis, dementia, depression, diabetes, epilepsy, heart failure, hypercholesterolemia, hypertension, motor neuron disease, multiple sclerosis, osteoporosis, Paget's disease of bone, Parkinson's disease, shingles, and stroke.

[0231] Fibrosis In some embodiments, the methods of the present disclosure relate to the treatment of fibrosis. In some embodiments, the fibrosis is pulmonary fibrosis, liver fibrosis, skin fibrosis, kidney fibrosis, pancreatic fibrosis, systemic sclerosis, cardiac fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, and / or macular degeneration.

[0232] Metabolic diseases In some embodiments, the present disclosure provides for the treatment of metabolic diseases and / or disorders. In some embodiments, the treatment relates to delaying or preventing the onset of a metabolic disorder. In some embodiments, the treatment relates to delaying or preventing the onset of complications associated with one or more metabolic disorders. In still further embodiments, the treatment relates to hormone therapy or enzyme therapy. In some embodiments, the metabolic disorder is caused by a genetic defect. In still further embodiments, the metabolic disorder is selected from the group consisting of familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), Niemann-Pick, phenylketonuria (PKU), porphyria, Tay-Sachs disease, and Wilson disease.

[0233] Cardiovascular diseases In some embodiments of the present disclosure, the methods of the present disclosure relate to the treatment of cardiovascular diseases. In some embodiments, the cardiovascular disease is atherosclerosis, congestive heart failure, vulnerable plaque, stroke, or ischemia. In still further embodiments, the cardiovascular disease is coronary artery disease, peripheral artery disease, or carotid artery disease. In further embodiments of the present disclosure, the methods relate to the treatment of cardiovascular diseases or disorder-related symptoms. In some embodiments, such symptoms include chest pressure or pain, shortness of breath, pain or discomfort in the arm or shoulder, pain or discomfort in the jaw, neck or back, weakness, lightheadedness, or nausea. In some embodiments, the symptoms to be addressed may include abnormal fatigue, sleep disorders, shortness of breath, and / or indigestion.

[0234] Endocrine-related disorders In some embodiments, the methods disclosed herein relate to the treatment of endocrine-related diseases or disorders. In some embodiments, the methods relate to the treatment of endocrine glands. In still further embodiments, endocrine glands include, but are not necessarily limited to, the adrenal glands, hypothalamus, ovaries, pancreatic islet cells, parathyroid glands, pineal gland, pituitary gland, testes, thymus, and / or thyroid gland. In some embodiments, treatment by the disclosed methods relates to diseases, problems with the endocrine feedback system, the inability of a gland to stimulate the hormone release of another gland, genetic disorders, infections, injury to the endocrine gland, and / or tumors of the endocrine gland. In some embodiments, endocrine-related disorders are adrenal insufficiency, Cushing's disease, gigantism (acromegaly), hyperthyroidism, hypothyroidism, hypopituitarism, multiple endocrine neoplasia I and II, polycystic ovary syndrome, and precocious puberty.

[0235] Genetic diseases In some embodiments, the method relates to a method for treating a genetic disorder. Genetic disorders that can accept the treatment method of the present disclosure include, but are not limited to, arrhythmogenic right ventricular dysplasia / cardiomyopathy, Alzheimer's disease, arthritis, autism spectrum disorder, Brugada syndrome, cancer, Charcot-Marie-Tooth disease, cleft lip and palate, craniosynostosis, cystic fibrosis, diabetes, Down syndrome, fragile X syndrome, familial adenomatous polyposis, Hirschsprung's disease, Huntington's disease, Klinefelter syndrome, Niemann-Pick syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, sickle cell disease, diplomyelia, Tay-Sachs disease, triple X syndrome, Turner syndrome, trisomy 18, trisomy 13, and von Hippel-Lindau. In some embodiments, the genetic disorder is chromosomal. In other embodiments, the genetic disorder is complex and results from a combination of gene mutations and other factors (e.g., diet, certain drugs, tobacco, alcohol use, etc.). In still further embodiments, the genetic disorder is a single-gene disorder.

[0236] Oncology In some embodiments of the present disclosure, the method relates to the treatment of cancer. Cancer treatments provided by the present disclosure include, but are not limited to, carcinomas, sarcomas, melanomas, lymphomas, and / or leukemias. In some embodiments, the method of the present disclosure can replace, advance, or follow other treatment regimens. In some embodiments, other treatment regimens include, but are not limited to, chemotherapy, radiation therapy, surgery, hormone therapy, biological response modifier therapy, immunotherapy, and / or bone marrow transplantation.

[0237] Immunity In some embodiments, the method of the present disclosure relates to immunity. In some embodiments, the immunity relates to bacteria, parasites, viruses, fungi, and / or cancer cells. In some embodiments, the immunity is autoimmune and is the result of the immune system attacking self-molecules. In some embodiments, the method relates to the treatment of immune tolerance.

[0238] Inflammation In some embodiments of the present disclosure, the method relates to the treatment of inflammation. In some embodiments, the inflammation is acute or relatively short-term and lasts from a few minutes to a few hours. In still other embodiments, the inflammation is chronic or longer-term, lasting for weeks to months, and in some cases, years. In some embodiments, the methods disclosed herein relate to the treatment or prevention of conditions associated with inflammation. In certain embodiments, such conditions include, but are not limited to, skin flushing, pain or tenderness, swelling, heat, fatigue, fever, joint pain or stiffness, stomatitis, and rash. In some embodiments, the inflammation is caused by other diseases or disorders.

[0239] Autoimmune In some embodiments, the disclosed method relates to the treatment of autoimmune diseases. In some embodiments, autoimmune diseases can include diseases of the joints and muscles (e.g., psoriatic arthritis, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus), diseases of the gastrointestinal tract (e.g., Crohn's disease, celiac disease, ulcerative colitis, inflammatory bowel disease), diseases of the endocrine system (e.g., Graves' disease, Hashimoto's thyroiditis, Addison's disease), diseases of the skin (e.g., dermatomyositis, psoriasis, scleroderma), diseases of the nervous system (e.g., chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, multiple sclerosis), and other diseases (e.g., myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, autoimmune lymphoproliferative syndrome, type 1 diabetes), but are not limited thereto.

[0240] Additional treatment In yet further embodiments, the methods of the present disclosure relate to the treatment of any indication for which any hormone, growth factor, or protein replacement is currently utilized. Those skilled in the art will understand that the therapeutic effect need not be complete so long as some benefit is provided to the subject.

[0241] In some embodiments, an endocrine organ is any organ that secretes a protein into the circulation.

[0242] In some embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease) metabolic activity, cell proliferation, cell metastasis, cell migration, autophagy, apoptosis, endocrine function, or a combination thereof.

[0243] In some embodiments, the effective amount is sufficient to decrease (e.g., inhibit) metabolic activity, cell proliferation, cell metastasis, cell migration, apoptosis, endocrine function, or a combination thereof. In certain embodiments, the effective amount is sufficient to decrease metabolic activity, cell proliferation, cell metastasis, cell migration, apoptosis, endocrine function, or a combination thereof by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, the decrease is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0244] In some embodiments, the effective amount is sufficient to increase (e.g., promote) cell growth, metabolic activity, autophagy, apoptosis, endocrine function, or a combination thereof. In certain embodiments, the effective amount is sufficient to increase metabolic activity, cell growth, cell metastasis, cell migration, apoptosis, endocrine function, or a combination thereof by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is about 10 - 99%, such as about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0245] In some aspects, the effective amount is sufficient to modulate (e.g., increase or decrease) organ and / or cell growth, cell proliferation, cell activation (e.g., T cell activation), cell migration, cell metabolic rate, cell death, cell autophagy, cell differentiation, cell polarization (e.g., polarization of epithelial cells or immune cells such as Th1, Th2, M1 / M2, etc.), cell maturation (e.g., maturation of stem cells), enzyme activity, or a combination thereof.

[0246] Immunity / Inflammation In some embodiments, the methods of the present disclosure relate to immunity. In some embodiments, the immunity relates to parasites, viruses, fungi, and / or cancer cells. In some embodiments, the immunity is autoimmune and is the result of the immune system attacking self - molecules. In some embodiments, the method relates to the treatment of immune tolerance.

[0247] In some embodiments of the present disclosure, the method relates to the treatment of inflammation. In some embodiments, the inflammation is acute or relatively short-term and lasts from a few minutes to a few hours. In still other embodiments, the inflammation is chronic or longer-term, lasting for weeks to months, and in some cases, years. In some embodiments, the methods disclosed herein relate to the treatment or prevention of conditions associated with inflammation. In certain embodiments, such conditions include, but are not limited to, skin flushing, pain or tenderness, swelling, heat, fatigue, fever, joint pain or stiffness, stomatitis, and rash. In some embodiments, the inflammation is caused by other diseases or disorders.

[0248] In some embodiments, the disclosed method relates to the treatment of autoimmune diseases. In some embodiments, autoimmune diseases can include diseases of the joints and muscles (e.g., psoriatic arthritis, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus), diseases of the gastrointestinal tract (e.g., Crohn's disease, celiac disease, ulcerative colitis, inflammatory bowel disease), diseases of the endocrine system (e.g., Graves' disease, Hashimoto's thyroiditis, Addison's disease), diseases of the skin (e.g., dermatomyositis, psoriasis, scleroderma), diseases of the nervous system (e.g., chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, multiple sclerosis), and other diseases (e.g., myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, autoimmune lymphoproliferative syndrome, type 1 diabetes), but are not limited thereto.

[0249] In some embodiments, the disease or condition is an inflammatory disease and / or an autoimmune disease. A wide variety of inflammatory and / or autoimmune diseases are treatable according to the methods described herein. In some embodiments, the inflammatory and / or autoimmune diseases include Alzheimer's disease, asthma, endometriosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease and ulcerative colitis), multiple sclerosis (MS), non-alcoholic fatty liver disease (e.g., non-alcoholic fatty liver disease (NAFLD)), obesity, Parkinson's disease cancer, psoriasis, rheumatoid arthritis (RA), scleroderma, systemic lupus erythematosus (SLE), type 1 diabetes, type 2 diabetes, or a combination thereof.

[0250] In some embodiments, the target protein activates an immune response. In certain embodiments, the target protein inhibits an immune response. In some embodiments, the immune response is an innate immune response (e.g., a humoral and / or cell-mediated immune response). In certain embodiments, the immune response is an adaptive immune response (e.g., a humoral and / or cell-mediated immune response). Non-limiting examples of immune responses include T cell-mediated immune responses (e.g., cytokine production and cytotoxicity), B cell-mediated immune responses, humoral immune responses, and activation of cytokine-responsive cells (e.g., macrophages).

[0251] In some embodiments, the target protein enhances signals involved in T cell activation and / or survival. In certain embodiments, the target protein activates a stimulatory checkpoint molecule. Non-limiting examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, glucocorticoid-induced TNFR family-related gene (GITR), inducible T cell co-stimulator (ICOS). In certain embodiments, the target protein is an agonist for CD28.

[0252] In some embodiments, the target protein reduces signals involved in T cell anergy and / or exhaustion. In certain embodiments, the target protein inhibits an inhibitory checkpoint molecule. Non-limiting examples of inhibitory checkpoint molecules include programmed cell death protein 1 (PD-1), PD-L1, PD-L2, T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), B and T lymphocyte attenuator (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), V domain Ig suppressor of T cell activation (VISTA), sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC 7), and sialic acid-binding immunoglobulin-type lectin 9 (SIGLEC 9). In certain embodiments, the target protein is an inhibitor of PD-1.

[0253] In some embodiments, the effective amount is as follows: a) Development of high endothelial venules (HEV) and / or tertiary lymphoid organs (TLO); b) Activation, degranulation, differentiation, maturation, migration, polarization, proliferation, and / or recruitment of immune cells (e.g., macrophages, monocytes, or dendritic cells); c) Egress and / or homing of immune cells to lymph nodes; d) Egress and / or homing of immune cells to tumors; e) Cytokine production; f) Antigen presentation; g) Target protein expression; or h) Autoantibody levels, or a combination thereof is sufficient to modulate (e.g., increase or decrease) them.

[0254] In some embodiments, the effective amount is as follows: a) Development of HEV and / or TLO; b) Activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) Exit and / or homing of immune cells from lymph nodes; d) Exit and / or homing of immune cells from tumors; e) Cytokine production; f) Antigen presentation; g) Target protein expression; or h) Autoantibody levels, or a combination thereof is sufficient to increase.

[0255] In certain embodiments, the increase is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is about 10 - 99%, e.g., about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0256] In some embodiments, the effective amount is as follows: a) Development of HEV and / or TLO; b) Activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) Exit and / or homing of immune cells from lymph nodes; d) Exit and / or homing of immune cells from tumors; e) Cytokine production; f) Antigen presentation; g) Target protein expression; or h) autoantibody levels, or a combination thereof is sufficient to reduce.

[0257] In certain embodiments, the reduction is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10 - 99%, e.g., about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0258] In some embodiments, the effective amount a) increases organ function; b) modulates inflammation (e.g., increases or decreases); c) reduces the level of autoantibodies; d) reduces the rate and / or number of relapses and / or recurrences; e) reduces the viral load; or f) reduces (e.g., controls) infection, or a combination of the foregoing is sufficient to effect.

[0259] In some embodiments, the effective amount is sufficient to increase organ function, inflammation, or a combination thereof. In certain embodiments, the increase is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0260] In some embodiments, the effective amount is sufficient to decrease inflammation, the level of autoantibodies, the rate and / or number of relapses and / or recurrences, the viral load or infection, or a combination of the foregoing. In certain embodiments, the decrease is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the decrease is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0261] In some embodiments, the target protein activates immune cells. In some embodiments, the target protein inhibits immune cells (e.g., inhibits activation of immune cells, induces immune cell death (e.g., apoptosis), or a combination thereof). Immune cells are cells that play a role in the immune response. Immune cells are cells of hematopoietic origin, including lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., basophils, eosinophils, granulocytes, macrophages, mast cells, and monocytes). The target protein can be expressed on cancer cells (e.g., metastatic cancer cells), in the tumor microenvironment (e.g., on stromal cells), or on non-malignant cells (e.g., immune cells).

[0262] In some embodiments, the effective amount is sufficient to increase the immune response. In certain embodiments, the increase is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, the increase is about 10-99%, e.g., about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0263] In some embodiments, the effective amount is sufficient to reduce the immune response. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10-99%, such as about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0264] In some embodiments, the effective amount is sufficient to reduce the inflammatory response. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10-99%, such as about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0265] In some embodiments, the effective amount is sufficient to reduce autoimmunity. In certain embodiments, the reduction is at least about 10%, for example, at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is from about 10 to 99%, for example, from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0266] In certain embodiments, the effective amount is sufficient to modulate the expression of the target protein in immune cells.

[0267] In some embodiments (e.g., for immune and / or inflammatory treatments), the effective amount is sufficient to modulate the migration of immune cells (e.g., antigen-presenting cells (e.g., dendritic cells and / or macrophages) and / or T cells), the proliferation of immune cells, the recruitment of immune cells (e.g., antigen-presenting cells (e.g., dendritic cells and / or macrophages), monocytes, T cells and / or B cells), the lymph node homing of immune cells (e.g., dendritic cells and / or T cells), the lymph node egress of immune cells (e.g., dendritic cells and / or T cells), the differentiation of immune cells, the activation of immune cells, the polarization of immune cells, cytokine production (e.g., increasing pro-inflammatory cytokines, decreasing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, decreasing anti-inflammatory cytokines), the degranulation of immune cells, the maturation of immune cells, antigen presentation, target protein expression, inflammation, autoantibody levels (e.g., increasing or decreasing); increasing organ function; reducing the rate and / or number of relapses or recurrences, viral load; controlling infection or performing the aforementioned combinations.

[0268] Cancer A variety of cancers can be treated according to the methods described herein. In some embodiments, the cancer includes solid tumors (e.g., tumors of the breast, lung, prostate, colon, bladder, ovary, kidney, stomach, colon, rectum, testis, head and / or neck, pancreas, brain, skin). Thus, in some embodiments, the cancer is a solid tumor cancer. Solid tumor cancers that can be treated according to the methods described herein include breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, gastric cancer, colorectal cancer, rectal cancer, colon-rectal cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, and skin cancer. In some embodiments, the cancer is a blood cancer (e.g., leukemia, lymphoma, myeloma). Blood cancers that can be treated according to the methods described herein include leukemia (e.g., acute leukemia, chronic leukemia), lymphoma (e.g., B-cell lymphoma, T-cell lymphoma), and multiple myeloma.

[0269] Examples of cancers treatable according to the methods described herein include acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; pediatric adrenocortical carcinoma; AIDS-related cancers (e.g., Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma); anal cancer; appendiceal cancer; pediatric astrocytoma; pediatric atypical teratoid / rhabdoid tumor, central nervous system; basal cell carcinoma of the skin; bile duct cancer; bladder cancer; pediatric bladder cancer; bone cancer (including Ewing sarcoma, osteosarcoma and malignant fibrous histiocytoma); brain tumor / cancer; breast cancer; Burkitt lymphoma; carcinoid tumor (gastrointestinal); pediatric carcinoid tumor; pediatric heart (cardiac) tumor; pediatric embryonal tumor; pediatric germ cell tumor; primary CNS lymphoma; cervical cancer; pediatric cervical cancer; cholangiocarcinoma; pediatric chordoma; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); chronic myeloproliferative tumor; colorectal cancer; pediatric colorectal cancer; pediatric craniopharyngioma; cutaneous T-cell lymphoma (e.g., mycosis fungoides and Sézary syndrome); ductal carcinoma in situ (DCIS); embryonal tumor, pediatric central nervous system,; endometrial cancer (uterine cancer); pediatric ependymoma; esophageal cancer; pediatric esophageal cancer; adherent neuroblastoma; Ewing sarcoma; pediatric extracranial embryonal tumor; extragonadal embryonal tumor; eye (ocular) cancer; pediatric intraocular melanoma; intraocular melanoma; retinoblastoma; fallopian tube cancer; fibrous histiocytoma, malignant and osteosarcoma of bone; gallbladder cancer; stomach (gastric cancer); pediatric stomach (gastric cancer); gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); pediatric gastrointestinal stromal tumor; germ cell tumor; pediatric central nervous system embryonal tumor (e.g., pediatric extracranial embryonal tumor, extragonadal embryonal tumor, ovarian embryonal tumor, testicular cancer); gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; pediatric heart tumor; hepatocellular (liver) cancer; histiocytosis, Langerhans cell; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; pediatric intraocular melanoma; islet cell tumor, pancreatic neuroendocrine tumor; Kaposi sarcoma; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia; lip and oral cavity cancer; liver cancer; lung cancer (non-small cell and small cell); pediatric lung cancer; lymphoma; male breast cancer; malignant fibrous histiocytoma of bone and osteosarcoma; melanoma; pediatric melanoma; melanoma, intraocular (ocular); pediatric intraocular melanoma; Merkel cell carcinoma; malignant mesothelioma; pediatric mesothelioma; metastatic cancer; metastatic squamous neck cancer of unknown primary; midline carcinoma with NUT gene alteration; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides;Myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms; chronic myeloid leukemia (CML); acute myeloid leukemia (AML); chronic myeloproliferative neoplasms; nasal and paranasal cavity cancers; nasopharyngeal cancer; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung cancer; oral cancer, lip cancer and oral cavity cancer as well as oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; pediatric ovarian cancer; pancreatic cancer; pediatric pancreatic cancer; pancreatic neuroendocrine tumors; papillomatosis (pediatric larynx); paraganglioma; pediatric paraganglioma; paranasal cavity cancer and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pediatric pheochromocytoma; pituitary tumor; plasma cell tumor / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; primary central nervous system (CNS) lymphoma; primary peritoneal cancer; prostate cancer; rectal cancer; recurrent cancer; renal cell (kidney) cancer; retinoblastoma; pediatric rhabdomyosarcoma; salivary gland cancer; sarcoma (e.g., pediatric rhabdomyosarcoma, pediatric hemangioma, Ewing sarcoma, Kaposi sarcoma, osteosarcoma (bone cancer), soft tissue sarcoma, uterine sarcoma); Sézary syndrome; skin cancer; pediatric skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; metastatic squamous neck cancer of unknown primary; stomach (gastric) cancer; pediatric gastric cancer; cutaneous T-cell lymphoma (e.g., mycosis fungoides and Sézary syndrome); testicular cancer; pediatric testicular cancer; laryngeal cancer (e.g., nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer); thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; endometrial uterine cancer; uterine sarcoma; vaginal cancer; pediatric vaginal cancer; hemangioma; vulvar cancer; and Wilms tumor and other pediatric kidney tumors are included.;

[0270] Metastases of the aforementioned cancers can also be treated according to the methods described herein. In some embodiments, the cancer is metastatic cancer.

[0271] In some embodiments, the cancer is selected from lung cancer, breast cancer, Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary central nervous system lymphoma, chronic lymphocytic leukemia, epithelial ovarian cancer, prostate cancer, squamous cell carcinoma, non-melanoma skin cancer, nasal polyp, basal cell carcinoma, keratinocyte carcinoma, multiple myeloma, serous invasive ovarian cancer, hepatocellular carcinoma, small cell lung carcinoma, adenocarcinoma, lung adenocarcinoma, non-small cell lung cancer, ovarian cancer or colorectal cancer.

[0272] In some embodiments, the treatment is: a) inhibiting cancer cell growth, proliferation, metastasis, invasion, or migration, or a combination of the foregoing; b) promoting cancer cell death; c) inducing autophagy in cancer cells, or a combination of the foregoing is.

[0273] In some embodiments, the effective amount is: a) inhibiting cancer cell growth, proliferation, metastasis, invasion, or migration, or a combination of the foregoing; b) promoting cancer cell death; c) inducing autophagy in cancer cells, d) or a combination of the foregoing is sufficient to effect.

[0274] In some embodiments, the effective amount is sufficient to reduce cancer (e.g., tumor) growth, proliferation, metastasis, invasion, migration, autophagy, or a combination of the foregoing. In certain embodiments, the reduction is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, the reduction is about 10 - 99%, e.g., about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0275] In some embodiments, the effective amount is sufficient to reduce cancer cell proliferation or tumor growth in a subject. In some embodiments, the reduction in cancer cell proliferation or tumor growth is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the reduction in cancer cell proliferation or tumor growth is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75% or 70 to 75%.

[0276] In some embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease) tumor autophagy, for example, by increasing at least one tumor inhibitory function of autophagy and / or decreasing at least one tumor promoting function of autophagy.

[0277] In some embodiments, the effective amount is sufficient to increase cancer autophagy. In certain embodiments, the increase is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0278] In some embodiments, the effective amount is sufficient to reduce autophagy in cancer. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10-99%, such as about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0279] In some embodiments, the effective amount is sufficient to prevent death of the subject and thereby reduce cancer (e.g., tumor) mortality. In certain embodiments, the reduction in cancer (e.g., tumor) mortality is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction in cancer (e.g., tumor) mortality is about 10-99%, such as about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0280] In some embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease) the expression of a target protein in cancer (e.g., tumor) cells.

[0281] In some embodiments, an effective amount is sufficient, for example, to inhibit cancer growth, reduce cancer malignancy, inhibit cancer metastasis, promote remission, modulate (increase and / or decrease) an immune-mediated response associated with cancer, or to modulate the body's response to cancer by any of the foregoing combinations.

[0282] Immuno - Oncology In some embodiments, the present disclosure provides treatments that harness the immune system. In some embodiments, the methods relate to immuno - oncology (e.g., cancer immunotherapy). In still further embodiments, the immune system is the innate immune system. In some embodiments, the immune system is the adaptive immune system. In still further embodiments, the treatment relates to humoral immunity or antibody - mediated immunity. In some embodiments, the treatment relates to cell - mediated immunity such as cancer. In some embodiments, the methods relate to treatment at the time of or prior to initial disease progression, while in other embodiments, the methods relate to treatment or prevention of late - stage disease progression at the time of or during late - stage disease progression. In still further embodiments, the immuno - oncology effect results from stimulation of the immune system.

[0283] In some embodiments, an effective amount is sufficient to modulate (e.g., increase) the immune system of a subject against cancer. In certain embodiments, the effective amount is the following: a) Immune cell - related readouts, immune cell activation, degranulation, maturation, migration, polarization, proliferation, and recruitment of immune cells (e.g., macrophages, monocytes, or dendritic cells); b) Lymph node activation, differentiation, egress, homing; c) Cytokine production; d) Antibody - dependent cell - mediated cytotoxicity (ADCC) and / or antibody - dependent cell - mediated phagocytosis (ADCP); e) Antigen presentation; f) Target protein expression, g) Or any combination of the foregoing is sufficient to modulate (e.g., increase or decrease) it.

[0284] In some embodiments, the adjustment is an increase. In certain embodiments, the increase is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0285] In some embodiments, the modulation is a decrease. In certain embodiments, the decrease is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the decrease is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0286] In some embodiments (e.g., immuno-oncology specific therapies), an effective amount is sufficient to modulate (e.g., increase or decrease) immune cell-related readouts, migration of immune cells (e.g., antigen presenting cells (e.g., dendritic cells and / or macrophages) and / or T cells), proliferation of immune cells, recruitment of immune cells (e.g., antigen presenting cells (e.g., dendritic cells and / or macrophages), monocytes, T cells and / or B cells), lymph node homing of immune cells (e.g., dendritic cells and / or T cells), lymph node egress of immune cells (e.g., dendritic cells and / or T cells), differentiation of immune cells, activation of immune cells, polarization of immune cells, cytokine production (e.g., increasing pro-inflammatory cytokines, decreasing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, decreasing anti-inflammatory cytokines), degranulation of immune cells, maturation of immune cells, ADCC of immune cells, ADCP of immune cells, antigen presentation, tumor homing of immune cells (e.g., T cells); increased tumor egress of immune cells (e.g., regulatory T cells), decreased tumor egress of immune cells (e.g., CD8+ T cells), target protein expression, or combinations of the foregoing.

[0287] Aging The methods described herein are applicable to the treatment of aging, aging-related conditions, and / or aging-related disorders or diseases. In some embodiments, the methods described herein include the treatment of overall health, body temperature, body weight, height, abdominal circumference, reproductive capacity, body fat, heart rate, blood pressure, pulse rate, blood oxygen level, respiratory rate, respiratory pattern, blood glucose level, blood pH, cardiac output, heart rhythm, and / or the concentration of certain substances in the blood. In some embodiments, the treatment can be evaluated by measurement of complete blood count, red blood cells, white blood cells, platelets, hemoglobin, hematocrit, mean corpuscular volume, basic metabolic panel, blood glucose, calcium, electrolyte panel, kidney function, blood enzyme tests, troponin, creatine kinase, lipoprotein panel, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, coagulation panel, and / or bone marrow examination.

[0288] In some embodiments, the effective amount is sufficient to delay aging. In certain embodiments, said delay is at least about 10%, for example, at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, said delay is about 10 - 99%, for example, about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0289] aging In some embodiments, the methods disclosed herein are for the treatment of aging. In still further embodiments, the methods disclosed herein relate to the treatment of aging-related diseases or disorders. In some embodiments, diseases or disorders include, but are not limited to, diabetes, metabolic syndrome and obesity. In other embodiments, the disease or disorder is related to photosensitivity or photoaging. In still further embodiments, the disease or disorder may be selected from arthritis, Alzheimer's disease, asthma, blindness, cancer, chronic bronchitis, chronic kidney disease, chronic obstructive pulmonary disease, coronary heart disease, deep vein thrombosis, dementia, depression, diabetes, epilepsy, heart failure, hypercholesterolemia, hypertension, motor neuron disease, multiple sclerosis, osteoporosis, Paget's disease of bone, Parkinson's disease, shingles, and stroke.

[0290] In some embodiments, the effective amount is sufficient to reduce aging. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0291] Fibrosis In some embodiments, the methods of the present disclosure relate to the treatment of fibrosis. In some embodiments, the methods relate to the treatment of conditions associated with or resulting from fibrosis. In some embodiments, the fibrosis is pulmonary fibrosis, hepatic fibrosis, dermal fibrosis, renal fibrosis, pancreatic fibrosis, systemic sclerosis, cardiac fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, and / or macular degeneration.

[0292] In some embodiments, the effective amount is sufficient to reduce fibrosis. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0293] Infectious disease In some embodiments, the present disclosure relates to the treatment of infectious diseases. In some aspects, the infectious disease is a bacterial infection, a protozoal infection, a viral infection, a fungal infection, or other pathogenic infection. In some embodiments, the infectious disease is AIDS or HIV, viral hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C), tuberculosis, salmonella, Lyme disease, meningococcal disease, influenza, measles, mumps, rubella (e.g., German measles), pneumonia, sexually transmitted infections (e.g., syphilis, chlamydia, gonorrhea), chronic rhinitis, whooping cough, pertussis, or a combination thereof.

[0294] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used in this specification are intended to have the meanings commonly understood by those skilled in the art related to this disclosure. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. It should be further understood that terms, such as those defined in commonly used dictionaries, should be construed to have meanings consistent with their meanings in the context of the relevant art and / or as otherwise defined in this specification.

[0295] The terms used in this specification are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0296] As used in this specification, the indefinite articles "a", "an", and "the" should be understood to include plural references unless the context clearly indicates otherwise.

[0297] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", mean, for example, including the stated integer or step or group of integers or steps but not excluding other integers or steps or group of integers or steps. As used in this specification, the term "comprising" can be replaced by the terms "containing" or "including".

[0298] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claims. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claims. Whenever used in the context of an aspect or embodiment of the present disclosure, the terms "comprising", "containing", "including", and "having" can all, in some embodiments, be replaced with "consisting of" or "consisting essentially of" to change the scope of the disclosure.

[0299] As used herein, the connecting term "and / or" between a plurality of recited elements is understood to encompass both the individual choices and the combined choices. For example, when two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any one of these options falls within the scope of meaning and is thus understood to meet the requirements of the term "and / or" as used herein. The simultaneous applicability of multiple options also falls within the scope of meaning and is thus understood to meet the requirements of the term "and / or".

[0300] When a list is presented, it should be understood that each individual element of the list, and all combinations of the list, are separate embodiments, unless otherwise stated. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

Examples

[0301] Illustration Example 1: Verification of a target protein as a circulating factor in human plasma This example demonstrates the ability to detect the target protein of the present disclosure in human plasma and verify that it is a circulating factor. In this example, plasma samples are obtained and an Agilent Multiple Affinity Removal Spin Cartridge is used for depletion of the top six most abundant proteins (albumin, IgG, IgA, antitrypsin, transferrin, and haptoglobin), followed by immunodepletion (ProteoPrep20 Plasma Immunodepletion Kit) of the 20 most abundant proteins (albumin, IgG, IgA, IgM, IgD, transferrin, fibrinogen, α2-macroglobulin, α1-antitrypsin, haptoglobin, α1-acid glycoprotein, ceruloplasmin, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, complement C1q, complement C3, complement C4, plasminogen, and prealbumin). The samples are depleted, digested, and measured in triplicate.

[0302] Sample preparation is performed as described (Geyer, et al., Cell Syst 2:185-195, 2016) using automated settings on an Agilent Bravo liquid handling platform. Plasma samples are diluted 1:10 in ddH 2 O and 10 μl of the sample is mixed with 10 μl of 2X concentrated SDC buffer. Reduction and alkylation are performed at 95 °C for 10 minutes. Trypsin and LysC (1:100 μg enzyme to microgram of protein ratio) are added to the mixture after a 5-minute cooling step at room temperature. Digestion is performed at 37 °C for 1 hour. The digest is acidified by adding 40 μl of 1% trifluoroacetic acid (TFA) in isopropanol. 20 μg amounts of peptides are loaded onto two 14-gauge StageTip plugs, followed by addition of 100 μl of 1% trifluoroacetic acid (TFA) in isopropanol and mixing vigorously. The StageTip is centrifuged at 1,500 × g using a 3D-printed in-house StageTip centrifuge device. The StageTip is washed twice with 100 μl of 1% trifluoroacetic acid (TFA) in isopropanol and ddH 2After washing once with 100 μl of 0.2% TFA in water, the purified peptide is eluted into an autosampler vial with 60 μl of elution buffer. The collected material is completely dried at 60 °C using a SpeedVac centrifuge (Eppendorf, Concentrator plus).

[0303] Samples are measured using an LC-MS instrument consisting of an EASY-nLC 1000 ultra-high pressure system (Thermo Fisher Scientific) combined with a Q Exactive HF Orbitrap (Thermo Fisher Scientific) and a nanoelectrospray ion source (Thermo Fisher Scientific). MS data are acquired using the Top15 data-dependent MS / MS scan method (topN method). The target value for the full-scan MS spectrum is 3 × 10 6 charges in the 300–1,650 m / z range, the maximum injection time is 55 ms, and the resolution at m / z 200 is 60,000. The proteins identified here are verified to be circulating in plasma.

[0304] Example 2: Validation of the target protein as a circulating factor (i.e., scRNAseq hashing) This example demonstrates the ability of the disclosed target proteins to act as circulating factors by single cell RNA sequencing and to look for shifts in cell populations after treatment with the target proteins. In this example, the target proteins discovered in the above examples are ordered through GenScript Biotech (Piscataway, NJ). The target proteins (e.g., those listed in the sequence listing and Table A) are synthesized by solid-phase peptide synthesis (SPPS) using fluorenylmethyloxycarbonyl (Fmoc) protecting group chemistry. Human primary blood mononuclear cells are seeded at 1 × 10 6 PBMC per well in each well of a 12-well plate containing 1 ml of RPMI-1640 (10% FBS). The PBMC are treated with 1 μg / mL of LPS (Sigma-Aldrich) for 24 h, with or without the target protein at a final concentration of 10 μM, or left untreated.

[0305] Collect the single cell suspension and centrifuge at 400×g for 5 minutes at 4°C. Discard the medium and resuspend the cells in 1 mL of cell staining buffer (BioLegend 420201). Add 5 μL of Human TruStain FcX (BioLegend 422301) to the cells and incubate at 4°C for 10 minutes. Add 1 μg of single cell hashing antibody (BioLegend TotalSeq-B#1~#10) to the cells together with approximately 50 μL of cell staining buffer to make the total volume 100 μL. Incubate this at 4°C for 30 minutes and then wash 3 times at 400×g. Pool 10 different cell hashing samples into one tube containing the desired number of cells (about 1000 cells / μL).

[0306] Process the single cells through the Chromium Next GEM Single Cell 3’_Reagent Kit (Dual Index) (10X Genomics CG000317 Rev C) according to the protocol provided by 10X Genomics. Briefly, process the sample through GEM generation and barcoding, GEM-RT cleanup and cDNA amplification, followed by 3’ gene expression library construction, cell surface protein library construction, and finally sequencing.

[0307] After sequencing, align the reads to the human reference genome (GENCODE34 / GRCH38). The reads are demultiplexed using DNA barcoded antibodies and quality control is performed (e.g., doublets are removed, singlet cells are selected with less than 15% mitochondrial contamination at the RNA level, and reads contain more than 500 genes). The raw data is normalized. Principal component analysis is performed and cell clusters are annotated. Differential gene expression analysis is performed between treatment with the control and the target protein. Shifts in cell population dynamics observed by principal component analysis and / or shifts in gene expression after treatment with the target protein in the supernatant demonstrate that the target protein acts as a circulating factor.

[0308] Example 3. Verification of target protein as a circulating factor in PBMCs treated with ORF (i.e., scRNAseq hashing) single-cell RNA sequencing. In other aspects, this example demonstrates the verification of target protein, SEQ ID NO: 38427, as a modulator of circulating factors. SEQ ID NO: 38427 is a novel secreted peptide that inhibits the innate immune response induced by other circulating factors, toll-like receptor agonists. Thus, SEQ ID NO: 38427 is suitable for the treatment of diseases caused by pathological innate immune responses such as lupus, multiple sclerosis, and rheumatoid arthritis.

[0309] To determine the effect of the target protein on TLR activity, the target protein, an irrelevant protein, and a control were applied to a commercially available reporter cell line that monitors two major signaling pathways in response to TLR activation, with or without an established TLR agonist. To determine which immune cells are most responsive to the target protein, the target protein was applied to peripheral blood mononuclear cells (PBMCs), and then, after 24 hours, the PBMCs were subjected to single-cell RNA-seq using cell hashing.

[0310] Methods and materials: Dual reporter assay: From the example: NF-kB-SEAP and IRF-Lucia luciferase reporter monocyte (THP1) cells (InvivoGen) are cultured in RPMI 1640 medium supplemented with HEPES buffer (10 mM), sodium pyruvate (1 mM), glucose (4.5 g / L), fetal bovine serum (10%), penicillin (100 U / mL), streptomycin (100 μg / mL), and 2-mercaptoethanol (0.05 mM). THP1 cells are treated with 100 ng / mL LPS (Sigma-Aldrich) for 24 hours, with the target protein (final concentration 10 μM), or left untreated.

[0311] After treatment, THP1 cells are cultured for 24 hours, and then the reporter activity is assayed. When using QUANTI-Blue (InvivoGen), a SEAP detection reagent, and QUANTI-Luc (InvivoGen), a luciferase detection reagent, both reporter proteins can be measured in the cell culture supernatant. QUANTI-Blue is a colorimetric enzyme assay developed to determine any alkaline phosphatase activity in biological samples such as cell culture supernatants. QUANTI-Luc is a lyophilized assay reagent containing all the components necessary to quantitatively measure the activity of other coelenterazine-utilizing luciferases in addition to Lucia Luciferase. An increase in reporter activity after treatment with the target protein is observed in cells where the target protein is immunostimulatory. Furthermore, a decrease in reporter activity after treatment with the target protein is observed in cells where the target protein is immunosuppressive.

[0312] scRNA-seq: The shift of the cell population after treatment with the target protein is evaluated by single-cell RNA-seq (scRNA-seq). The target proteins (e.g., those listed in Table 1) are synthesized by solid-phase peptide synthesis (SPPS) using fluorenylmethyloxycarbonyl (Fmoc) protecting group chemistry. Human primary blood mononuclear cells are seeded at 1×10 PBMC per well of a 12-well plate containing 1 mL of RPMI-1640 (10% FBS). The PBMCs are treated with 1 μg / mL of LPS (Sigma-Aldrich) for 24 hours with or without the target protein at a final concentration of 10 μM, or left untreated. The single-cell suspension is collected and centrifuged at 400×g for 5 minutes at 4°C. The medium is discarded and the cells are resuspended in 1 mL of cell staining buffer (BioLegend). 5 μL of Human TruStain FcX (BioLegend) is added to the cells and incubated at 4°C for 10 minutes. 1 μg of single-cell hashing antibody (BioLegend) is added to the cells together with approximately 50 μL of cell staining buffer to make a total volume of 100 μL. This is incubated at 4°C for 30 minutes and then washed 3 times at 400×g. 10 different cell hashing samples are pooled into one tube containing the desired number of cells (about 1000 cells / μL).

[0313] Single cells are processed through the Chromium Next GEM Single Cell 3’_Reagent Kit (Dual Index) (10X Genomics CG000317 Rev C) according to the protocol provided by 10X Genomics. Briefly, the samples are processed by GEM generation and barcoding, GEM-RT cleanup and cDNA amplification, followed by 3’ gene expression library construction, cell surface protein library construction, and finally sequencing.

[0314] After sequencing, reads are aligned to the human reference genome (GENCODE34 / GRCH38). Reads are demultiplexed using DNA barcoded antibodies and are run through quality control (e.g., doublets are removed, singlet cells are selected with less than 15% mitochondrial contamination at the RNA level, and reads contain more than 500 genes). Raw data are normalized. Principal component analysis is performed and cell clusters are annotated. Differential gene expression analysis is performed between treatment with control and target protein. Shifts in cell population dynamics observed by principal component analysis and / or shifts in gene expression after treatment with the target protein in the supernatant demonstrate that the target protein acts as a circulating factor.

[0315] SEQ ID NO: 38427 significantly blocks the IRF signaling pathway response from several TLRs. Upregulation of the IRF pathway is associated with many autoimmune diseases, particularly lupus. Consistent with its association with innate immune-related diseases such as lupus, SEQ ID NO: 38427 strongly affected activated monocytes and dendritic cells while having a relatively small effect on T cells, B cells, and NK cells (Figure 1, Figure 2, Figure 3).

[0316] [Table 1]

[0317] Example 4: Verification of the target protein as a circulating factor by morphological profiling. This example demonstrates the ability of the target protein of the present disclosure to act as a circulating factor (or secreted protein) through phenotypic screening against adipocytes. In this example, quantitative data is extracted from microscopic images of cells treated with the target protein of interest in the supernatant, and biologically relevant similarities and differences between samples are identified based on these profiles. This assay uses Cell Painting, a morphological profiling assay that multiplexes six fluorescent dyes imaged in five channels to reveal broadly relevant cellular components or organelles (Bray et al., Nat. Protoc. 2016 Sep;11(9):1757-1774.).

[0318] Briefly, human primary adipose-derived mesenchymal stem cells (AMSCs) are seeded into 96-well CellCarrier plates (Perkinelmer number 6005550). The AMSCs are differentiated for 14 days in the presence or absence of the target protein, and high-content imaging is performed on days 0, 3, 8, and 14 of adipogenic differentiation. On each day of the assay, the cell culture medium is removed, and each well is exchanged with 0.5 μM Mitotracker staining solution (1 mM MitoTracker Deep Red stock diluted in culture medium (Invitrogen #M22426)), followed by incubation for 30 minutes at 37 °C in the dark. After 30 minutes, the Mitotracker staining solution is removed, and the cells are washed twice with Dulbecco's phosphate-buffered saline (1X), DPBS (Corning® #21-030-CV), and 2.9 μM BODIPY staining solution (3.8 mM BODIPY 505 / 515 stock diluted in DPBS (Thermofisher #D3921) is added, followed by incubation for 15 minutes at 37 °C protected from light.

[0319] Subsequently, 16% paraformaldehyde without methanol, PFA (Electron Microscopy Sciences #15710-S), was directly added to the BODIPY staining solution to a final concentration of 3.2% to fix the cells, protected from light, and incubated at room temperature (RT) for 20 minutes. The PFA was removed, and the cells were washed once with Hank’s Balanced Salt Solution (1×), HBSS (Gibco #14025076). To permeabilize the cells, 0.1% Triton® X-100 (Sigma Aldrich #X100) was added, protected from light, and incubated at room temperature for 10 minutes. After permeabilization, a multi-staining solution (10 units of Alexa Fluor® 568 Phalloidin (ThermoFisher number A12380) diluted in HBSS, 0.01 mg / ml of Hoechst 33342 (Invitrogen #H3570), 0.0015 mg / ml of Wheat Germ Agglutinin, Alexa Fluor® 555 Conjugate (ThermoFisher #W32464), 3 μM of SYTO® 14 Green Fluorescent Nucleic Acid Stain (Invitrogen #S7576)) was added, and the cells were incubated at RT for 10 minutes, protected from light. Finally, the staining solution was removed, and the cells were washed three times with HBSS. The cells were imaged using an Opera Phenix high-content screening system using a confocal, 20× objective lens. For each well, 25 fields of view were imaged.

[0320] Generate morphological profiles at days 0, 3, 8, and 14 using LipocyteProfiler to verify differentiation at both depots by upregulation of adipogenic marker genes (LIPE, PPARG, PLIN1, GLUT4)). Simultaneously, use RNA sequencing to profile the transcriptome at the same time points of differentiation.

[0321] Example 5: Verification of the target protein as a circulating factor in which the target protein is mutated in a disease This example demonstrates the validation of the target protein of the present disclosure as a circulating factor containing mutations discovered by Genome Wide Association Study (GWAS) related to Th17 autoimmune diseases.

[0322] Synthesize circulating factors, target proteins, and mutant target proteins, and apply them to the medium of activated primary T cells (PHA blasts) containing or not containing TH17 cytokines, Th1 cytokines, and TH2 cytokines; IL-23 (Th17), IL-12 (Th1), and IL-6 (Th2). Evaluate the effects of the target protein and the mutant target protein on the activation states of STAT3, STAT1, and NF-kB.

[0323] Materials and Methods: Circulating proteins: Mutant and wild-type target proteins are synthesized by GenScript Inc. and purified in an LPS-free solution. IL-23, IL-12, and IL-6 are purchased from Abcam or Cell Signaling Technology.

[0324] Generation of PHA blasts: Fresh or frozen primary human PBMCs are placed in PBMax karyotyping medium, and 10 ng / ml of rhIL-2 is added for 3 - 4 days.

[0325] STAT3, STAT1, and NF-kB activation assays: PHA blast cells are placed in 80 μl of a 96-well plate at approximately 10 million cells / ml. The cells are serum-starved for 3 - 4 hours, and then IL-23, IL-12, or IL-6 is added for 30 minutes. The cells are harvested, and the cell extracts are assayed by phosphorylated-STAT3, STAT1, or NF-kB ELISA.

[0326] In summary, these experiments demonstrate that the target protein containing GWAS alleles related to autoimmune diseases has a more significant activation effect on STAT3 rather than STAT1 or NF-kB, strongly suggesting that the target protein is a potential target for the improvement of autoimmune diseases.

[0327] Example 6. Verification of Secretory Target Proteins that Stimulate or Inhibit Cytokine Release. A CBA (Cytometry) experiment was conducted to examine pro-inflammatory cytokines produced by PBMCs treated with secretory peptides. This example demonstrates the verification of a target protein, SEQ ID NO: 72416, as a modulator of circulating factors including cytokines. Such modulators are suitable for the treatment of autoimmune diseases such as psoriasis, arthritis, and multiple sclerosis.

[0328] Peripheral blood mononuclear cells were seeded for 24 - 48 hours in the presence or absence of the T cell activator CD3 / CD8 and in the presence or absence of the peptide. Cytokines were measured from the supernatant after incubation with a CBA Cytometer.

[0329] Methods and Materials: PBMC Thawing: PBMCs were thawed by preheating TexMACS medium (Miltenyi Biotech) in a conical 20 mL aliquot of 1 - 50 mL per donor. After warming the medium to 37°C, the cells were removed from liquid nitrogen and transferred to a tissue culture hood. For each donor, 1 mL of medium from the warmed aliquot was added to the cryovial and mixed up and down. Then, the medium + thawed cells were returned to a 50 mL conical. This was repeated until all cells were thawed and added to the 50 mL tube. Continue with other donors if used. The cells were spun at 400 x g for 5 minutes. The supernatant was aspirated and the cells were resuspended at approximately 10 × 10^6 cells / mL (refer to vial for cell count).

[0330] PBMC Seeding: Cryopreserved PBMCs from normal, healthy human volunteers (StemCell Technologies) were thawed and seeded at 250,000 cells / well. After thawing the PBMCs and resuspending them at 10×10^6 cells / mL, the cells were counted (Countess III, Thermo Scientific) and adjusted to 10×10^6 cells / well based on the count. The number of PBMCs determined by the number of wells for the experiment was taken. For example, 10 wells would require a total of 2.5×10^6 cells or 0.250 mL. The cells required for the experiment were then transferred to another conical tube. Warmed TexMACS medium was added such that the final concentration was 250,000 cells / 190 μL of medium. For example, for 10 wells, the total volume of medium (190 μL×10 wells) minus 0.250 mL of cells (1.9 mL - 0.250 mL), or 1.65 mL of medium needed to be added to 0.250 mL of cells. 190 μL of cell + medium was added to a 96-well U-bottom plate. It was placed in the incubator until dilution was carried out and it was ready.

[0331] Peptide Dilution: Ideally, all peptides should have a starting concentration of at least 10 mM, especially for peptides in DMSO (ATCC) to reach a final concentration of 1 μM (in H2O) or 0.5 μM (in DMSO). Dilution of all test substances should be done at 20-fold the final concentration. For example, a final concentration of 1 μM peptide should be diluted at 20 μM.

[0332] Reagent Dilution: Dilution sufficient to make 10 μL / well in triplicate (30 μL at 20-fold) was done with TexMACS medium. The activator - CD3 / CD28 (TransAct; Miltenyi Biotech) or cytokine was at 21-fold and was added after a 45-minute incubation with the peptide. The relevant controls - positive: TransAct + cells, negative: vehicle + cells must be included. Dilution should also be done in an ultra-low binding plate (Corning).

[0333] Assay Procedure: Peptide Treatment: As described above, start the assay by thawing the cells and seeding them in a 96-well U-bottom tissue culture plate (Corning). Place the cell plate in a tissue culture incubator (5% CO2, 37°C). While the cells are in the incubator, start the preparation of the dilution (optimally within 1 hour or less) as described above. Remove the cell plate from the incubator and add 10 μL of the 20-fold dilution to the relevant wells. After the addition of the inhibitor, place the cell plate in the tissue culture incubator for 45 minutes. Remove the plate from the incubator and add 10 μL of 21-fold TransAct to the relevant wells*. Then, return the plate to the incubator and incubate for 24 hours. After 24 hours, rotate the plate at 400 x g for 5 minutes.

[0334] *Activation: This assay can be performed in two different formats - activation and inhibition. The procedure is the same except that TransAct is not required in the peptide wells. TransAct is only required in the control wells. The purpose of this procedure is to determine the possible response from the peptide alone.

[0335] Cytometric Bead Array (CBA): After rotation, transfer the supernatant (approx. 150 μL) to a new ultra-low binding U-bottom plate. Then, freeze the supernatant at -20°C until thawed for CBA (BD) to determine the levels of human IL-2, TNFα, IL-1b, IP-10, IL-10, and IFNg. CBA was performed according to the manufacturer's protocol.

[0336] SEQ ID NO: 72416 significantly blocks the release of the cytokine TNF-α from activated PBMC, but does not block the release of the other inflammatory cytokine IL-1b. The specific inhibition of TNF-α release reduces the inflammatory response (Figures 4 and 5).

[0337] Table of Hits: X = Hits with significant activation or inhibition of associated cytokine release. Inhibition occurs when the peptide is applied together with anti-CD3 / CD28 T cell activating factor. When the peptide is applied alone to PBMC, activation occurs (see Materials and Methods). Significance = P = 0.05

[0338]

Table 2

[0339] Example 7. This example demonstrates the validation of four genotypes (V, A, G, D) of SEQ ID NO: 24296 as novel peptides capable of increasing glucose uptake in adipocytes. SEQ ID NO: 24296 contains human genetic variants associated with metabolic diseases, namely type 2 diabetes. These peptides may be suitable for the treatment of metabolic diseases such as type 2 diabetes.

[0340] The effect of putative stimulators of glucose uptake is evaluated using adipocytes differentiated from primary adipocytes. Preadipocytes are differentiated into mature adipocytes in vitro, treated with + / - peptide, and glucose uptake capacity is measured by a glucose uptake glow assay 24 hours after treatment.

[0341] Materials and Methods: Preadipocytes were seeded at a density of 10K / well in an opaque 96-well plate and differentiated using a standard adipogenic differentiation cocktail. Upon final differentiation, the adipocytes were serum-starved and treated with 0.5 uM peptide, 5 uM peptide, or a null control (DMSO) and incubated in a 37°C incubator with 5% CO2 for 24 hours. On the day of the assay, the medium was exchanged with 100 μl of DMEM without serum or glucose (Life Technologies, catalog number #11966) containing various insulin concentrations and incubated at 37°C in 5% CO2 for 1 hour. The medium was removed, 50 μl of 2DG (1 mM) in PBS was added, and the mixture was incubated at 25°C for 10 minutes. 2-Deoxyglucose (2DG) is transported into the cell and phosphorylated to produce 2-deoxyglucose-6-phosphate (2DG6P). 25 μl of stop buffer was added, and the plate was shaken briefly. 25 μl of neutralization buffer was added, and the plate was shaken briefly. 100 μl of 2DG6P detection reagent was added, the plate was shaken briefly, and the mixture was incubated at 25°C for 1 hour. The addition of the stop buffer stops 2DG transport, lyses the cells, destroys any intracellular NADPH, and inactivates the proteins. The addition of the neutralization buffer neutralizes the solution prior to the addition of the 2DG6P detection reagent. Glucose-6-phosphate dehydrogenase (G6PDH) in the reagent oxidizes 2DG6P to 6-phosphodeoxygluconic acid (6PDG) and reduces NADP+ to NADPH. Reductase uses NADPH to convert proluciferin to luciferin, which is then used by luciferase to generate light. Luminescence was recorded by integrating for 0.3 - 1 second with a luminometer.

[0342] As seen in Figure 6, the peptide of SEQ ID NO: 24296 was confirmed to increase glucose uptake in adipocytes at a concentration of 5 uM.

[0343] Example 8. This example demonstrates the ability to detect the target protein of the present disclosure in human plasma and verify that it is a circulating factor. Sample preparation is carried out as described in Keshishian et al Mol Cell Proteomics.2015 Sep;14(9):2375-93. A detailed description of the method is shown below.

[0344] Plasma depletion and enzymatic digestion. From 400 microliters of peripheral plasma from four patients collected at baseline and at 10, 60, and 240 minutes after alcohol removal, the 14 most abundant proteins were immunoaffinity depleted using IgY14 LC20 and Supermix LC10 columns (Sigma-Aldrich, St. Louis, MO), followed by depletion of approximately 50 moderately abundant proteins. Tandem depletion was performed on an Agilent 1100 HPLC (Agilent, Santa Clara, CA) system using dilution, stripping, and neutralization buffers provided according to the manufacturer and the manufacturer's instructions (Sigma-Aldrich). The flow-through of the Supermix column representing the depleted plasma was concentrated, and the buffer was exchanged to 50 mM ammonium bicarbonate at the original volume (400 μl) using an Amicon 3K concentrator (Millipore, Billerica, MA). The protein concentration of the depleted plasma was determined by the BCA protein assay (Thermo Fisher Scientific, Waltham, MA).

[0345] 400 microliters of IgY14 / Supermix-depleted peripheral plasma per time point and the patient were denatured with 6 M urea, reduced with 20 mM dithiothreitol at 37 °C for 30 minutes, and alkylated with 50 mM iodoacetamide in the dark at room temperature for 30 minutes. After diluting the urea concentration to 2 M with 50 mM ammonium bicarbonate, it was mixed on a shaker at 850 rpm for 2 hours at 30 °C with an enzyme ratio of 1:50 (w:w) to the substrate before Lys-C digestion (Wako, Richmond, VA). After further diluting the urea to less than 1 M, it was digested overnight with trypsin (Promega, Madison, WI) at an enzyme ratio of 1:50 (w:w) to the substrate at 37 °C while shaking at 850 rpm. The digestion was terminated with formic acid to a final concentration of 1%. The digest was desalted using an Oasis HLB 1 cc (30 mg) reversed-phase cartridge (Waters, Milford, MA) containing 0.1% formic acid and 0.1% formic acid / 80% acetonitrile as buffer A and B, respectively, using a vacuum manifold. The cartridge was conditioned with 3 × 500 μl of buffer B and then equilibrated with 4 × 500 μl of buffer A. After loading the digest at a reduced flow rate, it was washed with 3 × 750 μl of buffer A and eluted with 3 × 500 μl of buffer B. The eluate was frozen and dried by vacuum centrifugation. The digest was reconstituted in 400 μl of 0.1% formic acid, and the post-digestion concentration was determined by BCA. Based on the post-digestion concentration, an 80 μg aliquot was prepared, frozen, dried to dryness by vacuum centrifugation, and stored at -80 °C.

[0346] iTRAQ labeling of plasma samples. Eighty microgram dry aliquots of each PMI patient at four time points (baseline, 10, 60, and 240 minutes post-injury) were labeled with iTRAQ four-plex reagents according to the manufacturer's instructions (AB Sciex, Framingham, MA) for plasma labeling, which requires twice as much reagent for plasma than for other samples (cell lysates, tissues, etc.). Different iTRAQ channel layouts were used for different time points of four PMI patient samples to eliminate bias for any iTRAQ channel. After reconstituting the samples in 30 μl of 1 M triethylammonium bicarbonate (TEAB), 100 μl of ethanol was added to each sample. Pooled iTRAQ reagents from two vials were added to each sample, mixed, and incubated at room temperature for 1 hour. Three microliters of each sample was used to confirm label incorporation by LC-MS / MS and then the reaction was quenched. Once the labeling efficiency (label incorporation > 95%) was met, the reaction was quenched by adding Tris pH 8 at a final concentration of 100 mM and incubating at room temperature for 15 minutes. Labeled samples representing four different time points of PMI patients were mixed together, dried, and desalted using an Oasis HLB 1 cc (30 mg) reversed-phase cartridge as described above. The eluate was frozen, dried to dryness, and stored at -80 °C.

[0347] Fractionation of peptides by reversed-phase chromatography at high pH (basic pH RP). Digested iTRAQ-labeled plasma samples for each patient were reconstituted in 540 μl of 20 mM ammonium formate / 2% acetonitrile pH 10 and loaded onto a Zorbax 300 Extend 2.1×150 mm column (Agilent Technologies, Santa Clara, CA) and fractionated by basic reversed-phase chromatography on an Agilent 1100 Series HPLC apparatus at a flow rate of 200 μl / min. The mobile phase consisted of 20 mM ammonium formate / 2% acetonitrile pH 10 (buffer A) and 20 mM ammonium formate 90% acetonitrile pH 10 (buffer B). After loading 500 μl of sample (300 μg) onto the column, peptides were separated using the following gradient: isocratic hold at 0% B for 5 min, 0–15% solvent B in 8 min; 15–28.5% solvent B in 33 min; 28.5–34% solvent B in 5.5 min; 34–60% solvent B in 13 min, total gradient time was 64.5 min. Using a 96×2 ml well plate, fractions were collected every 0.6 min for a total of 84 fractions through the main elution profile of the fractionation. Additionally, the extreme initial and late portions of the gradient were collected in two further larger volume fractions. All fractions were acidified to a final concentration of 1% formic acid and then the internal 84 fractions were recombined by pooling the early, middle, and late fractions together to obtain a total of 28 fractions using a concatenation strategy. These 28 fractions, together with two further fractions corresponding to early and late eluting peptides, constructed a total of 30 fractions to be analyzed by LC-MS / MS. All fractions were dried down to dryness by vacuum centrifugation and stored at -80°C until mass spectrometry.

[0348] NanoLC-MS / MS analysis. For each plasma sample from individual patients, each of the 30 fractions was reconstituted with 16 μl of 5% formic acid / 3% acetonitrile, and 2 μl was analyzed by a Q Exactive mass spectrometer (Thermo Fisher Scientific) connected to an EASY-nLC 1000 UHPLC system (Thermo Fisher Scientific) equipped with a nanoflow ionization source (James A. Hill Instrument Services, Arlington, MA). Chromatography was performed on an ID 75 μm picolitre column (New Objective, Woburn, MA) in-house packed with Reprosil-Pur C18 AQ 1.9 μm beads (Dr. Maisch, GmbH, Entringen, Germany) to a length of 20 cm. To prevent overpressure of the column during UHPLC separation, the column was heated to 50 °C using a column heater sleeve (Phoenix-ST, Chester, PA). The LC system, column and platinum wire for delivering the electrospray power supply voltage were connected via a stainless steel cross (360 μm, IDEX Health & Science, UH-906x). The mobile phase consisted of 0.1% formic acid / 3% acetonitrile as solvent A and 0.1% formic acid / 90% acetonitrile as solvent B. Peptides were eluted at 200 nL / min with a gradient of 6 - 35% B for 150 min, 35 - 60% B for 8 min, 60 - 90% B for 3 min, held at 90% B for 10 min, 90% B - 50% B in 1 min, and then held at 50% B for 10 min under isocratic conditions. After a single Orbitrap MS scan of 300 - 1800 m / z at a resolution of 70,000 using an AGC set to 3e6, scans were performed at a resolution of 17,500 with a maximum of 12 ms / ms using an AGC set to 5e4. MS / MS spectra were collected with a normalized collision energy of 27 and an isolation width of 2.5 amu. Dynamic exclusion was set to 20 s and peptide matching was turned on.

[0349] For plasma samples pooled from multiple patients and used in iTRAQ / TMT comparisons, some of the above parameters were modified. The analysis was performed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) with an isolation width of 2.0 amu. For TMT-labeled peptides, the normalized collision energy was reduced to 26. For TMT-10-labeled peptides, MS / MS spectra were collected at a resolution of 35,000.

[0350] Processing of raw data for human proteins (ProFound Tx). The raw data was downloaded from ftp: / / MSV000079033:a@massive.ucsd.edu. We used the Spectromine search engine (Biognosys, version 3.2) to search for detected features in the raw MS files against the FL69ORF proteome database (updated on November 1, 2022; 800K protein entries). Only tryptic peptides at least 7 amino acids long and missing a maximum of two cleavages were considered. The initial tolerance for mass was set to 10 ppm at the MS level and 0.02 Da at the MS / MS level. We set N-terminal N-acetylation (42.010565 Da) and methionine oxidation (15.994915 Da) of proteins as variable modifications, and iTRAQ 4-plex labeling at the peptide N-terminus as a fixed modification (57.021464 Da). A 1% false discovery rate (FDR) was imposed for peptide spectral matches (PSMs) and protein identifications using the target-decoy approach. ITEAQ 4-plex quantification was performed using the default parameters of the Biognosys method (quantitative iTRAQ 4-plex).

[0351]

Table 3

[0352] Example 9. This example demonstrates the ability to detect the target protein of the present disclosure in human saliva and confirm that it is a circulating secretory factor. Sample preparation is performed as described in Grassl et al Genome Medicine volume 8, 44 (2016). A detailed description of the method is shown below.

[0353] Protein digestion and peptide purification. After collection, the swabs were transferred to Eppendorf tubes containing 200 μl of lysis buffer (1% sodium dodecyl carbonate (v / v), 10 mM tris(2-carboxyethyl)phosphine, 40 mM 2-chloroacetamide, 100 mM Tris buffer pH 8.5), squeezed well against the inner wall of the Eppendorf tubes, and removed. As estimated by the Bradford protein assay, more than 100 μg of protein was reproducibly recovered in this way. Sample preparation essentially followed the in-StageTip protocol. Briefly, 0.4 μg of trypsin and LysC were added to our lysis buffer, and a total of 20 μg of protein was digested by incubating at 37 °C for 60 minutes with shaking. After this short digestion, we acidified the peptides to a final concentration of 1% trifluoroacetic acid (TFA) and loaded them onto an SDB-RPS StageTip. The filter was then washed, and the peptides were finally eluted with 60 μl of 80% acetonitrile (ACN) (v / v) and 1% ammonium (v / v), dried in a SpeedVac concentrator, and resuspended in A* buffer (2% ACN (v / v), 0.1% TFA (v / v), pH 2) to a concentration of 1 g / l.

[0354] Single-run and preparative fraction liquid chromatography-MS measurements. To obtain a deep saliva proteome, eight wake samples were fractionated using basic reversed-phase chromatography prior to liquid chromatography (LC)-MS measurements. Approximately 15 μg of peptides were separated on a 20 cm, 75 μm inner diameter column in-house packed with ReproSil-Pur C18 beads (Dr. Maisch GmbH, Germany) with an 80-minute gradient. The concatenated fractions were dried with a SpeedVac concentrator and resuspended in buffer A to a concentration of 1 g / l. Both the fractionated samples and the single-run samples were subjected to a 100-minute chromatography gradient using an EASY-nLC 1000 ultrahigh-pressure system (Thermo Fisher Scientific) and an in-house-made 40 cm column of the type described above. Chromatography was coupled online to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) by applying a spray voltage of 2.2 kV. The MS scan resolution was set to 120,000 at m / z 200, the scan range was 300 - 1650 m / z, and the maximum injection time was 55 ms. Fifteen of the most intense ions per MS scan were selected for high-energy collision dissociation (HCD) fragmentation with a separation width of 1.5 m / z and measured at a resolution of 30,000. Dynamic exclusion was used with an exclusion time of 30 seconds.

[0355] Processing of raw data of human proteins (ProFound Tx). The raw data was downloaded from https: / / www.ebi.ac.uk / pride / archive / projects / PXD003028. The inventors used the Spectromine search engine (Biognosys, version 3.2) to search for detected features in the raw MS files against the FL69ORF proteome database (updated on November 1, 2022; 800K protein entries). Only tryptic peptides that were at least 7 amino acids long and missed at most 2 cleavages were considered. The initial tolerance for mass was set to 10 ppm at the MS level and 0.02 Da at the MS / MS level. The inventors set N-acetylation (42.010565 Da) at the N-terminus of the protein and oxidation of methionine (15.994915 Da) as variable modifications, and carbamidomethylation of cysteine (57.021464 Da) as a fixed modification. A 1% false discovery rate (FDR) was imposed for peptide spectrum matches (PSMs) and protein identifications using the target-decoy approach. Relative quantification was performed using the default parameters of the Biognosys method.

[0356]

Table 4

[0357] Example 10. Verification of target proteins that regulate the cellular uptake of disease-related circulating factors. This example demonstrates the validation of SEQ ID NO: 26888, SEQ ID NO: 36277, and SEQ ID NO: 75353 as novel peptides that can increase LDL uptake in hepatocytes. All three peptides contain human genetic variants associated with metabolic diseases, i.e., SEQ ID NO: 26888 contains variants associated with cholesterol, LDL cholesterol, fat-free mass, fat mass, and BMI; SEQ ID NO: 36277 contains gene variants associated with HDL cholesterol and triglycerides; SEQ ID NO: 75353 contains variants associated with HDL cholesterol levels. These peptides may be suitable for both primary prevention (reduction of CVD risk in patients without known CVD) and secondary prevention (prevention of subsequent heart attacks, strokes, and other CVD events in patients with established CVD).

[0358] The effect of the putative cholesterol-lowering peptide was evaluated using an LDL uptake assay in primary human hepatocytes derived from a pool of 10 healthy donors. Hepatocytes were treated with the peptide and LDL-labeled BODIPY for 4 hours. The cells were then fixed, permeabilized, and imaged on an Opera Phenix high-content imaging platform. LDL uptake was determined using the BODIPY staining intensity.

[0359] Materials and Methods: Human primary hepatocytes were seeded at a density of 37K / well in 96-well PhenoVue plates and allowed to recover overnight. Next, the hepatocytes were treated with 5 uM peptide or null control (DMSO) and BODIPY-labeled LDL (Invitrogen™ Image-iT™ Low Density Lipoprotein Uptake Kit, Bodipy FL, I34359). This assay was designed to specifically detect the binding and uptake of LDL via the LDL receptor internalization pathway and allows for maximum control and flexibility in experimental design. Two controls, unlabeled LDL and heparin, were incorporated into the system. Unlabeled LDL provides any pretreatment to block cell surface receptors prior to probing with the labeled construct, while heparin is provided as an additional control to chelate LDL in the solution phase outside the cell and thus prevent the binding and uptake of labeled LDL. Metformin, a type 2 diabetes drug with cholesterol-lowering effects, was used as a positive control for increased LDL uptake (Figure 1). After peptide, control, and LDL-BODIPY treatment, the cells were incubated in a 37°C incubator with 5% CO2 for 4 hours. After incubation, the cells were fixed with 4% paraformaldehyde for 15 minutes. The cells were then permeabilized and stained with DAPI to visualize the nuclei. The wells were then washed with HBSS and imaged at 20x magnification with 9 fields per well using a Perkin Elmer Opera Phenix high-content imaging platform (fluorescence). BODIPY spot intensity was quantified using Harmony image analysis software.

[0360] As seen in Figure 9, the peptides of SEQ ID NO: 26888, SEQ ID NO: 36277, and SEQ ID NO: 75353 were confirmed as increasing LDL uptake in hepatocytes at a concentration of 5 uM.

[0361] [Table 5]

[0362] Example 11. Discovery of Secreted Proteins Associated with Systemic Lupus Erythematosus (SLE) Disease. This example presents novel findings regarding the identification of multiple open reading frame (ORF) proteins such as SEQ ID NO: 75451, SEQ ID NO: 75452, SEQ ID NO: 75453, and SEQ ID NO: 74932, which exhibit differential expression patterns in plasma samples obtained from individuals with systemic lupus erythematosus (SLE) compared to those from healthy donors. The ORF proteins identified in this study hold significant potential as therapeutic agents or targets for the management of SLE.

[0363] This study aimed to perform deep plasma proteomics profiling of healthy and systemic lupus erythematosus (SLE) plasma samples using a highly optimized workflow. Low molecular weight proteins (LMWPs) were enriched by a combination of protein precipitation and sequential solubilization developed by ProFound Tx. The LMWP fraction was digested with a trypsin / Lys-C mixture. In the first test, two pooled samples were prepared, one from 5 healthy donors and the other from 5 SLE patients. Peptides from these two samples were fractionated using online reversed-phase liquid chromatography (RPLC) and analyzed using timsTOF Pro 2 MS combined with EvoSep nanoLC using data-independent acquisition (DIA) method. The MS spectra were searched against the FL69ORF_DB database containing approximately 800K ORF entries based on RiboSeq studies in ProFound Tx. The newly discovered ORF proteins were classified by ORF types including annotated, ncRNA, polycistronic, and without transcript. Quantitative analysis was performed using the combined peptide intensities from each protein, and differentially expressed proteins between SLE and healthy samples were identified based on a threshold of at least 2-fold change in plasma samples.

[0364] Materials and Methods: Sample collection: Plasma samples were collected from SLE patients according to standard procedures. Briefly, approximately 2 mL of whole blood samples from each patient were collected into EDTA-coated collection tubes. The tubes were gently inverted 8 - 10 times, and the tubes were placed upright on ice for 5 minutes to sediment the blood cells. The blood samples were centrifuged at 1000 x g (RCF) for 10 minutes. To prevent proteolysis, the upper plasma layer (approximately 1 mL) was isolated into a new sample tube containing 10 μL of a protease inhibitor cocktail (Thermo). For each SLE patient, healthy donors with matching age, ethnic group, and gender were selected. Plasma samples were collected using the same protocol. After on-site sample collection, the plasma samples were stored at -80 °C for proteomic analysis.

[0365] Sample preparation: Protein precipitation and differential solubilization (PP + DS): An aliquot of 50 μL of plasma sample was diluted 1:2 (v / v) with 100 μL of denaturing solution (8 M urea) and heat-denatured at 70 °C for 3 minutes. An additional 50 μL of water was added, and then it was slowly dripped into 1800 μL of ice-cold acetone, immediately stirred at -20 °C for 2 hours, and subsequently centrifuged at 19,000 g at 4 °C for 15 minutes. The precipitate was dissolved in 300 μL of 80% ACN containing 12 mM HCl and sonicated for 10 seconds at 40% power of the probe. Then, the sample was mixed overnight at 4 °C. The sample was centrifuged again at 19,000 g at 4 °C for 15 minutes. Low molecular weight proteins (LMWP) were extracted from the supernatant. The sample was dried in a SpeedVac to complete drying, and the sample was stored at 4 °C for subsequent use.

[0366] Protein denaturation, reduction, and alkylation: Add 1 / 25 volume of 25x volume of TCEP stock solution and 1 / 10 volume of 10x CAA stock solution to each sample vial. Heat the sample to 37 °C and incubate the sample for 1 hour to reduce and alkylate the proteins.

[0367] In-solution digestion: Denatured protein samples are diluted by adding 8 μL of digestion buffer such as 100 mM Tris-HCl (pH 8.0) in water. 2 μg of sequencing-grade trypsin / Lys-C mixture is added to each sample at an enzyme:protein ratio of 1:50. Samples are incubated overnight at 37 °C while mixing at 800 rpm on a tabletop thermomixer.

[0368] Basic reversed-phase (HpH) high-performance liquid chromatography (HPLC) fractionation: For HPLC fractionation, equal amounts (approx. 10 μg) of digested samples from 5 healthy or 5 SLE donors were pooled separately into two mixtures (representing healthy and SLE plasma digests). Samples were centrifuged at 16,000 × g for 10 minutes at room temperature. 50 μg of peptide mixture of each pooled peptide digest was fractionated into 48 fractions according to the following protocol. Briefly, approximately 50 μg of peptide mixture was loaded onto a Waters XBridge BEH130 C18 3.5 μm 3 mm ID × 150 mm Length column of an Agilent 1290 HPLC operating at 0.6 mL / min. Buffer A consisted of 0.1% ammonium hydroxide in water and buffer B consisted of 93% ACN and 7% water with 0.1% ammonium hydroxide. The same injection protocol and gradient were used for all fractionation experiments. All fractions were collected at 0.5-minute intervals using an Agilent fraction collector in a 96-deep well plate. Samples were first loaded onto the column at 0.8 mL / min for 10 minutes and then the fractionation gradient was started at 0.6 mL / min as follows: rising from 3% B to 45% B in 35 minutes, 60% B in 4 minutes, and 90% B in 2 minutes. Fraction collection was started 10 minutes after sample loading. After holding the gradient at 90% B for 5 minutes, it was returned to 3% B, then the column was washed and equilibrated. The total number of concatenated fractions was set to 96 throughout all experiments. For optimized results, the first 48 fractions were pooled in +49 well fractions (i.e., pool well A1+E1, A2+E2,...., B1+F1,..., D12+H12) to obtain the final 48 fractions. Before concatenation, ammonium hydroxide was evaporated in a SpeedVac operating at 40 °C.

[0369] NanoLC-MS analysis: Reconstitute the dried peptide fraction with 0.1% formic acid in water. Load 20% of each fraction (approx. 200 ng of digest) for each nanoLC injection onto an EvoTip. An IonOpticks column (Aurora ELITE, 75 um ID x 150 mm L) was used for peptide separation with a standard separation method 40 SPD from EvoSep. The EvoSep nanoLC coupled to a timTOF pro 2 MS (Bruker) was run in DIA mode for data acquisition.

[0370] Database search and data analysis: Using the Spectronaut search engine (Biognosys, version 17.1) and the directDIA search algorithm, search for the detected features in the raw MS files against the FL69ORF proteome database (updated November 2022; 800K protein entries). Consider only fully tryptic peptides that are at least 7 amino acids long and missed a maximum of 2 cleavages. The initial tolerance mass was set at 20 ppm at the MS level and 0.05 Da at the MS / MS level. The inventors set N-terminal N-acetylation (42.010565 Da) of proteins and oxidation of methionine (15.994915 Da) as variable modifications, and carbamidomethylation of cysteine (57.021464 Da) as a fixed modification. A 1% false discovery rate (FDR) was imposed for peptide spectral matches (PSMs) and protein identifications using the target-decoy approach. Label-free quantification was performed using the default parameters of the Biognosys quantification method (MS / MS-based peak integration and composite area). For each protein, the sum of the peak areas of all peptides identified from all fractions of healthy or SLE samples was combined. Differentially expressed proteins between SLE samples and healthy samples were identified based on a threshold of at least 2-fold change in plasma samples (i.e., the log2 fold change is greater than 1 for upregulation and less than -1 for downregulation).

[0371] Approximately 984 secreted proteins (or protein groups) were identified from the LMWP fraction of healthy or SLE plasma samples. After removal of the annotated proteins, 23 non-canonical proteins containing different types of ORFs were identified (11 polycistronic (2 dORFs, 9 others), 11 ncRNAs (1 lncRNA and 10 pseudogenes), and 1 without a transcript). Among these novel ORF proteins, 7 ORFs showed upregulated expression in SLE samples compared to healthy controls, and 3 ORF proteins showed downregulated expression. For example, SEQ ID NO: 75451 (dORF of SOD2), SEQ ID NO: 75452 (polycistronic ORF of CISH), and SEQ ID NO: 75453 (overlapping with the uORF of TMED2) showed higher expression levels in SLE plasma samples. These ORF proteins and the upregulated annotated proteins showed significantly enriched GO terms for interleukin-mediated adaptive immune response and neutrophil degranulation and signaling. Inhibition of these ORF targets may result in a weak immune response in SLE patients. The lncRNA of SEQ ID NO: 74932 (NIPBL-DT) was conserved in the protein sequence and was shown to be downregulated in SLE samples, which have been reported to be related to the immune response in mammals. SEQ ID NO: 74932 may potentially be used as a therapeutic agent for releasing SLE symptoms (Figure 10, Figures 11A and 11B).

[0372]

Table C

[0373] Example 12. Verification of two target proteins as novel G protein-coupled receptor (GPCR) ligands. This example demonstrates the validation of two novel peptides, SEQ ID NO: 49310 and SEQ ID NO: 42382, which act as GPCR ligands. SEQ ID NO: 49310 blocks CXCR4 and cancer cell migration, making it suitable for the treatment of multiple cancers. On the other hand, SEQ ID NO: 42382 agonizes C3AR1, an important modulator of immune response and inflammation. The target proteins of SEQ ID NO: 49310 and SEQ ID NO: 42382 were treated with gpcrMAX, an inclusive panel that encompasses 168 G protein-coupled receptors (GPCRs) from over 60 different receptor families. This panel utilizes the PathHunter® β-arrestin technology (Eurofins DiscoverX). The PathHunter β-arrestin GPCR cell lines are engineered to co-express a ProLink™ (PK)-tagged GPCR and an enzyme acceptor (EA)-tagged β-arrestin. Activation of GPCR-PK induces the recruitment of β-arrestin-EA, forcing the complementation of two β-galactosidase enzyme fragments (EA and PK). The resulting functional enzyme hydrolyzes the substrate to generate a chemiluminescent signal.

[0374] Materials and Methods: Cell treatment: PathHunter® cell lines were grown from freezer stocks according to standard procedures. Cells were seeded in white-wall 384-well microplates at a total volume of 20 μL and incubated at 37 °C for an appropriate time prior to testing.

[0375] Peptide Processing: The target protein sequence numbers 49310, 42382, and an irrelevant peptide were synthesized by solid-phase peptide synthesis (SPPS) using Fluorenylmethyloxycarbonyl (Fmoc) protecting group chemistry. For Screening: The target protein, sequence numbers 49310, 42382, and eight irrelevant peptides were processed on the gpcrMAX panel at a final top test concentration of 0.12 μM. For Hit Confirmation: The target protein sequence number 49310 and an irrelevant peptide were tested in a CXCR4 human chemokine GPCR cell-based antagonist arrestin assay at the highest test concentrations of 1 μM and 0.3 μM.

[0376] Assay Design: Agonist Format: For agonist determination, cells were incubated with the sample to induce a response. An intermediate dilution of the sample stock was performed to generate a 5× sample in assay buffer. 5 μL of the 5× sample was added to the cells and incubated at 37 °C or room temperature for 90 - 180 minutes. The vehicle concentration was 1%. Antagonist Format: For antagonist determination, cells were pre-incubated with the antagonist and subsequently challenged with the agonist at the EC80 concentration. An intermediate dilution of the sample stock was performed to generate a 5× sample in assay buffer. 5 μL of the 5× sample was added to the cells and incubated at 37 °C or room temperature for 30 minutes. The vehicle concentration was 1%. 5 μL of a 6× EC80 agonist in assay buffer was added to the cells and incubated at 37 °C or room temperature for 90 or 180 minutes.

[0377] CXCR4 Human Chemokine GPCR Cell-Based Antagonist Arrestin Assay: For hit confirmation, the PathHunter® β-arrestin cell line for the CXCR4 human chemokine GPCR antagonist assay was used with CXCL12 / SDF-1a as the activator and Plerixafor as the inhibitor.

[0378] C3aR Human Complement Peptide GPCR Cell-based Agonist Assay: For hit confirmation, the PathHunter® β-arrestin cell line for the C3AR1 human complement peptide GPCR agonist assay was used with a C3A receptor agonist (Short Fragment) as the control activator.

[0379] Signal Detection: The assay signal was generated by a single addition of 12.5 or 15 μL (50% v / v) of the PathHunter® Detection reagent cocktail, followed by a 1-hour incubation at room temperature. The microplate was read after signal generation using a PerkinElmer Envision® device for chemiluminescence signal detection.

[0380] Data Analysis: Compound activities were analyzed using the CBIS data analysis suite (ChemInnovation, C). For agonist mode assays, the percentage activity was calculated using the formula: % activity = 100% × (mean RLU of test sample - mean RLU of vehicle control) / (mean RLU of mean MAX control ligand - mean RLU of vehicle control). For antagonist mode assays, the % inhibition was calculated using the following formula: % inhibition = 100% × (1 - (mean RLU of test sample - mean RLU of vehicle control) / (mean RLU of EC80 control - mean RLU of vehicle control)).

[0381] Chemotaxis assay: NAMALWA cells (human Burkitt lymphoma cell line, ATCC) were cultured in serum-free ATCC-formulated RPMI for 24 hours before the assay. After starvation, the cells were harvested and pelleted by centrifugation at 1,000 × g for 5 minutes. The cells were resuspended in serum-free medium. Using Abcam's Cell Migration / Chemotaxis Assay Kit (96 well, 8 μm), 150 μL of serum-free medium containing the desired chemotactic agent was added to the bottom chamber. Then, 50,000 cells and the desired inhibitor (or peptide) were added to each well of the upper chamber. The plate was placed and incubated at 37 °C in a CO2 incubator for 24 hours. After incubation, 110 μL of Cell Dye + Cell Dissociation Solution mixture was added to each bottom well and incubated at 37 °C in a CO2 incubator for 1 hour. After incubation, the upper chamber was removed and the plate was read at Ex / Em = 530 / 590 nm.

[0382] Results: Two target peptides, SEQ ID NO: 49310 and SEQ ID NO: 42382, were identified as novel GPCR ligands using Eurofins' gpcrMAX panel. The target peptide of SEQ ID NO: 42382 is an agonist of C3AR1, an important anaphylatoxin receptor that plays an important role in inflammation. The target peptide, SEQ ID NO: 49310, blocked CXCR4, a chemokine receptor involved in cell migration and homing. Furthermore, since SEQ ID NO: 49310 was able to significantly inhibit the chemotactic migration of human Burkitt lymphoma cells, its potential as a chemokine for treating cancer was suggested (Figures 12, 13, 14, 15).

[0383] The teachings of all patents, published applications, and references cited herein are hereby incorporated by reference in their entirety.

[0384] While exemplary embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

[Claim 1] The invention described in the specification.

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