Compositions and methods for modulating circulating factors

Abstract

In various embodiments, the disclosure provides compositions, such as polypeptides, polynucleotides, gene editing systems, small molecules, vectors, or host cells, comprising and / or modulating the expression or activity of an immunomodulatory-related protein. In various embodiments, the disclosure also provides for the treatment of aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer, infections, immune diseases, and the like using agents comprising and / or modulating the expression or activity of an immunomodulatory-related protein. Methods of replacing an adaptation of therapy or combinations thereof with hormones, growth factors and / or proteins, as well as methods of identifying the agents.

Description

Materials Incorporated by Reference

[0001] 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. Incorporation of XML Materials by Reference

[0002] This application incorporates by reference the sequence listing contained in the following eXtensible Markup Language (XML) file filed simultaneously 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 homeostasis and metabolism of the body. The imbalance of these factors may be affected by genetic factors and diseases such as cancer or hormone imbalance. Therefore, endocrine circulating factors can be markers of disease states or biological / pre-disease phenotypes. Because the imbalance of endocrine circulating factors may affect the homeostasis of the body, such imbalance may cause wide-range symptoms, and affect growth, development, metabolism, sexual function and mood. Therefore, it is urgent to identify other novel endocrine circulating factors as biomarkers and therapeutic targets. Summary of the invention

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

[0005] On the one hand, the disclosure relates to a medicament comprising a target protein identified herein (e.g., a target protein listed in a sequence table or Table A, or a variant thereof) and / or regulating (e.g., increasing or decreasing) the expression and / or activity of the target protein. In some embodiments, the medicament comprises a target protein identified herein (e.g., a target protein listed in a sequence table or Table A, or a variant thereof). In certain embodiments, the medicament regulates (e.g., increases or decreases) the expression and / or activity of a target protein identified herein (e.g., a target protein listed in a sequence table or Table A, or a variant thereof). In some embodiments, the medicament comprises, is essentially composed of, or is composed of a polypeptide, a polynucleotide, a gene editing system, a small molecule, or a cell (e.g., a cell therapy). The medicament may be an inhibitor or activator of a target protein identified herein. In some embodiments, the medicament regulates the expression of a target protein identified herein. In some embodiments, the medicament regulates 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 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 that a subject suffers from the disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, wherein the expression or activity level of the target protein in the sample indicates the likelihood that the subject suffers from the disease or condition, wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infections, immune diseases (e.g., inflammatory and / or autoimmune diseases), indications for treatment with hormones, growth factors and / or protein replacement (e.g., enzyme replacement, antibody replacement), or a combination thereof.

[0010] In another aspect, the present disclosure provides a method of preparing a sample that can be used to determine the likelihood that a subject has a disease or condition, 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 that can be used to detect the possibility of suffering from cancer; and c) quantifying the expression or activity of the target protein in the sample prepared in step b), The disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infections, immune diseases (e.g., inflammatory and / or autoimmune diseases), indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

[0011] In some embodiments, the method further comprises treating a subject predicted to have a likelihood of developing the disease or condition, the treatment comprising administering to the subject an effective amount of an agent or a pharmaceutical composition comprising the agent, the agent comprising the target protein identified herein and / or modulating the expression or activity of the target protein.

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

[0013] In another aspect, the present disclosure provides a method for 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 from the subject, and selecting the subject suitable for treatment of the disease or condition based on the expression or activity level 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), infections, immune diseases (e.g., inflammatory and / or autoimmune diseases), indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

[0014] On the other hand, 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 thereof in a cell (e.g., a cancer cell, such as a cancer cell of a subject), the method comprising contacting the cell (e.g., in vitro, ex vivo, or in vivo) with an agent or a pharmaceutical composition comprising the agent, the agent comprising the target protein identified herein and / or modulating the expression or activity of the target protein.

[0015] In another aspect, the disclosure provides a method of identifying an agent that modulates the expression or activity of a target protein identified herein, the method comprising: a) contacting the target protein with an agent; and b) determining whether the agent modulates the expression or activity of the target protein, wherein a difference in the expression or activity of the target protein that has been contacted with the agent compared to a reference for the expression or activity of the target protein indicates that the agent modulates the expression or activity of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which 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 the embodiments.

[0017] Figure 1Shown is the inhibition of interferon regulatory factor (IRF) pathway by SEQ ID NO:38427 protein treatment in THP-1 dual reporter cells. In the presence of lipopolysaccharide (LPS), THP-1 dual reporter cells were incubated with increasing concentrations of protein (0, 2.5, 5, 7.5 and 10 μM) for 24 hours. Relative IRF activity was assessed by measuring reporter gene expression. Data are presented as the mean ± SD of 10 technical replicates. Asterisks indicate statistical significance (**p<0.01, ****p<0.0001) compared to cells treated with LPS alone.

[0018] Figure 2 Shown is the selective inhibition of IRF response by co-treatment of SEQ ID NO:38427 protein with specific TLR agonists in THP-1 dual reporter cells. In the presence of SEQ ID NO:38427 protein (10 μM) or scrambled protein, THP-1 dual reporter cells were treated with 10 different TLR agonists (TLR1 / 2, TLR2, TLR3, TLR4, TLR5, TLR6 / 2, TLR7, TLR8 and TLR9) for 24 hours. Relative IRF activity was assessed by measuring reporter gene expression. Data are presented as the mean ± SD of 10 technical replicates per treatment group from a representative experiment. Asterisks indicate statistical significance (****p<0.0001) compared to the corresponding TLR agonists in the case of cells treated with scrambled protein. The protein inhibited IRF responses when co-treated with TLR1 / 2, TLR4 and TLR6 / 2 agonists, whereas no significant effect was observed with TLR2, TLR3, TLR5, TLR7, TLR8 or TLR9 agonists.

[0019] Figure 3A shows a UMAP visualization of cluster analysis, which revealed 10 different PBMC cell subtypes; Figure 3B Shown is a bar graph demonstrating counts of up- and down-regulated genes when treated with SEQ ID NO: 38427 protein compared to a scrambled protein control.

[0020] Figure 4 Shown is a dose-dependent reduction of hTNFa on PBMCs activated by CD3 / CD28 (TransAct) when compared to control (TA_H2O). ***=p<0.0002 and ****=p<0.0001 compared to corresponding controls by two-way ANOVA.

[0021] Figure 5Shown is the lack of IL-1b inhibition on PBMCs activated by CD3 / CD28 (TransAct) when compared to control (TA_H2O). The positive control showed significant inhibition. ****=p<0.0001 compared to the corresponding control by two-way ANOVA.

[0022] Figure 6 A relative increase in glucose uptake in adipocytes is shown.

[0023] Figure 7 Representative results of novel ORF identification from plasma samples are shown - SEQ ID NO: 24651.

[0024] Figure 8 Representative results of novel ORF identification from saliva samples are shown - SEQ ID NO: 75388.

[0025] Fig. 9 Shown is the relative increase in LDL uptake in human primary hepatocytes.

[0026] Fig.10 Ranked bar graph of differentially secreted proteins in human plasma from SLE and healthy donors is shown. Differential ORF proteins in SLE compared to healthy plasma samples.

[0027] Fig.11A is a bar graph of enriched terms of proteins upregulated in SLE compared to healthy plasma samples (colored by p-value), and Fig. 11B Shown is a bar graph of enriched terms (colored by p-value) of proteins down-regulated in SLE compared to healthy plasma samples (via Metascape).

[0028] Fig.12 Bar graphs are shown showing agonism (presented as % activity) (upper graph) and antagonism (presented as % inhibition) (lower graph) of a pool of 9 peptides tested against 168 GPCR targets (including target proteins SEQ ID NO: 49310 and SEQ ID NO: 42382). % activity refers to β-arrestin recruitment compared to a known agonist at EC80 concentration. % inhibition refers to the reduction in β-arrestin recruitment induced by an agonist at EC80 concentration. CXCR4 (black) exhibits significant GPCR antagonist activity, as demonstrated by the percentage of inhibition and suppression of agonist activity against the peptide pool. C3AR1 (black) exhibits strong agonism when compared to a known agonist and exhibits a significant fold change relative to basal activity (black dots).

[0029] Fig.13A bar graph showing the results of the CXCR4 human chemokine GPCR antagonist assay is shown. % inhibition of target protein (SEQ ID NO: 49310) tested on β-arrestin cell line. Target protein SEQ ID NO: 49310 in black showed significant inhibition (about 80%) at both 1 μM and 0.3 μM when compared to irrelevant peptide in grey, irrelevant scrambled peptide, peptide pool with irrelevant peptide and target protein (SEQ ID NO: 30949). *p value < 0.05, ***p value < 0.001, ****p value < 0.0001, compared to irrelevant scrambled peptide_1 uM, one-way ANOVA, Dunnett's multiple comparison test.

[0030] Fig.14 A bar graph showing the results of the C3AR1 human complement GPCR antagonist assay is shown. Activity % of target protein (SEQ ID NO: 42382) tested on β-arrestin cell line. Target protein SEQ ID NO: 34229 in black showed significant activity at both 1 μM and 0.3 μM when compared to irrelevant peptide in grey, irrelevant scrambled peptide, peptide pool with irrelevant peptide and target protein (SEQ ID NO: 42382). Activity % of C3AR1 is relative to known agonist C3A receptor agonist. *p value < 0.05, ***p value < 0.001, ****p value < 0.0001, compared to irrelevant scrambled peptide_1 uM, one-way ANOVA, Dunnett's multiple comparison test.

[0031] Fig.15 The quantification of the chemotactic response of NAMALWA cells to SEQ ID NO: 49310 migration is shown. Cells were allowed to migrate in the lower chamber of a trans-well plate for 24 hours in the absence or presence of SEQ ID NO: 49310 or SDF-1 (CXCL-12). A migration inducer was used as a positive control for chemotaxis, and ADM3100 was used as a negative control for chemotaxis. Data are presented as mean ± standard error of the mean (SEM) of three independent experiments, each performed in triplicate. Statistical analysis was performed using a two-tailed t-test, *p<0.05. DETAILED DESCRIPTION

[0032] Following is a description of an exemplary embodiment. Target protein

[0033] In one aspect, the disclosure provides a target protein identified herein. As used herein, the expressions "target protein identified herein" and "target protein disclosed herein" include polypeptides disclosed in the sequence listing (e.g., a target protein comprising an amino acid sequence selected from the group consisting 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 any one of the following: SEQ ID NO: 75451-75473), variants thereof (e.g., a target protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36277_T31A, SEQ ID NO: 7353_P25L, SEQ ID NO: 74164_I36T, SEQ ID NO: 7354_P36T, SEQ ID NO: 7354_P36T, SEQ ID NO: 7354_P36T, SEQ ID NO: 7354_P36T, SEQ ID NO: NO: 26888_L8V, a target protein having an amino acid sequence of SEQ ID NO: 24296rs221797V to A, V to G or V to D) and the peptides disclosed in Table A herein. The target protein can be produced recombinantly (e.g., via DNA or mRNA) or synthetically.

[0034] In various embodiments, the target protein is an extracellular (secreted) protein.

[0035] In various embodiments, the target protein is a protein in the sequence listing or Table A. In some embodiments, the target protein is a protein comprising an amino acid sequence shown in the sequence listing or Table A. In some embodiments, the target protein consists of an amino acid sequence shown in the sequence listing or Table A. In some embodiments, the target protein comprises an amino acid sequence having an amino acid substitution relative to the amino acid sequence shown in the sequence listing or Table A, wherein the substitution is to replace the N-terminal residue in the amino acid sequence in the sequence listing or Table A with a methionine (Met) residue. In some embodiments, the target protein consists of an amino acid sequence having an amino acid substitution relative to the amino acid sequence shown in the sequence listing or Table A, wherein the substitution is to replace the N-terminal residue in the amino acid sequence in the sequence listing or Table A with a methionine (Met) residue. In some embodiments, the target protein comprises an amino acid sequence shown 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 an amino acid sequence shown in the sequence listing or Table A and a methionine (Met) residue at its N-terminus.

[0036] Table A. Target peptides of the present disclosure

[0037] Certain target proteins in the sequence listing and Table A have been identified as differentially expressed (e.g., upregulated or downregulated) in a disease and / or condition state selected from the following as compared to a reference state (e.g., a normal state): aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infections, immune diseases (e.g., inflammatory and / or autoimmune diseases), indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof, such that modulation of the level and / or activity of the target protein plays a role in treating, alleviating the disease or condition and / or preventing the onset of the disease or condition.

[0038] As used herein, the term "differential expression" refers to at least one identifiable difference in protein expression. It can be a quantitatively measurable, semi-quantitatively estimable or qualitatively detectable difference in protein expression. Therefore, a differentially expressed protein or "DEP" can have a higher expression level in a reference state (e.g., a normal state) than in a disease state where 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) where the DEP has a lower expression level or is not expressed at all. In addition, if a DEP is identifiably changed (e.g., mutated) between the two states under comparison, the expression can be considered to be differential. Identifiable changes can include amino acid substitutions, insertions and / or deletions (including N-terminal and C-terminal truncations) and modifications (e.g., post-translational modifications).

[0039] As used herein, the term "reference" refers to a standard for one or more comparison purposes. Those skilled in the art can select an appropriate reference for one or more specific comparison purposes. Thus, for example, a reference for a disease state can be a normal healthy state; a reference for a mutated protein can be a protein that has not been mutated; a reference for a disease treatment can be no treatment or can be a standard of care treatment. In some embodiments, particularly in embodiments involving methods for identifying agents that regulate 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 an agent. In some embodiments, the reference is based on a predetermined level, such as based on functional expression or empirical determination. In some embodiments, the reference is obtained from a cell, sample, or subject (e.g., a cell or sample from a healthy subject, a subject not suffering from a specific disease; a healthy subject, a subject not suffering from a specific disease). In some embodiments, the reference is obtained from more than one cell (e.g., a cell population), sample, or subject (e.g., a cell or sample from a healthy subject, a subject not suffering from a specific disease; a healthy subject, a subject not suffering from a specific disease), such as 2, 3, 4, 5, 10, 20, 30, 50, 100 or more or a statistically significant number of cells, samples, or healthy subjects. References obtained from more than one cell, sample, or subject can be expressed as a statistic (eg, a mean or median).

[0040] In some embodiments, proteins are used as markers of immune and / or disease states.

[0041] 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., α cells, β cells and / or δ cells). In some embodiments, the expression level of the target protein in endocrine organs and / or secretory cells is at least about 0.5 times higher, such as at least about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 times (e.g., 50 times, 100 times higher) compared to the target protein expression level in a reference organ and / or cell.

[0042] In certain embodiments, the target protein has a lower expression level in endocrine organs and / or secretory cells. In certain embodiments, the expression level of the target protein in endocrine organs and / or secretory cells is at least about 0.5 times lower than the target protein expression level in a reference organ and / or cell, for example, at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 times (e.g., 50 times lower, 100 times lower).

[0043] In some embodiments, the expression level of the target protein in the disease (e.g., as determined from a sample of cells or tissue from a subject having the disease) is at least about 0.5-fold higher, e.g., at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10-fold higher (e.g., 50-fold higher, 100-fold higher) compared to the expression level of the target protein in a reference (e.g., a sample of cells or tissue from a subject not having the disease).

[0044] In some embodiments, the expression level of the target protein in the disease (e.g., as determined from a sample comprising or obtained from cells or tissues of a subject suffering from the disease) is at least about 0.5-fold lower, such as at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) compared to the expression level of the target protein in a reference (e.g., a sample of cells or tissues from a subject not suffering from the disease). In some embodiments, the target protein is not expressed in the disease or is expressed at an undetectable level (e.g., as determined from a sample comprising or obtained from cells or tissues of a subject suffering from the disease).

[0045] In some embodiments, the transcript level of the target protein in the disease (e.g., as determined from a sample of cells or tissues from a subject suffering from the disease) is at least about 0.5 times higher, e.g., at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10 times higher (e.g., 50 times higher, 100 times higher) than the transcript level of the target protein in a reference (e.g., a sample of cells or tissues from a subject not suffering from the disease). In specific embodiments, an increase in the transcript level of the target protein contributes to (e.g., causes) a disease or disorder described herein.

[0046] In some embodiments, the transcript level of the target protein in the disease (e.g., as determined from a sample containing or obtained from cells or tissues of a subject suffering from the disease) is at least about 0.5 times lower, such as at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 times lower (e.g., 50 times lower, 100 times lower) compared to the transcript level of the target protein in a reference (e.g., a sample of cells or tissues from a subject not suffering from the disease). In some embodiments, the transcript of the target protein is not expressed in the disease or is expressed at an undetectable level (e.g., as determined from a sample containing or obtained from cells or tissues of a subject suffering from the disease). In specific embodiments, the reduction in the transcript level of the target protein contributes to (e.g., causes) a disease or disorder described herein.

[0047] In certain embodiments, the gene encoding the target protein in the diseases described herein comprises at least one mutation (eg, fusion, deletion, insertion, point mutation, and / or expansion of amino acid repeats).

[0048] 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 microbiopsy), pancreatic juice, chorionic villus samples, cells, and the like isolated from a subject.

[0049] 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).

[0050] In some embodiments, the target protein is translated from a non-exon element in an 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 a 5' untranslated region (5'UTR) in the pre-mRNA. In some embodiments, the non-exon element is a 3' untranslated region (3'UTR) in the pre-mRNA.

[0051] In some embodiments, the target protein is 2,000 or less amino acids in length, such as 1000 or less amino acids, 750 or less amino acids, 500 or less amino acids, 250 or less amino acids, 150 or less amino acids, or 100 or less amino acids. In some embodiments, the target protein is 7 or more amino acids in length, such as 8, 9, 10, 15, 18, 25, 50, 75 or 100 or more amino acids. In certain embodiments, the target protein is from about 50 to about 200 amino acids in length, such as from about 100 to about 150 amino acids. In a specific embodiment, the target protein is 7 or more amino acids in length. In a more specific embodiment, the target protein is about 18 amino acids in length.

[0052] In some embodiments, the target protein is an upstream signaling molecule of a metabolic pathway selected from: acyl-CoA hydrolysis, acylglyceride metabolism, alanine, aspartate and glutamate metabolism, amino sugar and nucleotide sugar metabolism, aminoacyl-tRNA biosynthesis, androgen metabolism, arachidonic acid metabolism, arginine and proline metabolism, ascorbic acid and aldarate metabolism, beta oxidation of branched-chain fatty acids (mitochondria), beta oxidation of diunsaturated fatty acids (n-6) (mitochondria), beta oxidation of diunsaturated fatty acids (n-6) (peroxisomes), beta oxidation of even-chain fatty acids (mitochondria), beta oxidation of even-chain fatty acids (peroxisomes), beta oxidation of odd-chain fatty acids (mitochondria), beta oxidation of phytanic acid (mitochondria), beta oxidation of phytanic acid (mitochondria), beta oxidation of acetylcholine (peroxisomes ... Oxidation (peroxisome), β-oxidation of polyunsaturated fatty acids (mitochondria), β-oxidation of polyunsaturated fatty acids (n-7) (mitochondria), β-oxidation of polyunsaturated fatty acids (n-7) (peroxisome), β-oxidation of polyunsaturated fatty acids (n-9) (mitochondria), β-oxidation of polyunsaturated fatty acids (n-9) (peroxisome), β-alanine metabolism, Bile acid biosynthesis, Bile acid recycling, Biopterin metabolism, Biotin metabolism, Blood group biosynthesis, Butyrate metabolism, C5-branched dicarboxylic acid metabolism, Carnitine shuttle (cytosol, endoplasmic reticulum, mitochondria and / or peroxisome), Cholesterol biosynthesis 1 (Bloch pathway), Cholesterol biosynthesis 2, Cholesterol biosynthesis 3 (Kandustch-R ussell pathway), cholesterol metabolism, chondroitin / heparan sulfate biosynthesis, chondroitin sulfate degradation, coenzyme A synthesis, cysteine ​​and methionine metabolism, drug metabolism, eicosanoid metabolism, estrogen metabolism, ether lipid metabolism, fatty acid activation (cytosolic and / or endoplasmic reticulum), fatty acid biosynthesis (even-chain and / or odd-chain), fatty acid biosynthesis (unsaturated), fatty acid desaturation (even-chain and / or odd-chain), fatty acid elongation (even-chain and / or odd-chain), fatty acid oxidation, folate metabolism, cholesterol ester formation and hydrolysis, fructose and mannose metabolism, galactose metabolism, glucocorticoid biosynthesis, glutathione metabolism, glycerolipid metabolism, glycerophospholipid metabolism, glycine, serine, and threonine metabolism, glycolysis / gluconeogenesis , Glycosphingolipid biosynthesis - ganglionic series, Glycosphingolipid biosynthesis - globular series, Glycosphingolipid biosynthesis - lactose and neolactose series, Glycosphingolipid metabolism, Glycosylphosphatidylinositol (GPI)-anchor biosynthesis, Heme degradation, Heme synthesis, Heparan sulfate degradation, Histidine metabolism, Phosphoinositide metabolism, Isolated 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, ω-3 fatty acid metabolism, ω-6 fatty acid metabolism, Oxidative phosphorylation, Pantothenic acid and CoA biosynthesis, Pentose and glucuronic acid interconversion, Pentose phosphate pathway, Phenylalanine metabolism,Phenylalanine, tyrosine and tryptophan biosynthesis, phosphatidylinositol phosphate metabolism, pool reactions, porphyrin metabolism, propionate metabolism, prostaglandin biosynthesis, protein assembly, protein degradation, 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 / dicarboxylic acid 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. ,

[0053] 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 proteins are agonists of one or more GPCRs. In some embodiments, the target proteins are antagonists of one or more GPCRs. In some embodiments, the target proteins are direct modulators of one or more GPCRs, such as ligands of one or more GPCRs. In some embodiments, the target proteins are indirect modulators of one or more GPCRs.

[0054] The expression and / or activity of various GPCRs have been linked to different diseases / disorders, conditions and indications, including those shown in Table B (see, e.g., Kenakin, T., Biased Receptor Signaling in Drug Discovery, Pharmacol Rev 71: 267-315, April 2019; Harmar, AJ 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, deGraaf C, Brown AJH and Maguire JJ. Advances in therapeutic peptides targeting G protein-coupled receptors. Nat Rev. 1998, 55(5): 115-123. DrugDiscov. [Nature Review: Drug Discovery] June 19, 2020 (6): 389-413; the contents of each are incorporated herein by reference in their entirety). Therefore, in some embodiments, the target proteins disclosed herein (which are modulators of GPCRs) can be used to treat and / or diagnose one or more diseases / disorders, conditions and / or indications, such as cancer or precancerous conditions, or any of the diseases / disorders, conditions and indications listed in Table B that are known to be associated with GPCR expression and / or activity.

[0055] Table B. Disease-related GPCRs Agents that modulate target proteins

[0056] Provided herein are agents for regulating the expression of a target protein disclosed herein (such as a target protein in the sequence listing, Table A) or a variant or a fragment thereof (e.g., a biologically active fragment of a target protein). The expression of a target protein or a variant or fragment thereof can be regulated directly or indirectly by a wide range of processes, resulting 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, extension or termination, RNA processing, RNA stability (e.g., mRNA stability), RNA degradation, translation initiation, post-translational modification of the protein, protein stability, protein degradation (e.g., cleavage, such as protease cleavage), or a combination of the foregoing.

[0057] In some embodiments, the agent regulates (e.g., increases or decreases) the expression of a gene or gene transcript encoding a target protein. In some embodiments, the agent regulates the expression or activity of a target protein. In some embodiments, the agent reduces (e.g., inhibits, reduces or neutralizes) the activity of a target protein. In some embodiments, the agent increases (e.g., activates) the activity of a target protein. In some embodiments, the agent reduces (e.g., inhibits or downregulates) the expression of a target protein. In other embodiments, the agent increases (e.g., activates or upregulates) the expression of a target protein.

[0058] As used herein, the term "increasing" or "increase" refers to a regulation that results in a higher level of expression, activity, function, or a combination thereof or a measure (e.g., cancer cell death or DNA methylation at a target site) of a target protein relative to a reference (e.g., a level before or in the absence of an agent). In some embodiments, the agent increases the expression or activity or measure of the target protein by at least about 5% relative to a reference, such as by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to a reference.

[0059] As used herein, the term "decreasing" or "decrease" refers to a regulation that results in a lower level of expression, activity, function, or a combination thereof or a measure (e.g., cancer cell death or DNA methylation at a target site) of a target protein relative to a reference (e.g., a level before or in the absence of an agent). In some embodiments, the agent reduces the expression or activity or measure of the target protein by at least about 5% relative to a reference, such as by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to a reference.

[0060] Non-limiting examples of metrics include energy production or energy conversion in the liver (e.g., regulation of ATP synthesis, B-oxidation, oxidation of metabolites derived from glycolysis, oxidation of metabolites derived from amino acids), mitochondrial transcription, mitochondrial ribosome assembly, mitochondrial translation, mitochondrial thermogenesis, hormone 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, unfolded protein stress response pathways, signaling processes (e.g., calcium signaling).

[0061] In some embodiments, the level of expression, activity, function, or a combination or metric thereof of the target protein is measured, e.g., after a treatment regimen has begun, after the agent is contacted with (e.g., a cell) or administered (e.g., to a subject) for at least about 1 day, e.g., 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.

[0062] In some embodiments, the agent comprises, consists essentially of, or consists of a polypeptide, a polynucleotide, a gene editing system, a small molecule, or a cell (e.g., a cell therapy).

[0063] In some embodiments, the target protein activates immune cells, and the agent regulates (e.g., increases or decreases) the level of expression, activity, function, or a 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 regulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of the target protein.

[0064] In certain embodiments, the agent regulates (e.g., increases or decreases) the expression, activity, function, or combination thereof of the target protein in the following cells: cancer cells (e.g., metastatic cancer cells), cells in the tumor microenvironment (e.g., stromal cells), target cells of 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 lymphocytes (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 a combination thereof. In certain embodiments, the agent regulates the expression, activity, function, or combination thereof of the target protein in a tumor, a tumor microenvironment, a metastatic site, a lymph node, a spleen, a secondary lymphoid organ, a tertiary lymphoid organ, a barrier tissue, skin, intestines, airways, wounds, another immune tissue, a non-immune tissue, or a combination thereof.

[0065] In some embodiments, the agent modulates (e.g., increases or decreases) inflammation, reduces levels of autoantibodies, increases organ function, reduces the rate or number of relapses or flare-ups, reduces viral load, controls infection, or a combination of the foregoing.

[0066] In some embodiments, the agent induces downregulation of the target protein (e.g., increases degradation of the target protein); prevents multimerization (e.g., dimerization) of the target protein; sequesters the target protein (e.g., secreted target protein); regulates (e.g., excites, antagonizes, or destroys) the known function of the target protein; reduces the binding between the target protein and the binding partner (e.g., via steric hindrance); regulates (e.g., increases or decreases) downstream cell signaling; induces antibody-dependent cell killing, phagocytosis, and / or opsonization of cells expressing the target protein; or a combination of the foregoing. In certain embodiments, the agent lacks agonistic activity against the target protein. In certain embodiments, the agent has agonistic activity against the target protein. In some embodiments, the agent lacks antagonistic activity against the target protein. In some embodiments, the agent has antagonistic activity against the target protein. In certain embodiments, the agent binds to at least one residue of the target protein, and the at least one residue is 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, and the one or more binding sites and / or domains are involved in binding of the target protein to the binding partner.

[0067] 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 thyroid stimulating hormone receptor.

[0068] In some embodiments, the agent induces downregulation of the binding partner of the target protein; sequesters the binding partner of the target protein (e.g., secreted binding partner); prevents multimerization (e.g., dimerization) of the binding partner of the target protein; sequesters the binding partner of the target protein (e.g., secreted binding partner); regulates (e.g., excites, antagonizes, or destroys) the known function of the binding partner of the target protein; reduces the binding between the target protein and the binding partner (e.g., via steric hindrance); regulates (e.g., increases or decreases) downstream cell signaling; induces antibody-dependent cell killing, phagocytosis, and / or opsonization of cells expressing the binding partner of the target protein; or a combination of the foregoing. In certain embodiments, the agent lacks agonistic activity for the binding partner of the target protein. In certain embodiments, the agent has agonistic activity for the binding partner of the target protein. In some embodiments, the agent lacks antagonistic activity for the binding partner of the target protein. In some embodiments, the agent has antagonistic activity for the binding partner of the target protein. In a specific embodiment, the agent further binds to at least one residue of a binding partner of the target protein, and the at least one residue is involved in the binding between the target protein and the binding partner. In a more specific embodiment, the agent further binds to one or more binding sites and / or domains of a binding partner of the target protein, and the one or more binding sites and / or domains are involved in the binding between the target protein and the binding partner.

[0069] In some embodiments, the agent modulates (eg, activates or inhibits) immune signaling, cytokine signaling, inflammatory signaling, or a combination of the foregoing.

[0070] In some embodiments, the agent enhances the signal 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 a specific embodiment, the agent is an agonist for CD28.

[0071] 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 SIGLEC9. In a particular embodiment, the agent is an inhibitor (e.g., blocking antibody) for PD-1.

[0072] In certain embodiments, the agent regulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of a variant of a 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 may 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.

[0073] As used herein, the term "sequence identity" refers to the degree 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, a sequence is typically designated as a reference sequence to which a test sequence is compared. The sequence identity between a reference sequence and a test sequence is expressed as the percentage of the position over the entire length of the reference sequence, at which the reference sequence and the test sequence have the same nucleotide or amino acid when aligning the reference sequence and the test sequence to achieve the maximum level of identity. As an example, when the test sequence has the same nucleotide or amino acid residue at 70% of the same position over the entire length of the reference sequence when the alignment is to achieve the maximum level of identity, the two sequences are considered to have 70% sequence identity.

[0074] Sequence alignment for comparison to achieve maximum identity level can be easily performed by those of ordinary skill in the art using appropriate alignment methods or algorithms. In some cases, alignment can include the introduction of gaps to provide maximum identity level. Examples include the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin) and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology).

[0075] When using a sequence comparison algorithm, a test sequence and a reference sequence are input into a computer, subsequent coordinates are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of one or more test sequences relative to the reference sequence based on the specified program parameters. A common tool for determining sequence identity percentage is the protein basic local alignment search tool (BLASTP), which can be obtained by the National Center for Biotechnology Information of the National Library of Medicine of the National Institutes of Health (National Institutes of Health). (Altschul et al., 1990).

[0076] In some embodiments, the amino acid sequence of the 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 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 relative to the amino acid sequence of the target protein disclosed herein. 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.

[0077] One or more amino acid substitutions in a variant can be substitutions with classical amino acids or non-classical amino acids. Non-classical amino acids include, but are not limited to, D amino acids, such as the D forms of classical L-amino acids.

[0078] In some embodiments, the amino acid substitution is a conservative substitution.The term "one or more conservative amino acid substitutions" or "one or more conservative substitutions" refers to an amino acid substitution that has a value of 0 or greater in BLOSUM62.

[0079] In some embodiments, the amino acid substitutions are highly conservative substitutions.The term "one or more highly conservative amino acid substitutions" or "one or more highly conservative substitutions" refers to an amino acid substitution that has a value of at least 1 (eg, at least 2) in BLOSUM62.

[0080] In some embodiments, relative to the amino acid sequence of the target protein disclosed herein, the variant of the target protein disclosed herein comprises about 5-60 amino acid substitutions. 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. A. Peptide Agents

[0081] The term "polypeptide", "peptide" or "protein" refers to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Proteins, peptides or polypeptides can contain 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). The amino, carboxyl and / or other functional groups on the peptide can be free (e.g., unmodified) or protected with suitable protecting groups. 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. Functional groups of proteins, peptides, or polypeptides may also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label such as a fluorophore or a hapten) using methods known in the art. If desired, the protein, peptide, or polypeptide may include one or more modifications (e.g., an amino acid linker, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclization modification), N-methyl-α-amino group substitution). In addition, the protein, peptide, or polypeptide may be an analog of a known and / or naturally occurring peptide, such as a peptide analog having one or more conservative amino acid residue substitutions.

[0082] 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 of expression and / or activity of a target protein disclosed herein (e.g., a direct inhibitor or an indirect inhibitor). In some embodiments, the polypeptide is an activator of expression and / or activity of a target protein disclosed herein (e.g., a direct activator or an indirect activator). In some embodiments, the polypeptide reduces 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 portion thereof (e.g., a biologically active portion thereof, such as a biologically active fragment of a target protein).

[0083] 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 modulate the expression or activity of a target protein.

[0084] In some embodiments, the polypeptide is an antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule that is capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through 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 or multimer thereof (e.g., IgM) comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (including domains CH1, hinge, CH2 and CH3). Each light chain contains a light chain variable region (V L ) and the light chain constant region (CL). H and V L The V region can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed within framework regions (FRs). H and V L Each comprises three CDRs and four FR segments arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The antibody may be of any species, such as a rodent (e.g., mouse, rat, guinea pig) antibody, a human antibody, or the antibody may be a humanized antibody or a chimeric antibody.

[0085] 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 kappa light chain constant region. In some embodiments, the antibody comprises a lambda light chain constant region.

[0086] 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, such as bispecific, trispecific, or tetraspecific. In some embodiments, the antibody is a heterologous conjugate antibody.

[0087] 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 V H Area, V L region, Fab fragment, F(ab′) 2 fragment, Fd fragment, Fv fragment and a V H domain or a V L Domain antibodies (dAbs) composed of domains, etc. H and V L The domains can be linked together via synthetic linkers to form various types of single-chain antibody designs, where V H / V L The domains can be paired intramolecularly or intermolecularly, in the latter case, V H and V L The domains are expressed from separate chains to form monovalent antigen binding sites, such as single-chain Fv (scFv) or diabodies. In some embodiments, the polypeptides disclosed herein are antigen binding fragments selected from the group consisting of Fab, Fab′, F(ab′) 2 , Fd, Fv, disulfide-linked Fv (sdFv, such as diabodies, triabodies or tetrabodies), scFv, SMIP or rIgG. In some embodiments, the polypeptide is a scFv. Antigen-binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some cases, by chemical peptide synthesis procedures known in the art.

[0088] 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 antigenic epitope. Multispecific antibodies (such as bispecific antibodies or trispecific antibodies) are included in the term monoclonal antibody.

[0089] "Multispecific" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes within an antigen (e.g., three, four, or five different antigens or epitopes). "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen.

[0090] An "isolated antibody" refers to an antibody or antigen-binding fragment thereof that is substantially free of other antibodies having different antigenic 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 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, a polypeptide agent (e.g., a monoclonal antibody) is at least 80% pure, such as about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0091] In some embodiments, the polypeptide is an antagonist antibody that binds to a target protein (e.g., a target protein whose expression or activity increases in a cancer state relative to a reference state). In some embodiments, the antibodies described herein are antagonist antibodies that bind to proteins that can regulate the expression or activity of a target protein. As used herein, the term "antagonist antibody" refers to an antibody that reduces (e.g., inhibits) the function of an antigen after binding to an antigen (e.g., a target protein or a protein that can regulate the expression or activity of a target protein). 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 weakens the signal transduction pathway mediated by the signaling molecule. In some embodiments, the antagonist antibody reduces antigen function by at least about 10%, such as by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 99%.

[0092] 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 cancer state relative 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 a target protein. As used herein, the term "agonist antibody" refers to an antibody that increases the function of an antigen after binding to an antigen (e.g., a target protein or a protein that can regulate the expression or activity of a target protein). 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 signal transduction pathway mediated by a signaling molecule. In some embodiments, the agonist antibody increases antigen function by at least about 10%, for example, by 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%.

[0093] In some embodiments, the agonist antibody does not exert at least one of the following functional properties: reducing (e.g., inhibiting) the activity of an antigen; inducing antibody-dependent cellular killing 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 down-regulation of the antigen on the cell surface (e.g., by hyper-crosslinking or clustering the antigen to induce internalization and degradation).

[0094] Suitable techniques, assays, and reagents for preparing and using therapeutic antibodies directed against an antigen are known in the art. For methods of 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, see, e.g., Therapeutic Monoclonal Antibodies: From Bench to Clinic (Edited by Zhiqiang An, 1st edition 2009); Antibodies: A Laboratory Manual (Edited by Fdward A. Greenfield, 2d edition 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).

[0095] 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 simulate the ability of an antibody to bind an antigen but is structurally different from a natural antibody structure. Non-limiting examples of antibody mimetic include Adnectin, Affibody, Affilin, Affimer, Affitin, Alphabody, Anticalin, Avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, nanobody, nanoCLAMP, and Versabody.

[0096] In some embodiments (e.g., when the expression or activity of the target protein is reduced in a disease state relative to a 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 (e.g., a biologically active portion or fragment) of the target protein. For example, the percent identity to the full-length target protein or its biologically active portion or fragment may 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%. 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.

[0097] 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.

[0098] In some embodiments, the 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 is capable of regulating the expression or activity of a target protein. For example, the identity percentage may 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 an amino acid sequence of a protein that is capable of regulating the expression or activity of a target protein.

[0099] In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence having at least one amino acid substitution relative to a protein capable of regulating 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 substitution is a conservative substitution. In some embodiments, the amino acid substitution is a highly conservative substitution.

[0100] In some embodiments, the polypeptide is a cell penetrating peptide. In certain embodiments, the polypeptide is connected 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), transmembrane peptides, pVEC, MPG8, transportan, transportan 10, PepFect3, PepFect 6, PepFect 14, polyarginine, stearoyl-polyarginine, Pep-1, Pep-3, CADY, YTA2, YTA4, SynB1, SynB3, Maurocalcine, and PTD4.

[0101] In some embodiments, the polypeptide is a circulating factor (eg, a cytokine).

[0102] In some embodiments, the biological properties (e.g., biological activity or half-life) of the polypeptide (e.g., separated polypeptide) and the target protein are similar. Non-limiting examples of biological activity include 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., functional groups are transferred from one molecule to another molecule), oxidoreductase activity (e.g., catalytic oxidation-reduction reaction), hydrolase activity (e.g., cutting chemical bonds via hydrolysis), lyase activity (e.g., producing double bonds), ligase activity (e.g., connecting two molecules via covalent bonds) and isomerase activity (e.g., catalyzing the structural change from one isomer to another isomer within a molecule).

[0103] 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 and David C James, ed., 2005); Pharmaceutical Biotechnology: Fundamentals and Applications (Daan JACrommelin, Robert D. Sindelar and Bernd Meibohm, ed., 2013). Mammalian cells, insect cells, yeast or bacteria, etc. can be used to recombinantly express polypeptides under the control of appropriate promoters.

[0104] In some embodiments, the polypeptides described herein (e.g., target proteins or portions thereof, polypeptide agents regulating target proteins) are modified, for example, by cleavage (e.g., protease cleavage) or post-translational modification. In certain embodiments, one or more modifications will affect the activity of the polypeptide, for example, by making an inactive polypeptide active or by changing (e.g., increasing, reducing) the activity level of the polypeptide. In a particular embodiment, the polypeptides described herein are provided as prodrugs, for example, they can be converted (e.g., by proteolytic cleavage, post-translational modification) into active polypeptides in vivo. In some embodiments, the polypeptides include post-translational modifications or other chemical modifications. 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.

[0105] In some embodiments, the polypeptide is linked to a heterologous peptide or protein (e.g., via a covalent bond (such as a peptide bond) or a non-covalent bond), such as in a conjugate or fusion protein. In some embodiments, the polypeptide comprises a tag (e.g., a detectable label (such as a fluorophore or an enzyme) or a purification tag (such as an epitope tag)).

[0106] In some embodiments, the polypeptide comprises one or more neoantigens selected from the sequence listing, Table A, or variants thereof. As used herein, the term "neoantigen" refers to a tumor antigen produced by a target protein described herein. In some embodiments, the neoantigen is a cancer-specific neoantigen. There are a variety of ways to produce neoantigens. For example, the neoantigen can be produced in vitro as a polypeptide before being formulated into a tumor vaccine or an immunogenic pharmaceutical composition. In some embodiments, the immunogenic pharmaceutical composition comprises an effective amount of one or more neoantigens or one or more pharmaceutically acceptable salts thereof. In some embodiments, the immunogenic pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, adjuvant or additive.

[0107] Alternatively, a new antigen can be produced in vivo by introducing a polynucleotide or expression vector (e.g., a viral expression vector) encoding a new antigen into a cell or tissue (e.g., a subject in need thereof). In certain embodiments, the polypeptide comprises at least two new antigens. 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: a constant chain, a leader sequence of a tissue-type plasminogen activator, a PEST sequence, a cyclin destruction box, a ubiquitination signal, and a SUMOylation signal. B. Polynucleotide Agents

[0108] In some embodiments, the medicament comprises a polynucleotide or its analog or derivative. In some embodiments, the polynucleotide or its analog or derivative is an inhibitor of the target protein. In some embodiments, the polynucleotide or its analog or derivative is an activator of the target protein. In some embodiments, the polynucleotide or its analog or derivative reduces (e.g., reduces or neutralizes) the expression or activity of the target protein. In other embodiments, the polynucleotide or its analog or derivative increases the expression or activity of the target protein.

[0109] The polynucleotide may have a sequence containing naturally occurring ribonucleotides or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or a combination thereof. Thus, the polynucleotide may 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 comprises at least one modified nucleotide. Non-limiting examples of modified nucleotides include 2'-fluoro, 2'-o-methyl, 2'-deoxy, non-locked nucleic acids, 2'-hydroxyl, thiophosphate, 2'-thiouridine, 4'-thiouridine, and 2'-deoxyuridine. In some embodiments, the modification increases nuclease resistance, increases serum stability, reduces immunogenicity, or a combination of the foregoing.

[0110] 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).

[0111] In some embodiments, polynucleotides include analogs or derivatives of polynucleotides. In some embodiments, analogs or derivatives are peptide nucleic acids (PNA). In some embodiments, analogs or derivatives are locked nucleic acids (LNA). In some embodiments, analogs or derivatives are morpholino oligonucleotides. In some embodiments, analogs or derivatives include one or more thiophosphates. In some embodiments, medicaments include deoxyribonucleic acid guanidine (DNG) nucleotides. In some embodiments, medicaments include ribonucleic acid guanidine (RNG) nucleotides.

[0112] In some embodiments, the polynucleotide modulates 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 portion thereof (e.g., a biologically active portion or fragment thereof).

[0113] In some embodiments, the polynucleotide comprises a nucleotide sequence that is complementary (e.g., fully or partially complementary) to at least a portion of a gene or gene transcript encoding a target protein disclosed herein, such that the polynucleotide sequence is capable of hybridizing or annealing 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 portion of a gene or gene transcript encoding a protein that can regulate the expression or activity of a target protein disclosed herein.

[0114] In some embodiments, the polynucleotide encodes a target protein disclosed herein or a variant thereof (e.g., a bioactive variant) or a portion thereof (e.g., a bioactive portion or fragment).

[0115] In some embodiments, the nucleic acid encoding the target protein or a variant or portion thereof (e.g., a fragment) is a gene sequence or a portion thereof. In some embodiments, the encoding nucleic acid is an unprocessed RNA transcript (e.g., pre-mRNA) or a portion thereof (e.g., 5′UTR, 3′UTR, intron). In some embodiments, the encoding nucleic acid is an mRNA molecule or a portion 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).

[0116] The encoding nucleic acid can comprise a classical open reading frame (ORF) or a non-classical ORF. In certain embodiments, the encoding nucleic acid comprises a non-classical ORF.

[0117] 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 double-stranded polynucleotide is about 15 - 50 base pairs, such as about: 15 - 45, 15 - 40, 15 - 35, 15 - 30, 15 - 25, 18 - 50, 18 - 45, 18 - 40, 18 - 35, 18 - 30, 18 - 25, 20 - 50, 20 - 45, 20 - 40, 20 - 35, 20 - 30, 20 - 25, 25 - 50, 25 - 45, 25 - 40, 25 - 35, 25 - 30, 30 - 50, 30 - 45, 30 - 40, 30 - 35, 35 - 50, 35 - 45, 35 - 40 or 40 - 50 base pairs. In some embodiments, the length of the polynucleotide is about 19 - 23 base pairs. In some embodiments, the length of the polynucleotide is about 21 base pairs.

[0118] In some embodiments, the polynucleotide is single-stranded (ss). In some embodiments, the single-stranded polynucleotide has a length of about 15-50 nucleotides, such as about: 15-45, 15-40, 15-35, 15-30, 15-25, 18-50, 18-45, 18-40, 18-35, 18-30, 18-25, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40, or 40-50 nucleotides.

[0119] In some embodiments, the polynucleotide prevents maturation of newly generated nuclear RNA transcripts into mRNA for transcription.In some embodiments, the polynucleotide comprises a nucleotide sequence that is complementary to a sequence at an intron and exon boundary.

[0120] In some embodiments, the polynucleotide (e.g., antisense oligonucleotide) can hybridize to an mRNA encoding a target protein (e.g., under physiological conditions). In some embodiments, the polynucleotide is at least about 10 nucleotides in length, 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 target transcript, such as at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0121] In some embodiments, the polynucleotide further comprises an overhang sequence (e.g., an unpaired overhang nucleotide that is not directly involved in the formation of a double helix structure with 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 a modified ribonucleotide or deoxynucleotide, such as a thiophosphate, a phosphorothioate, or a deoxynucleotide reverse (3' to 3' linked) nucleotide.

[0122] 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), micro RNA (miRNA), antagomir, antisense DNA, antisense RNA, morpholino nucleic acids (MNA), locked nucleic acids (LNA), peptide nucleic acids (PNA), aptamers and guide RNA (gRNA).

[0123] In some embodiments, the polynucleotide inhibits gene expression (e.g., a biological process via RNA interference (RNAi)). Polynucleotides suitable for RNA interference can be readily designed and generated by one of ordinary skill using techniques, assays, and reagents known in the art, including computational tools. See, e.g., 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.

[0124] In some embodiments, the polynucleotide is a miRNA. In some embodiments, the length of the miRNA is about 22 nucleotides. The miRNA binds to a target site on an mRNA molecule and silences the mRNA, such as by causing the cutting of the mRNA, the destabilization of the mRNA, or the inhibition of mRNA translation.

[0125] In some embodiments, the polynucleotide is siRNA. In some embodiments, the siRNA comprises a nucleotide sequence identical to about 15-25 consecutive mRNA sequences encoding a 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 GC content of the siRNA is about 30%-70%, for example, 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%.

[0126] In some embodiments, the polynucleotide is shRNA. shRNA is an RNA molecule comprising a hairpin turn that reduces target gene expression via RNAi. shRNA can be delivered to cells in the form of a plasmid (e.g., a viral or bacterial vector), for example, by transfection, electroporation, or transduction.

[0127] siRNA and shRNA 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, siRNA functions as miRNA; in other embodiments, miRNA functions as siRNA (see, e.g., Zeng et al., Mol Cell 9:1327-33 (2002); Doench et al., Genes Dev 17:438-42 (2003)). Like siRNA, microRNA uses RISC to downregulate target genes, but unlike siRNA, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein output by translational repression or poly A removal and mRNA degradation (see, e.g., Wu et al., Proc Natl Acad Sci USA 103:4034-39 (2006)). The known miRNA binding site is within the mRNA 3'UTR; miRNAs appear to target a site that has almost perfect complementarity to the 2-8 nucleotides at 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 referred to as the seed region. Because 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 produce stronger downregulation (see, e.g., Doench et al., Genes Dev 17: 438-42 (2003)).

[0128] In some embodiments, the polynucleotide is a messenger RNA (mRNA) or circular RNA (circRNA) encoding a target protein disclosed herein or a variant thereof (e.g., at least about 70% identical (e.g., at least about: 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the wild-type protein). In some embodiments, the mRNA is codon-optimized (e.g., to improve the efficiency of protein synthesis and limit mRNA destabilization caused 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)).

[0129] In some embodiments, polynucleotides including RNA are chemically synthesized. In some embodiments, polynucleotides including RNA are recombinantly expressed. In some embodiments, RNA is transcribed in vitro. The preparation and use of RNA therapeutics are known in the art. See, for example, RNA Therapeutics: Function, Design, and Delivery (Mouldy Sioud, ed., 2010) and Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Medicine 9: 60 (2017).

[0130] 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 a modified base, a 5' cap, a 5' cap analog, an anti-reverse cap analog (ARCA), or a combination thereof.

[0131] In some embodiments, the mRNA comprises a poly(A) tail. In some embodiments, the poly(A) tail is about 100-200 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)).

[0132] 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-dithiodiphosphate-modified cap (see, e.g., Strenkowska et al., Nucleic Acids Res. 44:9578-90 (2016)).

[0133] In some embodiments, the mRNA comprises a modified 3′ untranslated region (UTR), 5′UTR, or both. In some embodiments, the modified UTR comprises sequences responsible for recruiting RNA binding proteins (RBPs) and miRNAs (e.g., to enhance the level of protein products (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, a miRNA binding site, or a combination thereof to control RNA expression in a cell-specific manner (see, e.g., Wroblewska et al., Nat Biotechnol. 33:839-41 (2015)).

[0134] 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 immunostimulatory activity and enhance translation initiation (see, e.g., Andries et al., J Control Release [Journal of Controlled Release] 217: 337-44 (2015) and Svitkin et al., Nucleic Acids Res. [Nucleic Acids Research] 45: 6023-36 (2017)).

[0135] In some embodiments, the RNA (e.g., mRNA) is a circular RNA.

[0136] Compositions and methods for producing mRNA are disclosed in, for example, WO 2016011306, WO 2016014846, WO 2016022914, WO 2016077123, WO 2016164762, WO 2016201377, WO 2017049275, US9937233, US 8710200, US10022425, US 9878056, US 9572897, WO 2010084371, US 9353153, WO 2015034925, and WO 2019236673. See also, 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 (e.g., for in vivo delivery, see, e.g., U.S. Pat. No. 9,764,036, U.S. Pat. No. 9,682,139, Kauffman et al., Nano Lett. 15:7300-6 (2015), and Fenton et al., Adv Mater. 28:2939-43 (2016)).

[0137] In some embodiments, the polynucleotide is an aptamer. In certain embodiments, the aptamer binds to a target protein disclosed herein. In specific embodiments, the aptamer binds to a binding partner of a target protein disclosed herein.

[0138] In some embodiments, the polynucleotide is connected to the delivery polymer (e.g., covalently). In some embodiments, the connection between the polynucleotide and the delivery polymer is reversible. In some embodiments, the polynucleotide is connected to the delivery polymer via a physiologically unstable linker. In some embodiments, the physiologically unstable linker is a disulfide bond.

[0139] In some embodiments, the polynucleotide is conjugated to the polymer in the presence of excess polymer. In some embodiments, excess polymer is removed prior to administration (eg, to a cell or subject).

[0140] One of ordinary skill in the art can readily prepare suitable polynucleotide agents for use in the compositions, kits, and methods described herein using locus information (such as chromosomal location, start and stop nucleotide positions, and polymorphism identification) of the protein sequences contained in the Sequence Listing and Table A incorporated herein.

[0141] C. Agents Comprising Gene Editing Systems

[0142] In some embodiments, the agent comprises a gene editing system. In some embodiments, the gene editing system creates a deletion of nucleotides, a substitution of nucleotides, an addition of nucleotides, or a combination of the foregoing in a gene encoding a target protein.

[0143] 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 type II CRISPR / Cas system.

[0144] In some embodiments, the gene editing system (e.g., a CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates (e.g., via gene knockout) the expression of a target protein. In some embodiments, the gene editing system (e.g., a CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates (e.g., via gene knockout) the expression of a protein capable of regulating the expression or activity of a target protein. In some embodiments, the gene editing system (e.g., a CRISPR / Cas system) increases (e.g., via gene knock-in or gene replacement) the expression of a target protein. In some embodiments, the gene editing system (e.g., a CRISPR / Cas system) increases (e.g., via gene knock-in or gene replacement) the expression of a protein capable of regulating the expression or activity of a target protein.

[0145] In some embodiments, the CRISPR system specifically catalyzes cleavage in a gene encoding a target protein, thereby inactivating the gene. Repair of the nucleic acid strand break by non-homologous end joining (NHEJ) often results in changes in the DNA sequence at the cleavage site, leading to small insertions or deletions (insertions and deletions (Indels)). In some embodiments, NHEJ is used to knockout a gene encoding a target protein. In some embodiments, homologous directed repair (HDR) is used to simultaneously inactivate a gene encoding a target protein and insert a heterologous sequence into 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.

[0146] 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 (from Acidaminococcus sp.) or LbCpf1 (from Lachnospiraceae sp.). The selection of nuclease and one or more gRNAs will typically be determined based on whether a deletion, substitution or addition of one or more nucleotides of the target sequence is required.

[0147] In some embodiments, the type II Cas endonuclease is Cas9 (e.g., of Streptococcus pyogenes). In some embodiments, the modified Cas9 is a nickase Cas9, an inactive Cas9 (dCas9), or an 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 a double nickase Cas9 (e.g., to achieve more accurate genome editing, see, e.g., Ran et al., Cell [Cell] 154: 1380-89 (2013)). Wild-type Cas9 produces double-strand breaks (DSBs) at specific DNA sequences targeted by gRNA. Nickase Cas9 only produces single-strand breaks. dCas9 is catalytically inactive. In some embodiments, dCas9 is fused to a nuclease (e.g., FokI to produce 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 / ).

[0148] 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) CRISPR RNA ("crRNA"); and c) trans-activating crRNA ("tracrRNA").

[0149] In some embodiments, the crRNA comprises at least one "guide RNA" (sgRNA), for example, 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, for example, 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.

[0150] The design of gRNA sequences for gene editing is known in the art. See, for example, Cong et al., Science [Science], 339: 819-23 (2013) and Ran et al., Nature Protocols [Natural · Program] 8: 2281-308 (2013). Cas9 requires at least about 16 or 17 nucleotides of the gRNA sequence to cut DNA, and Cpfl requires at least about 16 nucleotides of the gRNA sequence to cut DNA. In fact, the length of the gRNA sequence is 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 sgRNA has also been shown to be effective in genome editing (see, for example, Hendel et al., Nature Biotechnol. [Natural · Biotechnology], 985-91 (2015)).

[0151] In some embodiments, crRNA further comprises a sequence capable of binding to tracrRNA.After binding, the partial double-stranded structure is cut by RNase III, and the resulting crRNA / tracrRNA hybrid guides the Cas9 endonuclease to recognize and cut the target DNA sequence.

[0152] In some embodiments, the target DNA sequence is close to a "protospacer adjacent motif" ("PAM") that is specific for the Cas endonuclease. PAM sequences appear to be found throughout a given genome. CRISPR endonucleases of various prokaryotic species have unique 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 (e.g., Cas9 endonucleases) associate with G-rich PAM sites (e.g., 5'-NGG) and blunt-end cleave the target DNA at a position 3 nucleotides upstream (5') of the PAM site.

[0153] 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; and b)crRNA.

[0154] The Cpf1-associated CRISPR array is processed into mature crRNA without the need for tracrRNA. The Cpf1 endonuclease associates with T-rich PAM sites (e.g., 5′-TTN). Cpf1 can also recognize 5′-CTA PAM motifs. Cpfl cuts target DNA by introducing misplaced or staggered double-strand breaks with 5′ overhangs of 4 or 5 nucleotides, for example, cutting such target DNA, where the misplaced or staggered cuts of 5 nucleotides are 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 generated by such misplaced cuts allow DNA insertion by homologous recombination to be more precisely edited than insertions at blunt-end cut DNA. See, e.g., Zetsche et al., Cell 163: 759-71 (2015).

[0155] In some embodiments, the gene editing system activates or inhibits 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.

[0156] In some embodiments, the chimeric protein inhibits the expression of the target protein (CRISPRi). In some embodiments, the chimeric protein activates the 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.

[0157] The effector domain comprises a biologically active portion of an effector protein (e.g., a transcriptional activator or repressor). In some embodiments, the gene editing system comprises 1 effector domain. In some embodiments, the gene editing system comprises at least 2 effector domains, such as 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 embodiments, dCas9 is H840A.

[0158] Because dCas9 is catalytically inactive, dCas9 does not cut the target DNA, but interferes with transcription through steric hindrance. The dCas9 chimeric protein (e.g., dCas9-VPR) (guided by one or more gRNAs to the upstream sequence of the transcription start site (TSS) of the target gene) regulates the transcription of the target gene. See, e.g., 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 fluorescenceimaging [Protein labeling system for signal amplification of gene expression and fluorescence imaging], Cell [Cell] 159: 635-46 (2014); Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex [Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex], Nature [Nature] 517: 583-88 (2015); Chavez et al., Highly efficient Cas9-mediated transcriptional programming [Highly efficient Cas9-mediated transcriptional programming], Nat. Methods.[Nature 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., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation, eLife. 5: e19760 (2016); Chavez et al., Comparison of Cas9 activators in multiple species, Nat Methods. 7: 563-67 (2016). .

[0159] CRISPR technology for editing genes in eukaryotic organisms is disclosed in U.S. Patent Application Publication Nos. 2016 / 0138008A1 and 2015 / 0344912A1 and U.S. Patent Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965 and 8,906,616. Cpf1 endonuclease and corresponding guide RNA and PAM site are disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1. CRISPR technology for generating mtDNA dysfunction in the mitochondrial genome is disclosed in Jo et al., BioMed Res. Int. [International Biomedical Research] 2015: 305716 (2015). Co-delivery of Cas9 and sgRNA using nanoparticles is disclosed in Mout et al., ACS Nano [American Chemical Society Nano] 11 (3): 2452-58 (2017).

[0160] In some embodiments, the agent comprises a transposon-based gene editing system. An example of a suitable transposon-based gene editing system for use in the present disclosure provided herein is a gene writer system described in International Publication No. WO 2020 / 047124, published on March 5, 2020, the contents of which are incorporated herein by reference in their entirety.

[0161] In some embodiments, the agent comprises a transcription activator-like effector nuclease (TALEN) system. The TALEN-based system comprises a protein containing a TAL effector DNA binding domain and an enzyme domain. They are prepared by fusing the TAL effector DNA binding domain with a DNA cleavage domain (a nuclease that cuts the DNA chain). The above-mentioned FokI restriction enzyme is an exemplary enzyme domain suitable for use in a TALEN-based gene regulation system.

[0162] TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when the bacteria infects plants. The DNA binding domain contains a repeated, highly conserved sequence of 33-34 amino acids, with the 12th and 13th amino acids being different. These two positions, called repeat variable diresidues (RVDs), are highly variable and strongly associated with specific nucleotide recognition. Therefore, TAL effector domains can be engineered to bind to specific target DNA sequences by selecting a combination of repeat segments containing appropriate RVDs. The nucleic acid specificity of the RVD combinations is as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (although, in some embodiments, NN can also bind to adenine with lower specificity).

[0163] 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 to, or 100% identical to, a target DNA sequence of a 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 to, or 100% identical to, a target DNA sequence defined by a set of genomic coordinates.

[0164] In some embodiments, the gene regulation system comprises two or more TAL effector fusion proteins (each comprising a TAL effector domain), wherein at least one TAL effector domain binds to a target DNA sequence of a 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 to a target DNA sequence defined by a set of genomic coordinates, or 100% identical thereto.

[0165] Methods and compositions for assembling TAL effector repeat sequences 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 constructing TAL effector repeat sequences are commercially available, for example, from Advantgene.

[0166] In some embodiments, the agent comprises a zinc finger nuclease (ZFN) system. Commercially available plasmids (e.g., plasmid pair (CSTZFN-1KT) from Sigma Aldrich (St. Louis, Missouri) can be used. Custom zinc finger nuclease (ZFN) (R-3257609)) to generate ZFN domains. Plasmids can be prepared using a commercial system according to the manufacturer's protocol (e.g., NEB Monarch Miniprep (Cat. No. T1010), New England Biolabs, Ipswich, MA).

[0167] In some embodiments, the medicament comprises a vector intended to deliver conventional gene therapy (e.g., gene knockout or knock-in via homologous recombination). Non-limiting examples of the vector include retroviruses (e.g., lentivirus 5), adenoviruses, adeno-associated viruses, herpes simplex viruses, nanoparticles, and DNA transposons. D. Small molecule drugs

[0168] 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 a target protein. In some embodiments, the small molecule is an inhibitor (e.g., a direct inhibitor, an indirect inhibitor) of a target protein. In some embodiments, the small molecule is an activator (e.g., a direct activator and an indirect activator) of a target protein.

[0169] Examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic compounds, organometallic compounds or inorganic compounds. The atoms in small molecules are typically linked together via covalent bonds and / or ionic bonds. In certain embodiments, small molecules are small organic molecules. The arrangement of atoms in small organic molecules can represent chains (e.g., carbon-carbon chains or carbon-heteroatom chains), or can represent rings containing carbon atoms (e.g., benzene or polycyclic systems) or rings of combinations of carbon and heteroatoms (i.e., heterocycles, such as pyrimidine or quinazoline). Although small molecules can have a wide range of molecular weights, they generally include molecules less than about 5,000 daltons. For example, such small molecules can be less than about 1000 daltons, and preferably less than about 750 daltons, or more preferably less than about 500 daltons. Small molecules can be found in nature (e.g., identification, separation, purification) and / or synthetically produced (e.g., by traditional organic synthesis, biologically mediated synthesis or a combination thereof). See, e.g., 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 their derivatives.

[0170] In certain embodiments, the agent comprises a proteolysis targeting chimera (PROTAC).

[0171] 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. E. Therapeutic cells and cell-based therapies

[0172] In some embodiments, the medicament comprises a therapeutic cell. In certain embodiments, the therapeutic cell expresses and / or is engineered to express a target protein as described herein (e.g., a target protein in a sequence table, Table A, or a variant thereof), a polypeptide (e.g., an antibody, an antigen binding fragment, or a polypeptide comprising an amino acid sequence at least 70% identical to at least a portion of a target protein), a polynucleotide (e.g., a recombinant DNA, an RNA (such as an mRNA or an siRNA)), and / or a gene editing system (e.g., a CRISPR / Cas system).

[0173] In some embodiments, the polypeptide disclosed herein (e.g., an antibody or antigen binding fragment) is incorporated into a cell-based therapy. 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 macrophages (CAR-M)). In some embodiments, CAR comprises a transmembrane domain and an antigen recognition portion, wherein the antigen recognition portion 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 a specific embodiment, the polypeptide is a cytokine receptor expressed on the membrane of a CAR-T, CAR-NK, or CAR-M cell. In a more specific embodiment, the polypeptide is a cytokine secreted from a CAR-T, CAR-NK, or CAR-M cell.

[0174] Therapeutic cells suitable for use in the compositions, kits and methods disclosed herein can be produced, identified and / or enriched using methods known to those of ordinary skill in the art. Non-limiting examples of the method include purifying, propagating and / or differentiating cells from subjects (e.g., humans) into specific cell products; engineering somatic cells for gene therapy; cell immortalization; ex vivo genetic modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technology); in vivo genetic modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technology); genome editing; cell plasticity technology; genetic modification; and flow cytometry. In some embodiments, therapeutic cells are autologous or isogenic. In other embodiments, therapeutic cells are allogenic. Expression vectors and hosts

[0175] In another aspect, the present disclosure provides an expression vector comprising a polynucleotide described herein.

[0176] The term "expression vector" refers to a replicable nucleic acid from which one or more proteins can be expressed when the expression vector is transformed into an appropriate expression host cell.

[0177] In some embodiments, the expression vector comprises an expression control polynucleotide sequence operably connected to the 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 DNA region to which RNA polymerase binds and initiates gene transcription. The term "operably connected" means that nucleic acid is located in a recombinant polynucleotide (e.g., a vector) in such a way that nucleic acid can be expressed under the control of the element to which it is connected (e.g., a promoter). The term "selectable marker element" is an element that imparts a trait suitable for artificial selection. The selectable marker element can be a negative or positive selection marker. Non-limiting examples of expression vectors for use with bacteria, fungi, yeast, and mammalian cell hosts are described in Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual] (Michael R. Green and Joseph Sambrook, ed., 4th edition 2012).

[0178] In another aspect, the present disclosure provides an expression host cell comprising any one or more of the polynucleotides or expression vectors described herein.

[0179] The term "expression host cell" refers to a cell that can be used to receive, maintain, replicate and / or amplify a vector.

[0180] Non-limiting examples of expression host cells include mammalian cells, such as hybridoma cells, baby hamster kidney fibroblasts (BHK cells), Chinese hamster ovary (CHO) cells, COS cells, HeLa cells, and human embryonic kidney (HEK) cells; yeast cells, such as Pichia pastoris cells; or bacterial cells, such as DH5α, etc. For methods of host cell culture for producing protein therapeutics, see, e.g., Mammalian Cell Cultures for Biologics Manufacturing (Weichang Zhou and Anne Kantardjieff, eds., 2014); for purification of protein therapeutics, see, e.g., Protein Biotechnology: Isolation, Characterization, and Stabilization (Felix Franks, ed., 2013) and Protein Purification Protocols (Paul Cutler, ed., 2010); and for formulation of protein therapeutics, see, e.g., Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic (Brian K Meyer, ed., 2012).

[0181] Techniques known in the art (including transformation, electroporation and transduction) can be used to introduce polynucleotides or expression vectors described herein into suitable or desired host cells. The introduced nucleic acid can be extrachromosomal in the host cell, or can be integrated into the genome of the host cell. Pharmaceutical composition

[0182] In another aspect, the disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutical agent disclosed herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a composition or its final dosage form or formulation having pharmacological activity or other direct effect in alleviating, treating or preventing cancer.

[0183] In some embodiments, the composition (e.g., pharmaceutical composition) comprises a pharmaceutically acceptable carrier, excipient, stabilizer, diluent or tonifier (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents or supplements 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 TM and polyethylene glycol (PEG).

[0184] In some embodiments, the agent (e.g., polypeptide, polynucleotide, or small molecule) of the pharmaceutical composition is modified, for example, conjugated to a heterologous moiety. The term "conjugated" refers to attachment via covalent or non-covalent interactions. Conjugation can employ any suitable linker; non-limiting examples include peptide linkers, compound linkers, and chemical cross-linkers.

[0185] In some embodiments, the heterologous moiety is a label (e.g., a fluorescent label or a radioactive label), a molecule that stabilizes the agent, a molecule that targets the agent (e.g., to specific cells or tissues to promote or prevent crossing the blood-brain barrier), or a combination thereof.

[0186] In some embodiments, the heterologous part is polyethylene glycol (PEG), hexadecene acid, hydrogel, nanoparticle, polymerized domain and carrier peptide. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is a polymer nanoparticle. In some embodiments, the polymer is an amphipathic polymer. In other embodiments, the polymer is a hydrophobic or hydrophilic polymer. The non-limiting examples of polymers include poly (lactic acid)-poly (ethylene glycol), poly (lactic acid-co-glycolic acid)-poly (ethylene glycol), poly (lactic acid-co-glycolic acid) (PLGA), poly (lactic acid-co-glycolic acid)-d-α-tocopheryl polyethylene glycol succinate, poly (lactic acid-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 nanoparticle comprises poly (lactic acid-co-glycolic acid) (PLGA).

[0187] In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for suitable administration regimens and routes. Non-limiting examples of routes of administration include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical. In some embodiments, the composition (e.g., 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).

[0188] In some embodiments, the composition is formulated to be administered together with one or more additional therapeutic agents (e.g., with a second therapeutic agent) as a combination therapy. As used herein, "combination therapy" or "combined administration" means that two (or more) different medicaments or treatments are administered to a subject as part of a defined treatment regimen for a specific disease or condition. Non-limiting examples of additional medicaments or treatments include biological agents (e.g., antibodies, peptides), steroid hormones, protein replacement therapy, substrate therapy, enzyme therapy, cell therapy, gene therapy, small molecules, and medicaments that affect metabolic activity.

[0189] The treatment regimen defines the dosage and cycle of each medicament used so that the effect of a single medicament on a subject overlaps. In certain embodiments, two or more medicaments are used in a sequential manner as a part of a prescription regimen. In other embodiments, the delivery of two or more medicaments is simultaneous or parallel. In certain embodiments, two or more medicaments are co-prepared. In certain embodiments, two or more medicaments or treatments are used in combination so that the decline of symptoms or other parameters related to the disorder is greater than the situation observed when a single medicament or treatment is delivered or when there is no other. The effects of the two treatments can be partially accumulated, completely accumulated or greater than accumulated (for example, collaboratively). Each of the two or more therapeutic agents can be administered by any appropriate route including but not limited to oral route, intravenous route, intramuscular route and directly absorbed by mucosal tissue. Two or more therapeutic agents can be administered by the same route or by different routes.

[0190] In some embodiments, the pharmaceutical agents or compositions of the present disclosure are delivered via a viral vector (e.g., by contacting a cell with a viral vector), administered locally (e.g., injected) to a tumor, or administered systemically (e.g., intravenously or orally) to a subject (e.g., a human patient).

[0191] Viral genomes provide a rich source of vectors that can be used to effectively deliver foreign genes to mammalian cells. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained in such genomes are typically incorporated into the nuclear genome of mammalian cells by universal or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Non-limiting examples of viral vectors include retroviruses (e.g., Retroviridae virus 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 virus)), positive-strand RNA viruses (such as picornaviruses and alphaviruses), and double-stranded DNA viruses (including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus, replication-defective herpes virus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox virus)). Additional 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 virus, avian C virus, mammalian C virus, B virus, D virus, oncorretrovirus, HTLV-BLV group viruses, lentiviruses, alpharetroviruses, gammaretroviruses, foamy viruses (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). Additional non-limiting examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, monkey sarcoma virus, Rous sarcoma virus, and lentivirus. Additional non-limiting examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the teachings of which are incorporated herein by reference.

[0192] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered in vivo, in vitro, ex vivo or in situ by a membrane-based carrier. In some embodiments, the membrane-based carrier is a cell-based carrier (e.g., a mammalian (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 described herein (e.g., a plasmid, a virus, a virus-like particle, or a virosome).

[0193] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more liposomes. Liposomes are spherical vesicle structures consisting of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and load and transport them across biological membranes and blood-brain barriers (BBB) ​​(see, e.g., Spuch and Navarro, J Drug Deliv. [Drug Delivery Magazine] 2011: 469679 (2011)).

[0194] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. Pat. No. 6,693,086, which is incorporated herein by reference for its teachings on the preparation of multilamellar vesicle lipids). Although vesicle formation can be spontaneous when the lipid film is mixed with an aqueous solution, it can also be accelerated by applying force in the form of oscillation using a homogenizer, sonicator, or extrusion device (see, e.g., Spuch and Navarro, J Drug Deli v [Journal of Drug Delivery] 2011: 469679 (2011). Extruded lipids can be prepared by extrusion through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15: 647-52 (1997), which is incorporated herein by reference for its teachings on the preparation of extruded lipids.

[0195] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by lipid nanoparticles (LNP). In one embodiment, the LNP preparation comprising the medicament or pharmaceutical composition of the present disclosure has one or more of the following features: (a) the LNP preparation comprises a cationic lipid, a neutral lipid, cholesterol and a PEG lipid, (b) the average particle size of the LNP preparation is between 80nm and 160nm.

[0196] Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of SLNs, improve drug stability and drug loading, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of 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 that combines 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. Therefore, the two components increase drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs. See, for example, Li et al., Nanomaterials 7(6): 122 (2017).

[0197] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by a carbohydrate carrier (e.g., anhydride-modified phytoglycogen or glycogen-based material). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin.

[0198] In certain embodiments, the medicament 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 polyribonucleotide). Non-limiting examples of protein carriers include human serum albumin (HAS), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and globulin.

[0199] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by a cationic carrier (e.g., a cationic lipopolymer or a transfection reagent). Non-limiting examples of cationic carriers include lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, 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-oleyl-3-( The products were 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propane ammonium trifluoroacetate (DOSPA), 3B-[N-('\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol hydrochloride), diheptadecylamido glycylidene 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).

[0200] In some embodiments, the pharmaceutical agents or compositions of the present disclosure are formulated for delivery via exosomes, adipocytes, and / or erythrocytes. See, e.g., Ha et al., Acta Pharm Sin B. [Pharmaceutical Journal B] 6(4): 287-96 (2016).

[0201] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more fusosomes. Fusosomes have been engineered to confer target cell specificity for fusion and payload delivery, allowing the generation of delivery vehicles with programmable cell specificity. See, for example, patent application WO 2020014209, which is incorporated herein by reference in its teachings on fusosome design, preparation and use.

[0202] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated for delivery via ex vivo differentiated erythrocytes. See, e.g., WO 2015073587; WO 2017123646; WO 2017123644; WO 2018102740; WO2016183482; WO 2015153102; WO 2018151829; WO 2018009838; Shi et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 111(28):10131-36 (2014); U.S. Patent 9,644,180; Huang et al., Nature Communications [Nature Communications] 8:423 (2017).

[0203] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more microsomes, virus-like particles (VLPs) or plant nanovesicles and plant messenger packages (PMPs). See, for example, WO 2011097480, WO2013070324, WO 2017004526 and WO 2020041784.

[0204] In certain embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more anellosomes. The preparation and use of anellosomes for delivering therapeutic products are described in U.S. Patent No. 11,166,996, and its teaching about anellosome design, preparation and use is incorporated herein by reference. Methods for regulating target proteins

[0205] On the other hand, the present disclosure provides a method for regulating the expression or activity of a target protein identified in the sequence listing, Table A, or the aforementioned variants 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 or a pharmaceutical composition comprising an agent, the agent comprising the target protein identified herein and / or regulating the expression or activity of the target protein.

[0206] The target cell can be of any cell type. In some embodiments, the target cell is a liver cell (e.g., a hepatocyte (HC), a hepatic stellate cell (HSC), a Kupffer cell (KC), and / or a liver sinusoidal endothelial cell (LSEC)); a pancreatic cell (e.g., an α cell, a β cell, a δ cell, and / or a PP cell); a thyroid cell; a glandular cell; or a combination thereof. In a specific embodiment, the target cell is a hepatocyte (HC), a Kupffer cell (KC), a pancreatic β cell, a muscle cell, a heart cell, a brain cell, a kidney cell, a fat cell, or a combination thereof.

[0207] In some embodiments, the target cell is involved and / or participates in inflammation. In certain embodiments, the target cell is an epithelial cell, an endothelial cell, a stem cell, a non-immune cell, or a combination thereof.

[0208] In some embodiments, the target cell is involved and / or participates in fibrosis, aging, senescence, or a combination thereof. In certain embodiments, the target cell is a stem cell, an epithelial cell, an endothelial cell, a non-immune cell, or a combination thereof.

[0209] In certain embodiments, target cell is an immune cell. In certain embodiments, target cell is effector T cell, helper T cell, Th1 cell, Th2 cell, Th17 cell, B cell, natural killer (NK) cell, innate lymphocyte (for example, ILC1 cell, ILC2 cell, ILC3 cell), macrophage (for example, M1 macrophage, M2 macrophage), monocyte and / or antigen presenting cell (for example, dendritic cell) or aforesaid combination.

[0210] In some embodiments, the target protein of the present disclosure is used to mediate the depletion of a cell population (e.g., a cancer cell (such as a tumor cell) population; an immune cell population). In some embodiments, the target protein of the present disclosure facilitates cell targeting (e.g., delivering a therapeutic agent in a cell type-specific manner), for example as a conjugate of a surface marker.

[0211] The target tissue can be any tissue in the body.

[0212] The target tissue can be any gland of the body (e.g., adrenal glands, pituitary glands, parathyroid glands, and / or pineal glands) or reproductive tissue (e.g., ovaries, testicles). In certain embodiments, the target tissue comprises liver, pancreas, thyroid gland, ovaries, testicles, muscle, heart, brain, kidney, adipose tissue, or a combination thereof. In some embodiments, the target tissue comprises adrenal glands, pituitary glands, parathyroid glands, pineal glands, or a combination thereof.

[0213] In some embodiments, the target tissue is an immune tissue. In some embodiments, the target tissue is a non-immune tissue. In some embodiments, the target tissue comprises a lymph node, a spleen, a secondary lymphoid organ, a tertiary lymphoid organ, a barrier tissue, skin, an intestinal tract, an airway, a wound, an immune tissue, a non-immune tissue, or a combination thereof.

[0214] 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%.

[0215] 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%, 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 increase 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%. 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.

[0216] 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 a combination of the foregoing. In some embodiments, the effective amount is sufficient to modulate such a signaling pathway, which involves a Janus kinase (JAK)-signal transduction factor family (e.g., JAK1, JAK2, JAK3, and TYK2), a member of the signal transduction factor and transcription activator (STAT) protein family (e.g., STAT1, STAT3), a member of the protein kinase B family (e.g., RAC-α, RAC-β, or RAC-γ serine / threonine protein kinase), a member of the interferon regulatory factor (IRF) family, a mitogen-activated protein kinase (MAPK), or a combination of the foregoing. Diagnosis and treatment methods

[0217] 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 a subject suffering from a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, wherein the expression or activity level of the target protein in the sample indicates the likelihood of the subject suffering from the disease or condition, wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infections, immune diseases (e.g., inflammatory and / or autoimmune diseases), indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

[0218] In another aspect, the present disclosure provides a method for classifying a subject based on a predicted likelihood of developing a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample 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 disease, cardiovascular disease, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infection, immune diseases (e.g., inflammatory and / or autoimmune diseases), indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

[0219] In another aspect, the present disclosure provides a method for stratifying a group of subjects having a disease or condition, the method comprising: quantifying the expression and / or activity of a target protein in a sample from an individual subject in the group; and stratifying the group of subjects for treatment according to the expression and / or activity level of the target protein in the sample from the individual subject, wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic disease, cardiovascular disease, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infection, immune disease (e.g., inflammatory and / or autoimmune disease), indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

[0220] In some embodiments, a higher level of expression or activity of a target protein in a sample from a subject relative to an appropriate control (e.g., a reference standard) indicates a disease or disorder or the likelihood of developing a disease or disorder. In some embodiments, a lower level of expression or activity of a target protein in a sample from a subject relative to an appropriate control (e.g., a reference standard) indicates a disease or disorder or the likelihood of developing a disease or disorder.

[0221] In some embodiments, the method further comprises administering an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein to a subject determined to have or predicted to have a likelihood (or risk) of developing a disease or condition.

[0222] In some embodiments, the method further comprises administering an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein to a subject determined to have or predicted to have a likelihood of developing the disease or condition.

[0223] In another aspect, the present disclosure provides a method for preparing a sample that can be used to detect the likelihood of a subject suffering from a disease or condition, the method comprising: a) obtaining or having obtained a sample from a subject; b) adding a protease inhibitor, a control peptide, a standard peptide, or a combination thereof to the sample to prepare a sample that can be used to detect the possibility of having cancer; and c) quantifying the expression or activity of the target protein in the sample prepared in step b); The disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer (e.g., tumors), infections, immune diseases (e.g., inflammatory and / or autoimmune diseases), indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

[0224] In another aspect, the disclosure provides a method of treating a disease or disorder 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.

[0225] In another aspect, the present disclosure provides a method for 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 expression and / or activity level of a target protein disclosed herein in the subject is altered.

[0226] As used herein, "treatment" and "treating" refer to the medical management of a subject that is intended to improve, alleviate, stabilize (i.e., not worsen), prevent, or cure a disease, pathological condition, or disorder. "Treatment" includes active treatment (treatment intended to improve a disease, pathological condition, or disorder), etiological treatment (treatment directed at the cause of the relevant disease, pathological condition, or disorder), palliative treatment (treatment intended to relieve symptoms), preventive treatment (treatment intended to minimize or partially or completely inhibit the development of the relevant disease, pathological condition, or disorder); and supportive treatment (treatment used to supplement another therapy). Treatment also includes reducing the extent of a disease or disorder; preventing the spread of a disease or disorder; delaying or slowing the progression of a disease or disorder; alleviating or relieving a disease or disorder; and relief (whether partial or complete), whether detectable or undetectable. "Relieve" or "mitigate" a disease or disorder means that the extent of the disease, disorder, or disorder and / or the reduction of undesirable clinical manifestations and / or the time course of progression are slowed or prolonged compared to the extent or time course in the absence of treatment. "Treatment" also includes prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0227] In some embodiments, the subject is an animal. In other embodiments, the subject is a bird, such as a hen, a rooster, a turkey, or a 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 cattle (e.g., dairy cows or beef cattle), sheep, goats, pigs, horses, dogs, cats, mice, rats, etc. 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 teenager. In some embodiments, the human is an adult. In some embodiments, the human is less than 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, for example, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old.

[0228] As used herein, the term "effective amount", "therapeutically effective amount" or "sufficient amount" refers to an amount sufficient to achieve treatment (e.g., produce a beneficial or desired result) (including effects at the cellular, tissue or clinical level, etc.) when administered to a subject (e.g., a mammal, such as a human cancer patient). Therefore, the term depends on the context in which it is applied. For example, in the context of treating cancer, it is an amount of drug sufficient to achieve a response compared to a response obtained without the administration of the agent. The amount of a given composition described herein corresponding to such an amount will vary according to various factors such as the following: a given agent, a pharmaceutical formulation, an administration route, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or the host receiving treatment, etc., but can still be routinely determined by those skilled in the art. In some embodiments, the "therapeutically effective amount" of the composition disclosed herein is an amount that produces a beneficial or desired result (e.g., compared to a control) in a subject. The therapeutically effective amount of the composition disclosed herein can be easily determined by ordinary technicians by conventional methods known in the art. The dosage regimen can be adjusted to provide the best therapeutic response.

[0229] The therapeutic agents described herein can be administered via a variety of 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., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration, depending on the compound and the specific disease or condition to be treated. Administration can be local or systemic, as indicated. The preferred mode of administration can vary depending on the specific compound selected.

[0230] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more additional therapeutic agents (eg, a second therapeutic agent).

[0231] Administration of two or more therapeutic agents encompasses co-administration of therapeutic agents in a substantially simultaneous manner (such as in a drug combination). Alternatively, such administration encompasses co-administration of each therapeutic agent in multiple containers or separate containers (e.g., capsules, powders, and liquids). Such administration also encompasses the use of therapeutic agents in a sequential manner at approximately the same time or at different times. When two or more therapeutic agents are administered, the therapeutic agents may be administered via the same route of administration or via different routes of administration.

[0232] In another aspect, the disclosure provides a method for regulating the expression or activity of a target protein identified in the sequence listing, Table A, or a variant thereof 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 in a subject.

[0233] In another aspect, the present disclosure provides a method for identifying an agent that modulates the expression and / or activity of a target protein (e.g., a target protein in the sequence listing, Table A, or a variant of the foregoing), the method comprising: a) contacting a sample (e.g., a biological sample, such as a cell or tissue) comprising a target protein with an agent (e.g., a candidate agent to be tested for its ability to modulate expression and / or activity of the target); and b) determining whether the agent modulates the expression or activity of a target protein, A difference in the expression or activity of a target protein that has been contacted with the agent compared to a reference indicates that the agent modulates the expression or activity of the target protein.

[0234] In some embodiments, a difference of at least about 10% in the expression or activity of a protein that has been contacted with an agent 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.

[0235] In some embodiments, a decrease in the expression or activity of a target protein that has been contacted with an agent 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 compared to a reference indicates that the agent activates the expression or activity of the protein. Indications aging

[0236] The methods described herein are suitable for treating aging, age-related conditions and / or age-related disorders or diseases. In some embodiments, the methods described herein include treating general health, body temperature, weight, height, waist circumference, fertility, body fat, heart rate, blood pressure, pulse rate, blood oxygen level, respiratory rate, breathing pattern, blood sugar level, blood pH, cardiac output, heart rhythm, and the concentration of certain substances in the blood. In some embodiments, treatment can be assessed by measuring complete blood count, red blood cells, white blood cells, platelets, hemoglobin, hematocrit, mean corpuscular volume, basic metabolic panel, blood sugar, 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 test. senescence

[0237] In some embodiments, the methods disclosed herein are used to treat aging. In still other 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 associated with photosensitivity or photoaging. In still other 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, high cholesterol, hypertension, motor neuron disease, multiple sclerosis, osteoporosis, Paget's disease of bone, Parkinson's disease, herpes zoster and stroke. Fibrosis

[0238] 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. Metabolic diseases

[0239] In some embodiments, the disclosure provides treatment of metabolic diseases and / or disorders. In some embodiments, treatment relates to delaying or preventing the onset of metabolic disorders. In some embodiments, treatment relates to delaying or preventing the onset of complications associated with one or more metabolic disorders. In still other embodiments, treatment relates to hormone or enzyme therapy. In some embodiments, metabolic disorders are caused by genetic defects. In still other embodiments, metabolic disorders are selected from the group consisting of: familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease and Wilson disease. Cardiovascular disease

[0240] In some embodiments of the present disclosure, the method of the present disclosure relates to the treatment of cardiovascular disease. In some embodiments, cardiovascular disease is atherosclerosis, congestive heart failure, vulnerable plaque, stroke or ischemia. In still other embodiments, cardiovascular disease is coronary artery disease, peripheral artery disease or carotid artery disease. In other embodiments of the present disclosure, the method relates to the treatment of cardiovascular disease or disorder-related symptoms. In some embodiments, such symptoms include chest tightness or chest pain, shortness of breath, pain or discomfort in the arm or shoulder, pain or discomfort in the jaw, neck or back, feeling weak, dizzy or nausea. In some embodiments, symptoms to be addressed may include abnormal fatigue, sleep disorders, shortness of breath and / or indigestion. Endocrine-related disorders

[0241] In some embodiments, the method disclosed herein relates to the treatment of endocrine-related diseases or disorders. In some embodiments, the method relates to the treatment of endocrine glands. In still other embodiments, endocrine glands include but are not necessarily limited to adrenal glands, hypothalamus, ovaries, islet cells of the pancreas, parathyroid glands, pineal glands, pituitary glands, testicles, thymus and / or thyroid gland. In some embodiments, the treatment according to the disclosed method relates to diseases, problems with the endocrine feedback system, glands that cannot stimulate another gland to release hormones, hereditary disorders, infections, endocrine gland damage and / or tumors of endocrine glands. In some embodiments, endocrine-related disorders are adrenal insufficiency, Cushing's disease, gigantism (acromegaly), hyperthyroidism, hypothyroidism, hypopituitarism, multiple endocrine tumors I and II, polycystic ovary syndrome and precocious puberty. Genetic disorders

[0242] In some embodiments, the method relates to a method for treating a genetic disease. The genetic diseases applicable to the methods of treatment of the present disclosure include but are not limited to arrhythmogenic right ventricular dysplasia / cardiomyopathy, Alzheimer's disease, arthritis, autism spectrum disorder, Bruga syndrome, cancer, Charcot-Marie-Tooth disease, cleft lip and palate, cranio-clavicular dysplasia, cystic fibrosis, diabetes, Down syndrome, fragile X syndrome, familial adenomatous polyposis, Hirschsprung disease, Huntington's disease, Klinefelter syndrome, Kneist syndrome (Kneist Syndrome), Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, sickle cell disease, spina bifida, Tay-Sachs disease, trisomy X syndrome, Turner syndrome, trisomy 18, trisomy 13 and Von Hippel-Lindau disease (Von Hippel-Lindau). In some embodiments, the genetic disease is chromosomal. In other embodiments, the genetic disease is complex and is derived from a combination of gene mutations and other factors (e.g., diet, certain drugs, smoking, drinking, etc.). In still further embodiments, the genetic disease is a single gene genetic disease. Oncology

[0243] In some embodiments of the present disclosure, the method relates to the treatment of cancer. The cancer treatment provided by the present disclosure includes carcinoma, sarcoma, melanoma, lymphoma and / or leukemia. In some embodiments, the method of the present disclosure can replace another treatment regimen, before another treatment regimen, or after another treatment regimen. In some embodiments, another treatment regimen can include, but is not limited to, chemotherapy, radiotherapy, surgery, hormone therapy, biological response modifier therapy, immunotherapy and / or bone marrow transplantation. immunity

[0244] In some embodiments, the methods of the present disclosure relate to immunity. In some embodiments, immunity relates to bacteria, parasites, viruses, fungi and / or cancer cells. In some embodiments, immunity is autoimmunity and is the result of the immune system attacking its own molecules. In some embodiments, the methods relate to the treatment of immune tolerance. Inflammation

[0245] In some embodiments of the present disclosure, the method relates to the treatment of inflammation. In some embodiments, inflammation is acute, or has a relatively short duration, lasting for minutes to hours. In still other embodiments, inflammation is chronic, or has a longer duration, lasting for weeks to months, and may last for 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, fever, fatigue, fever, joint pain or stiffness, mouth sores, rash. In some embodiments, inflammation is caused by another disease or disorder. Autoimmunity

[0246] In some embodiments, the disclosed methods relate to the treatment of autoimmune diseases. In some embodiments, autoimmune diseases may include, but are not limited to, joint and muscle diseases (e.g., psoriatic arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus), digestive tract diseases (e.g., Crohn's disease, celiac disease, ulcerative colitis, inflammatory bowel disease), endocrine system diseases (e.g., Graves' disease, Hashimoto's thyroiditis, Addison's disease), skin diseases (e.g., dermatomyositis, psoriasis, scleroderma), nervous system diseases (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). Other treatments

[0247] In still further embodiments, the methods of the present disclosure relate to the treatment of any indication currently utilizing any hormone, growth factor, or protein replacement.One skilled in the art will appreciate that the therapeutic effect need not be complete, as long as some benefit is provided to the patient.

[0248] In some embodiments, an endocrine organ is any organ that secretes proteins into the circulation.

[0249] In some embodiments, the effective amount is sufficient to modulate (eg, increase or decrease) metabolic activity, cell proliferation, cell metastasis, cell migration, autophagy, apoptosis, endocrine function, or a combination thereof.

[0250] In some embodiments, the effective amount is sufficient to reduce (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 reduce metabolic activity, cell proliferation, cell metastasis, cell migration, apoptosis, endocrine function, or a combination thereof by at least about 10%, for example, by 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%.

[0251] In some embodiments, the effective amount is sufficient to increase (e.g., promote) cell proliferation, metabolic activity, autophagy, apoptosis, endocrine function, or a combination thereof. In certain embodiments, the effective amount is sufficient to increase metabolic activity, cell proliferation, cell metastasis, cell migration, apoptosis, endocrine function, or a combination thereof by at least about 10%, for example, by 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%, 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%.

[0252] In some embodiments, 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)), cell maturation (e.g., stem cell maturation), enzyme activity, or a combination thereof. Immune / Inflammation

[0253] In some embodiments, the methods of the present disclosure relate to immunity. In some embodiments, immunity relates to bacteria, parasites, viruses and / or cancer cells. In some embodiments, immunity is autoimmunity and is the result of the immune system attacking its own molecules. In some embodiments, the methods relate to the treatment of immune tolerance.

[0254] In some embodiments of the present disclosure, the method relates to the treatment of inflammation. In some embodiments, inflammation is acute, or has a relatively short duration, lasting for minutes to hours. In still other embodiments, inflammation is chronic, or has a longer duration, lasting for weeks to months, and may last for 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, fever, fatigue, fever, joint pain or stiffness, mouth sores, rash. In some embodiments, inflammation is caused by another disease or disorder.

[0255] In some embodiments, the disclosed methods relate to the treatment of autoimmune diseases. In some embodiments, autoimmune diseases may include, but are not limited to, joint and muscle diseases (e.g., psoriatic arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus), digestive tract diseases (e.g., Crohn's disease, celiac disease, ulcerative colitis, inflammatory bowel disease), endocrine system diseases (e.g., Graves' disease, Hashimoto's thyroiditis, Addison's disease), skin diseases (e.g., dermatomyositis, psoriasis, scleroderma), nervous system diseases (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).

[0256] In certain embodiments, disease or illness is inflammatory disease and / or autoimmune disease.Various inflammatory and / or autoimmune diseases are treatable according to the methods described herein.In certain embodiments, 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-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 its combination.

[0257] In some embodiments, the target protein activates an immune response. In certain embodiments, the target protein inhibits an immune response. In certain 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 activation of T cell-mediated immune responses (e.g., cytokine production and cytotoxicity), B cell-mediated immune responses, humoral immune responses, and cytokine response cells (e.g., macrophages).

[0258] In some embodiments, the target protein enhances the signal involved in T cell activation and / or survival. In certain embodiments, the target protein activates stimulatory checkpoint molecules. Non-limiting examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, glucocorticoid-induced TNFR family-related genes (GITR), inducible T cell co-stimulators (ICOS). In a specific embodiment, the target protein is an agonist for CD28.

[0259] In some embodiments, the Target Protein reduces signals involved in T cell anergy and / or exhaustion. In certain embodiments, the Target Protein inhibits inhibitory checkpoint molecules. 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 attenuation factor (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), V domain Ig inhibitor 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 a specific embodiment, the target protein is an inhibitor for PD-1.

[0260] In some embodiments, an effective amount is sufficient to modulate (e.g., increase or decrease): a) Development of high endothelial venules (HEV) and / or tertiary lymphoid organs (TLOs); b) immune cell activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) Immune cell lymph node egress and / or homing; d) immune cell tumor egress and / or homing; e) cytokine production; f) Antigen presentation; g) target protein expression; or h) Autoantibody levels, or a combination thereof.

[0261] In some embodiments, the effective amount is sufficient to increase: a) Development of HEV and / or TLO; b) immune cell activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) lymph node egress and / or homing of immune cells; d) immune cell tumor egress and / or homing; e) cytokine production; f) Antigen presentation; g) target protein expression; or h) Autoantibody levels, or a combination thereof.

[0262] In certain embodiments, the increase 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 increase 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%.

[0263] In some embodiments, the effective amount is sufficient to reduce: a) Development of HEV and / or TLO; b) immune cell activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) lymph node egress and / or homing of immune cells; d) immune cell tumor egress and / or homing; e) cytokine production; f) Antigen presentation; g) target protein expression; or h) Autoantibody levels, or a combination thereof.

[0264] 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 particular 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, an effective amount is sufficient to: a) increase organ function; b) modulate (e.g., increase or decrease) inflammation; c) reduce the level of autoantibodies; d) reduce the rate and / or frequency of recurrence and / or flare; e) reduce viral load; or f) reduce (e.g., control) infection, or a combination of the foregoing.

[0266] In some embodiments, an 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 particular 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%.

[0267] In some embodiments, the effective amount is sufficient to reduce inflammation, autoantibody levels, relapse and / or outbreak rate and / or number, viral load or infection, or a combination of the foregoing. 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 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%.

[0268] In some embodiments, the target protein activates immune cells. In some embodiments, the target protein inhibits immune cells (e.g., inhibits the activation of immune cells, induces immune cell death (e.g., apoptosis) or a combination thereof). Immune cells are cells that play a role in immune responses. Immune cells have hematopoietic origins, 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 a tumor microenvironment (e.g., on stromal cells) or on non-malignant cells (e.g., immune cells).

[0269] In some embodiments, the effective amount is sufficient to increase the immune response. In certain embodiments, the increase 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 increase 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%.

[0270] In some embodiments, the effective amount is sufficient to reduce the immune response. 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 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%.

[0271] In some embodiments, the effective amount is sufficient to reduce the inflammatory response. 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 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%.

[0272] 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 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%.

[0273] In certain embodiments, the effective amount is sufficient to modulate expression of the target protein in an immune cell.

[0274] In some embodiments (e.g., immune and / or inflammatory treatments), the effective amount is sufficient to modulate (e.g., increase or decrease) migration of immune cells (e.g., antigen presenting cells (such as dendritic cells and / or macrophages) and / or T cells), proliferation of immune cells, recruitment of immune cells (e.g., antigen presenting cells (such as 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 outflow 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., increase pro-inflammatory cytokines, decrease pro-inflammatory cytokines, increase anti-inflammatory cytokines, decrease anti-inflammatory cytokines), degranulation of immune cells, maturation of immune cells, antigen presentation, target protein expression, inflammation, autoantibody levels; increase organ function; reduce the rate and / or number of relapses or flare-ups, viral load; control infection, or a combination of the foregoing. cancer

[0275] A wide variety of cancers are treatable according to the methods described herein. In some embodiments, cancers include solid tumors (e.g., breast tumors, lung tumors, prostate tumors, colon tumors, bladder tumors, ovarian tumors, kidney tumors, stomach tumors, colon tumors, rectal tumors, testicular tumors, head and / or neck tumors, pancreatic tumors, brain tumors, skin tumors). Therefore, in some embodiments, 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, stomach cancer, colon cancer, rectal cancer, colorectal cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, and skin cancer. In some embodiments, 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.

[0276] Examples of cancers that can be treated according to the methods described herein include acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; adrenocortical carcinoma, children; AIDS-related cancers (e.g., Kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma); anal cancer; appendix cancer; astrocytoma, children; atypical teratoid / rhabdoid tumor, children, central nervous system; basal cell carcinoma of the skin; biliary tract cancer; bladder cancer; bladder cancer, children; bone cancer (including Ewing's sarcoma, osteosarcoma and malignant fibrous histiocytoma); brain tumor / carcinoma; breast cancer; Burkitt's lymphoma; carcinoid tumor (gastrointestinal tract); carcinoid tumor, children; cardiac / heart tumor, children; embryonal tumors, children; Fetal tumor, child; Germ cell tumor, child; Primary CNS lymphoma; Cervical cancer; Cervical cancer, child; Cholangiocarcinoma; Chordoma, child; Chronic lymphocytic leukemia (CLL); Chronic myeloid leukemia (CML); Chronic myeloproliferative neoplasms; Colorectal cancer; Colorectal cancer, child; Craniopharyngioma, child; Cutaneous T-cell lymphoma (e.g., mycosis fungoides and Sezary syndrome); Ductal carcinoma in situ (DCIS); Embryonal tumor, CNS, child; Endometrial cancer (uterine cancer); Ependymoma, child; Esophageal cancer; Esophageal cancer, child; Nasal glioma; Ewing's sarcoma; Extracranial germ cell tumor, child; Extragonadal germ cell tumor; Eye (Eye / Ocular) cancer; Intraocular melanoma, child; Intraocular melanoma ; Retinoblastoma; Fallopian tube cancer; Malignant fibrous histiocytoma and osteosarcoma of bone; Gallbladder cancer; Gastric (gastric / stomach) cancer; Gastric (gastric / stomach) cancer, childhood; Gastrointestinal carcinoid tumors; Gastrointestinal stromal tumors (GIST); Gastrointestinal stromal tumors, childhood; Germ cell tumors; Central nervous system germ cell tumors, childhood (eg, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, testicular cancer); Gestational trophoblastic disease; Hairy cell leukemia; Head and neck cancer; Cardiac tumors, childhood; Hepatocellular (liver) cancer; Histiocytosis, Langerhans cell; Hodgkin lymphoma; Hypopharyngeal cancer; Intraocular melanoma; Intraocular melanoma, childhood; Islet cell tumors, pancreatic neuroendocrine Neoplasms; Kaposi's sarcoma; Kidney (renal cell) carcinoma; Langerhans cell histiocytosis; Laryngeal cancer; Leukemia; Lip and oral cancer; Liver cancer; Lung cancer (non-small cell and small cell); Childhood lung cancer; Lymphoma; Male breast cancer; Malignant fibrous histiocytoma of bone and osteosarcoma; Melanoma; Childhood melanoma; Melanoma, intraocular (eye); Childhood intraocular melanoma; Merkel cell carcinoma; Mesothelioma, malignant; Childhood mesothelioma; Metastatic cancer; Metastatic squamous neck carcinoma with occult primary; Midline tract cancer with NUT gene alterations; Oral cancer; Multiple endocrine neoplasm syndrome; Multiple myeloma / plasma cell neoplasms; Mycosis fungoides; Myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms; Myeloid leukemia, chronic (CML);Myeloid leukemia, acute (AML); Myeloproliferative neoplasms, chronic; Nasal and sinus cancer; Nasopharyngeal cancer; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral, lip and oral cavity cancer, and oropharyngeal cancer; Osteosarcoma and malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian cancer, childhood; Pancreatic cancer; Pancreatic cancer, childhood; Pancreatic neuroendocrine tumors; Papillomatosis (children, laryngeal); Paraganglioma; Paraganglioma, childhood; Nasal and sinus cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pheochromocytoma, childhood; Pituitary tumors; Plasma cell neoplasms / multiple myeloma; Pleuropulmonary blastoma; Breast cancer, gestational; Primary central nervous system (CNS) lymphoma; Primary peritoneal cancer; Prostate cancer; Rectal cancer; Recurrent cancer; Renal cell (kidney) cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcomas (e.g., rheumatoid arthritis, childhood) striamus, childhood vascular tumors, Ewing sarcoma, Kaposi sarcoma, osteosarcoma (bone cancer), soft tissue sarcoma, uterine sarcoma); Sezary syndrome; skin cancer; childhood skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; squamous neck cancer with occult primary, metastatic; stomach (stomach / gastric) cancer; stomach (stomach / gastric) cancer, childhood; T-cell lymphoma, skin (e.g., mycosis fungoides and Sezary syndrome); testicular cancer; childhood testicular cancer; laryngeal cancer (e.g., nasopharyngeal, oropharyngeal, hypopharyngeal); thymoma and thymic cancer; thyroid cancer; renal pelvis and ureteral transitional cell carcinoma; ureter and renal pelvis, transitional cell carcinoma; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vaginal cancer, childhood; vascular tumors; vulvar cancer; and Wilms tumor and other childhood kidney tumors. ;

[0277] Metastasis of the above cancers can also be treated according to the methods described herein. In some embodiments, the cancer is a metastatic cancer.

[0278] 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 polyps, basal cell carcinoma, keratinocyte carcinoma, multiple myeloma, serous invasive ovarian cancer, hepatocellular carcinoma, small cell lung cancer, adenocarcinoma, lung adenocarcinoma, non-small cell lung cancer, ovarian cancer, or colorectal cancer.

[0279] In some embodiments, the treatment of: a) inhibiting cancer cell growth, proliferation, metastasis, invasion or migration, or a combination thereof; b) Promote cancer cell death; c) Induce autophagy of cancer cells, or a combination of the foregoing.

[0280] In some embodiments, the effective amount is sufficient to: a) inhibiting cancer cell growth, proliferation, metastasis, invasion or migration, or a combination thereof; b) Promote cancer cell death; c) Induce autophagy of cancer cells, d) or a combination of the foregoing.

[0281] In some embodiments, the effective amount is sufficient to reduce cancer (e.g., tumor) growth, proliferation, metastasis, infiltration, migration, autophagy, or a combination of the foregoing. 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%, 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%.

[0282] 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 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%.

[0283] In some embodiments, the effective amount is sufficient to modulate (eg, increase or decrease) tumor autophagy, such as by increasing at least one tumor suppressor function of autophagy and / or decreasing at least one tumor promoting function of autophagy.

[0284] 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 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%.

[0285] In some embodiments, the effective amount is sufficient to reduce cancer autophagy. 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%, 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%.

[0286] In some embodiments, the effective amount is sufficient to prevent the death of the subject, thereby reducing 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%.

[0287] In some embodiments, the effective amount is sufficient to modulate (eg, increase or decrease) expression of the target protein in cancer (eg, tumor) cells.

[0288] In some embodiments, the effective amount is sufficient to modulate the body's response to cancer, such as by inhibiting the growth of cancer, reducing the malignancy of cancer, inhibiting the metastasis of cancer, promoting remission, modulating (increasing and / or decreasing) immune-mediated responses associated with cancer, or a combination of the foregoing. Immuno-oncology

[0289] In some embodiments, the disclosure provides treatments utilizing the immune system. In some embodiments, the methods are directed to immuno-oncology (e.g., cancer immunotherapy). In still other embodiments, the immune system is an innate immune system. In some embodiments, the immune system is an adaptive immune system. In still other embodiments, treatment is directed to humoral immunity or antibody-mediated immunity. In some embodiments, treatment is directed to cell-mediated immunity, such as cancer. In some embodiments, the methods are directed to treatment at or before early disease progression, and in other embodiments, the methods are directed to treatment at or before late disease progression or prevention of late disease progression. In still other embodiments, immuno-oncology effects are caused by stimulation of the immune system.

[0290] In some embodiments, the effective amount is sufficient to modulate (e.g., increase) the subject's immune system against cancer. In certain embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease): 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, outflow, and homing; c) cytokine production; d) antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP); e) Antigen presentation; f) Target protein expression, g) or a combination of the foregoing.

[0291] In some embodiments, the modulation 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 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%.

[0292] In some embodiments, the modulation is a reduction. 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%, 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%.

[0293] In some embodiments (e.g., immuno-oncology-specific treatments), the effective amount is sufficient to modulate (e.g., increase or decrease) immune cell-related readouts, migration of immune cells (e.g., antigen presenting cells (such as dendritic cells and / or macrophages) and / or T cells), proliferation of immune cells, recruitment of immune cells (e.g., antigen presenting cells (such as 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 homing of immune cells (e.g., dendritic cells and / or T cells), lymphocyte proliferation of immune cells, and / or proliferation of immune cells. Nodal outflow, 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); increasing tumor outflow of immune cells (e.g., regulatory T cells), decreasing tumor outflow of immune cells (e.g., CD8+ T cells), target protein expression, or a combination of the foregoing. aging

[0294] The methods described herein are suitable for treating aging, age-related conditions and / or age-related disorders or diseases. In some embodiments, the methods described herein include treating general health, body temperature, weight, height, waist circumference, fertility, body fat, heart rate, blood pressure, pulse rate, blood oxygen level, respiratory rate, breathing pattern, blood sugar level, blood pH, cardiac output, heart rhythm, and the concentration of certain substances in the blood. In some embodiments, treatment can be assessed by measuring complete blood count, red blood cells, white blood cells, platelets, hemoglobin, hematocrit, mean corpuscular volume, basic metabolic panel, blood sugar, 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 test.

[0295] In some embodiments, the effective amount is sufficient to slow down aging. In certain embodiments, the slowing down 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 mitigation 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%. senescence

[0296] In some embodiments, the methods disclosed herein are used to treat aging. In still other embodiments, the methods disclosed herein relate to the treatment of aging-related diseases or disorders. In some embodiments, the diseases or disorders include, but are not limited to, diabetes, metabolic syndrome, and obesity. In other embodiments, the diseases or disorders are associated with photosensitivity or photoaging. In still other embodiments, the diseases or disorders can 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, high cholesterol, hypertension, motor neuron disease, multiple sclerosis, osteoporosis, Paget's disease of bone, Parkinson's disease, herpes zoster, and stroke.

[0297] 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 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%. Fibrosis

[0298] 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 a condition associated with or caused by 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.

[0299] 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 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%. Infect

[0300] In some embodiments, the disclosure relates to the treatment of infection. In some aspects, the infection is a bacterial infection, a protozoan infection, a viral infection, a bacterial infection, or another pathogenic infection. In some embodiments, the infection is AIDS or HIV, viral hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C), tuberculosis, salmonellosis, Lyme disease, meningococcal disease, influenza, measles, mumps, rubella (e.g., German measles), pneumonia, sexually transmitted diseases (e.g., syphilis, chlamydia, gonorrhea), chronic sinusitis, whooping cough, pertussis, or a combination thereof.

[0301] Unless otherwise defined, all art terms, symbols and other scientific terms or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this disclosure belongs. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be interpreted as representing substantial differences from what is commonly understood in the art. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having the meanings in the context of the relevant art and / or as otherwise defined herein.

[0302] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0303] As used herein, the indefinite articles “a,” “an,” and “the” should be understood to include plural referents, unless the context clearly indicates otherwise.

[0304] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of, for example, one stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprise" may be replaced by the terms "contains" or "includes".

[0305] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of" does not exclude materials or steps that have no substantial effect on the basic and novel features of the claim. Whenever used herein in the context of a certain aspect or embodiment of the present disclosure, any of the terms "comprising," "containing," "including," and "having" may be replaced in some embodiments by the terms "consisting of" or "consisting essentially of" to change the scope of the present disclosure.

[0306] As used herein, the connection term "and / or" between multiple listed elements is understood to cover both single and combined options. For example, where 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 of these options are understood to fall within the meaning and therefore meet the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one option is also understood to fall within the meaning and therefore meet the requirements of the term "and / or".

[0307] When presenting a list, it is understood that each individual element of the list and each combination of the list is a separate embodiment unless otherwise stated. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C." Examples Example 1: Validation of the target protein as a circulating factor in human plasma

[0308] This example shows the target protein of the present disclosure in the detection of human plasma to verify its ability of circulating factors. In this example, plasma samples are obtained, and the first six high-abundance proteins (albumin, IgG, IgA, antitrypsin, transferrin and haptoglobin) are exhausted using Agilent multiple affinity removal spin column, and then 20 proteins with the highest abundance (albumin, IgG, IgA, IgM, IgD, transferrin, fibrinogen, α2-macroglobulin, α1-antitrypsin, haptoglobin, α1-acid glycoprotein, ceruloplasmin, apolipoprotein AI, apolipoprotein A-II, apolipoprotein B, complement C1q, complement C3, complement C4, plasminogen and prealbumin) are immunodepleted (ProteoPrep20 plasma immunodepletion kit). Samples are exhausted, digested, and measured in triplicate.

[0309] Sample preparation was performed as described (Geyer et al., Cell Syst 2:185-195, 2016) on an Agilent Bravo liquid handling platform using an automated setup. Plasma samples were washed with ddH 2 O was diluted 1:10 and 10 μl of sample was mixed with 10 μl of twice concentrated SDC buffer. Reduction and alkylation were performed at 95°C for 10 min. After a 5 min cooling step at room temperature, trypsin and LysC (ratio of enzyme to microgram protein was 1:100 μg) were added to the mixture. Digestion was performed for 1 h at 37°C. The digest was acidified by adding 40 μl of 1% trifluoroacetic acid (TFA) in isopropanol. 20 μg of peptides were loaded onto two No. 14 StageTip plugs, followed by the addition of 100 μl of 1% trifluoroacetic acid (TFA) in isopropanol and intense mixing. The StageTip was centrifuged at 1,500 x g using a 3D printed, in-house manufactured StageTip centrifugation device. The StageTip was washed twice with 100 μl of 1% trifluoroacetic acid (TFA) in isopropanol and 100 μl of ddH 2 After washing once with 0.2% TFA in 4% 4H2O, the purified peptides were eluted with 60 μl elution buffer into autosampler vials. The collected material was completely dried at 60°C using a SpeedVac centrifuge (Eppendorf, Concentrator plus).

[0310] Samples were measured using an LC-MS instrument consisting of an EASY-nLC 1000 ultrahigh pressure system (Thermo Fisher Scientific) combined with a Q Exactive HF Orbitrap (Thermo Fisher Scientific) and a nano-electrospray ion source (Thermo Fisher Scientific). MS data were acquired using the Top15 data-dependent MS / MS scan method (topN method). The target value for the full scan MS spectrum was 3×10 6 The PCR product was characterized by a 500 nm PCR amplification system with a maximum injection time of 55 ms and a resolution of 60,000 at m / z 200. The proteins identified here were verified to circulate in plasma. Example 2: Validation of target proteins as circulating factors (i.e., scRNAseq hashing)

[0311] This example shows the ability of the target protein of the present disclosure to act as a circulating factor by single cell RNA sequencing to find changes in the cell population after treatment with the target protein. In this example, the target protein found in the above example was ordered by GenScript Biotech (Piscataway, New Jersey). The target protein (e.g., listed in the sequence table and Table A) was synthesized using solid phase peptide synthesis (SPPS) by fluorenylmethoxycarbonyl (Fmoc) protecting group chemistry. Primary human blood mononuclear cells were 1x 10 6 PBMCs were seeded in each well of a 12-well plate with 1 ml RPMI-1640 (10% FBS).PBMCs were treated with 1 μg / mL LPS (Sigma-Aldrich) plus or minus 10 μM final concentration of target protein for 24 h or left untreated.

[0312] The single cell suspension was harvested and centrifuged at 400 x g for 5 min at 4 ° C. The culture medium was discarded and the cells were resuspended with 1 ml of cell staining buffer (BioLegend 420201). 5 μl of human TruStain FcX (BioLegend 422301) was added to the cells and incubated at 4 ° C for 10 min. 1 μg of single cell hash antibody (BioLegend TotalSeq-B#1-#10) was added to the cells together with about 50 μl of cell staining buffer to obtain a total volume of 100 μl. This was incubated at 4 ° C for 30 min and then washed three times at 400 x g. 10 different cell hash samples were combined into one tube with the desired number of cells (about 1000 cells / μl).

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

[0314] After sequencing, the reads are compared with the human reference genome (GENCODE34 / GRCH38). The reads are demultiplexed using DNA barcoded antibodies, and run through quality control (e.g., removing double cells, selecting single cells with less than 15% mitochondrial contamination at the RNA level, and the 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 control and treatment with target protein. The changes in cell population dynamics observed by principal component analysis after treatment with the target protein in the supernatant and / or changes in gene expression verify that the target protein acts as a circulating factor. Example 3. Validation of target proteins as circulating factors (ie, scRNAseq profiling), single cell RNAseq analysis of PBMCs treated with ORFs.

[0315] On the other hand, this example demonstrates the validation of the 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 caused by other circulating factors (toll-like receptor agonists). Therefore, SEQ ID NO: 38427 will be suitable for treating diseases caused by pathological innate immune responses, such as lupus, multiple sclerosis and rheumatoid arthritis.

[0316] To determine the effect of target proteins on TLR activity, target proteins, irrelevant proteins, and controls + / - already used TLR agonists were applied to commercial reporter cell lines to monitor two major signal transduction pathways in response to TLR activation. To determine which immune cells are most responsive to target proteins, target proteins were applied to peripheral blood mononuclear cells (PBMCs), which were then subjected to single-cell RNA-seq using cell hashing 24 hours later.

[0317] Methods and Materials:

[0318] Dual reporter assay: From the example: NF-kB-SEAP and IRF-Lucia luciferase reporter monocytic (THP1) cells (Anvivogen) were cultured in RPMI1640 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 were treated with 100 ng / mL LPS (Sigma-Aldrich) for 24 h, treated with target protein (final concentration 10 μM), or left untreated.

[0319] After treatment, THP1 cells were cultured for 24h and then the reporter activity was determined. When using QUANTI-Blue (Anvivogen) (SEAP detection reagent) and QUANTI-Luc (Anvivogen) (luciferase detection reagent), two reporter proteins can be measured in the cell culture supernatant. QUANTI-Blue is a colorimetric enzyme assay developed for determining any alkaline phosphatase activity in biological samples (such as cell culture supernatants). QUANTI-Luc is a freeze-dried assay reagent containing all components required for the activity of Lucia luciferase and other luciferases utilizing coelenterazine. In cells where the target protein is immunostimulatory, an increase in the activity of the reporter after treatment with the target protein is observed. In addition, in cells where the target protein is immunosuppressive, a reduction in the activity of the reporter after treatment with the target protein is observed.

[0320] scRNA-seq: Single-cell RNA-seq (scRNA-seq) was used to evaluate changes in cell populations after treatment with target proteins. Target proteins (e.g., listed in Table 1) were synthesized using solid phase peptide synthesis (SPPS) by fluorenylmethoxycarbonyl (Fmoc) protecting group chemistry. Human primary blood mononuclear cells were seeded in each well of a 12-well plate with 1 ml RPMI-1640 (10% FBS) at 1 x 10 PBMCs. PBMCs were treated with 1 μg / mL LPS (Sigma-Aldrich) plus or minus 10 μM final concentration of target protein for 24 h, or left untreated. Single cell suspensions were harvested and centrifuged at 400 x g for 5 min at 4 ° C. The culture medium was discarded and the cells were resuspended with 1 ml cell staining buffer (Baijin Company). 5 μl of human TruStainFcX (Baijin Company) was added to the cells and incubated at 4 ° C for 10 min. 1 μg of single cell hash antibody (Baijin Company) was added to the cells together with about 50 μl of cell staining buffer to give a total volume of 100 μl. This was incubated at 4°C for 30 min and then washed three times at 400 x g. 10 different cell hash samples were combined into one tube with the desired cell number (about 1000 cells / μl).

[0321] Single cells were processed by Chromium Next GEM Single Cell 3' Kit (Dual Index) (10X Genomics CG000317 Rev C) according to the protocol provided by 10X Genomics. Briefly, samples were processed by GEM generation and barcoding, GEM-RT post-cleanup and cDNA amplification, 3' gene expression library construction, cell surface protein library construction and finally sequencing.

[0322] After sequencing, the reads are compared with the human reference genome (GENCODE34 / GRCH38). The reads are demultiplexed using DNA barcoded antibodies, and run through quality control (e.g., removing double cells, selecting single cells with less than 15% mitochondrial contamination at the RNA level, and the 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 control and treatment with target protein. The changes in cell population dynamics observed by principal component analysis after treatment with the target protein in the supernatant and / or changes in gene expression verify that the target protein acts as a circulating factor.

[0323] SEQ ID NO: 38427 significantly blocks IRF signaling pathway responses from several TLRs. Upregulation of the IRF pathway has been linked to many autoimmune diseases, especially lupus. Consistent with its association with innate immunity-related diseases like lupus, SEQ ID NO: 38427 strongly affects activated monocytes and dendritic cells, while having relatively little effect on T cells, B cells, and NK cells ( Figure 1 , Figure 2 , Figure 3). ORF ID Relative activity SEQ ID NO: 54278 +++ Example 4: Validation of target protein as circulating factor by morphological profiling.

[0324] This example shows the ability of the target protein of the present disclosure to act as a circulating factor (or secretory protein) by phenotypic screening of adipocytes. In this example, quantitative data is extracted from the microscope image of the cell treated with the target protein of interest in the supernatant, to identify the biologically relevant similarities and differences between samples based on these spectra. This determination uses cell drawing (Cell Painting) (morphological spectrum analysis determination), and the determination multiplexes six kinds of fluorescent dyes imaged in five channels, to reveal widely related cellular components or organelles (Bray et al., Nat.Protoc. [Natural · Scheme] September 2016; 11 (9): 1757-1774.).

[0325] In brief, human primary adipose-derived mesenchymal stem cells (AMSCs) were plated in 96-well CellCarrier plates (Perkinelmer #6005550). AMSCs were differentiated for 14 days in the presence or absence of target proteins, and high-content imaging was performed on days 0, 3, 8, and 14 of adipogenic differentiation. On the corresponding day of the assay, the cell culture medium was removed and replaced with 0.5 μM Mitotracker staining solution (1 mM MitoTracker deep red stock solution (Invitrogen #M22426) diluted in the culture medium) to each well, followed by incubation at 37 ° C in the dark for 30 min. After 30 min, the Mitotracker staining solution was removed, and the cells were incubated with Dulbecco's phosphate-buffered saline (1X) DPBS ( #21-030-CV) were washed twice, and 2.9 μM BODIPY staining solution (3.8 mM BODIPY 505 / 515 stock solution (Thermofisher #D3921) diluted in DPBS) was added, followed by incubation at 37°C in the dark for 15 min.

[0326] Subsequently, cells were fixed by adding 16% methanol-free paraformaldehyde PFA (Electron Microscopy Sciences #15710-S) directly to the BODIPY staining solution to a final concentration of 3.2% and incubated at room temperature (RT) in the dark for 20 min. PFA was removed and cells were washed once with Hank's Balanced Salt Solution (1x) HBSS (Gibco #14025076). To permeabilize the cells, 0.1% Triton X-100 (Sigma Aldrich #X100) was added and incubated at RT in the dark for 10 min. After adding the permeabilization multiple staining solution (10 units of Alexa Fluor TM 568 phalloidin (Thermo Fisher Scientific #A12380), 0.01mg / ml Hoechst 33342 (Invitrogen #H3570), 0.0015mg / ml wheat germ agglutinin Alexa Fluor TM 555 conjugate (Thermo Fisher Scientific #W32464), 3μM SYTO TM14 green fluorescent nucleic acid stain (Invitrogen #S7576), diluted in HBSS), the cells were incubated at room temperature in the dark for 10 minutes. Finally, the staining solution was removed and the cells were washed three times with HBSS. The cells were imaged using the OperaPhenix high-content screening system using a confocal 20x objective. Each well imaged 25 fields of view.

[0327] Morphological profiles were generated using LipocyteProfiler at days 0, 3, 8 and 14 to verify differentiation in both depots by increases in adipogenic marker genes (LIPE, PPARG, PLIN1, GLUT4). Concomitantly, RNA sequencing was used to profile the transcriptome at the same differentiation time points. Example 5: Validation of a target protein as a circulating factor where the target protein is mutated in a disease

[0328] This example demonstrates the validation of a target protein of the present disclosure as a circulating factor, which contains mutations found to be associated with Th17 autoimmune disease in a genome-wide association study (GWAS).

[0329] Circulating factor target proteins and mutant target proteins were synthesized and applied to culture media of activated primary T cells (PHA blasts) with or without TH17, Th1 and TH2 cytokines (IL-23 (Th17), IL-12 (Th1) and IL-6 (Th2). The effects of target proteins and mutant target proteins on the activation state of STAT3, STAT1 and NF-kB were evaluated.

[0330] Materials and Methods

[0331] Circulating proteins: Mutant and wild-type target proteins were synthesized and purified into LPS-free solutions by GenScript Inc. IL-23, IL-12, and IL-6 were purchased from Abcam or Cell Signaling Technology.

[0332] PHA blast generation: Fresh or frozen primary human PBMCs were placed in PBMax Karyotyping Medium supplemented with 10 ng / ml rhIL-2 for 3-4 days.

[0333] STAT3, STAT1 and NF-kB activation assays: PHA blasts were plated at approximately 10 million cells / ml in 96-well plates (80 μl). Cells were serum starved for 3-4 hours and then IL-23, IL-12 or IL-6 were added for 30 minutes. Cells were harvested and cell extracts were assayed by phospho-STAT3, STAT1 or NF-kB ELISA.

[0334] Together, these experiments demonstrate that target proteins containing GWAS alleles associated with autoimmune diseases have a more pronounced activation of STAT3 than STAT1 or NF-kB and strongly suggest that target proteins are potential targets for ameliorating autoimmune diseases. Example 6. Validation of secreted target proteins that stimulate or inhibit cytokine release. CBA (cytometry) experiments were performed to study the production of pro-inflammatory cytokines by PBMCs treated with secreted peptides.

[0335] This example demonstrates the validation of a target protein (SEQ ID NO: 72416) as a modulator of circulating factors, including cytokines. Such modulators would be suitable for the treatment of autoimmune diseases such as psoriasis, arthritis and multiple sclerosis.

[0336] PBMCs were inoculated with + / - T cell activators CD3 / CD8 and + / - peptides for 24-48 hours. Cytokines were measured in the supernatant after incubation by CBA cytometer.

[0337] Methods and Materials:

[0338] Thawing PBMC: PBMCs were thawed by pre-warming TexMACS culture medium (Miltenyi Biotech) in 20ml aliquots (1 50ml conical flask per donor). After the culture medium was warmed to 37°C, the cells were taken out of liquid nitrogen and brought to the tissue culture hood. For each donor, 1ml culture medium from the warmed aliquot was added to the frozen vial and mixed up and down. The culture medium plus the thawed cells were then added back to the 50ml conical flask. This process was repeated until all cells were thawed and added to the 50ml tube. Continue with other donors (if used). The cells were spun at 400x g for 5min. The supernatant was aspirated and the cells were resuspended at approximately 10*10^6 cells / ml (for cell number, see vial).

[0339] PBMC plating: Frozen PBMCs from normal healthy human volunteers (StemCell Technologies) were thawed and plated at 250,000 cells / well. After PBMCs were thawed and resuspended at 10*0^6 cells / ml, cells were then counted (Countess III, Thermo Scientific), and adjusted to 10*10^6 cells / well based on the count. Taking the number of PBMCs determined by the number of wells used for the experiment as an example, for example, 10 wells would require a total of 2.5*10^6 cells or 0.250ml. The cells required for the experiment were then transferred to another conical tube. Add warm TexMACS medium to a final concentration of 250,000 cells / 190ul medium, for example, 10 wells would require a total of 1.9ml (190ul x 10 wells) medium minus 0.250ml cells (1.9ml - 0.250ml) or 1.65ml medium added to 0.250ml cells. Add 190ul cells plus medium to a 96-well U-bottom plate. Place in an incubator until dilutions are prepared and ready.

[0340] Peptide dilutions: Ideally, the starting concentration of all peptides should be at least 10 mM, especially for those in DMSO (ATCC), to achieve a final concentration of 1 uM (H2O) or 0.5 uM (DMSO). All dilutions of the test article should be prepared at 20x final concentration, for example, a final concentration of 1 uM peptide would need to be prepared at 20 uM.

[0341] Reagent dilutions: Prepare enough dilutions for triplicate 10ul / well (30ul, 20x) in TexMACS medium. Activators - CD3 / CD28 (TransAct; Miltenyi Biotec) or cytokines will be 21x and added after 45 minutes of incubation with peptides. Be sure to include relevant controls - positive: TransAct+ cells, negative: vehicle+ cells. Dilutions should also be prepared in ultra-low binding plates (Corning).

[0342] Assay Procedure: Peptide Treatment: The assay was started as described above by thawing the cells and plating them in 96-well U-bottom tissue culture plates (Corning). The cell plates were placed in a tissue culture incubator (5% CO2, 37°C). While the cells were in the incubator, dilutions were prepared as described above (optimally 1 h or less). The cell plates were removed from the incubator and 10 ul of 20x dilutions were added to the relevant wells. After the addition of the inhibitors, the cell plates were placed in the tissue culture incubator for 45 minutes. The plates were removed from the incubator and 10 ul of 21x TransAct was added to the relevant wells*. The plates were then placed back in the incubator and incubated for 24 hours. After 24 hours, the plates were spun at 400x g for 5 minutes.

[0343] *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. Only TransAct is required in the control wells. The purpose of this procedure is to determine the possible response from the peptide alone.

[0344] Cytometric Bead Array (CBA): After spinning, transfer the supernatant (approximately 150ul) to a new ultra-low binding U-bottom plate. The supernatant was then frozen at -20°C until thawed for CBA (BD) to determine the levels of human IL-2, TNFa, IL-1b, IP-10, IL-10, and IFNg. CBA was performed according to the manufacturer's protocol.

[0345] SEQ ID NO: 72416 significantly blocks the release of the cytokine TNF-α from activated PBMCs, but does not block the release of another inflammatory cytokine IL-1b. Specific inhibition of TNF-α release will reduce the inflammatory response ( Figure 4 and Figure 5 ). Hit table: X = hits with significant activation or inhibition of relevant cytokine release. Inhibition occurred when peptides were applied together with anti-CD3 / CD28 T cell activators. Activation occurred when peptides were applied to PBMCs alone (see Materials and Methods). Significance = P = 0.05 Example 7.

[0346] This example demonstrates the validation of 4 genetic forms (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 (i.e., type 2 diabetes). These peptides may be suitable for treating metabolic diseases, such as type 2 diabetes.

[0347] The effects of putative stimulators of glucose uptake were assessed using adipocytes differentiated from primary adipocytes. Preadipocytes were differentiated into mature adipocytes in vitro and treated with + / - peptides and glucose uptake capacity was determined via a glucose uptake glow assay 24 hours after treatment.

[0348] Materials and Methods

[0349] Preadipocytes are seeded into opaque 96-well plates with 10K / well density, and standard adipogenesis differentiation mixture is used to differentiate them. When terminal differentiation, adipocytes are serum starved, and treated with 0.5uM peptide, 5uM peptide or blank (DMSO), and incubated for 24h at 37°C incubators with 5%CO. On the day of determination, the DMEM (Life Technologies, catalog number 11966) without serum or glucose containing a certain range of insulin concentrations of 100 μl of culture medium was replaced, and incubated for 1 hour at 37°C at 5%CO. Culture medium was removed, and 50 μl 2DG (1mM) was added in PBS, and incubated for 10 minutes at 25°C. 2-Deoxyglucose (2DG) is transported to cells and phosphorylated to produce 2-deoxyglucose-6-phosphate (2DG6P). 25 μl stop buffer was added, and the plate was vibrated for a short time. 25 μ l neutralization buffer is added, and the plate is shaken briefly. 100 μ l 2DG6P detection reagent is added, the plate is shaken briefly, and it is incubated at 25 ℃ for 1 hour. Stop buffer is added to stop 2DG transport, lyse cells, destroy any NADPH in the cell, and inactivate protein. Neutralization buffer is added to neutralize the solution, and then 2DG6P detection reagent is added. Glucose-6-phosphate dehydrogenase (G6PDH) in the reagent oxidizes 2DG6P to 6-phosphotyroxygluconic acid (6PDG), and NADP+ is reduced to NADPH. Reductase uses NADPH to convert proluciferin into luciferin, which is then used to produce light by luciferase. Luminescence is recorded with 0.3-1 second integral on a photometer.

[0350] like Figure 6 As shown, peptide SEQ ID NO: 24296 was demonstrated to increase glucose uptake in adipocytes at a concentration of 5 uM. Example 8.

[0351] This example demonstrates the ability to detect a target protein of the present disclosure in human plasma to verify that it is a circulating factor. Sample preparation was performed as described by Keshishian et al. Mol Cell Proteomics. 2015 Sep; 14(9): 2375-93. Below is a detailed description of the method.

[0352] Plasma depletion and enzyme digestion.Immunoaffinity depletion was performed using IgY14 LC20 and Supermix LC10 columns (Sigma-Aldrich, St. Louis, Missouri) to 14 most abundant proteins of four hundred microliters of peripheral plasma collected from four patients at baseline and 10, 60 and 240min after alcohol ablation, followed by the next approximately 50 proteins of medium abundance.Dilution buffer, stripping buffer and neutralization buffer provided by the manufacturer were used and depleted in series on Agilent1100HPLC (Agilent, Santa Clara, California) system according to the manufacturer's instructions (Sigma-Aldrich).The flow-through representing the Supermix column depleted of plasma was concentrated, and the buffer was exchanged to 50mm ammonium bicarbonate to the original volume (400 μl) using Amicon 3K concentrator (Millipore, Billerica, Massachusetts). The protein concentration of depleted plasma was determined by BCA protein assay (Thermo Fisher Scientific, Waltham, MA).

[0353] Four hundred microliters of IgY14 / Supermix depleted peripheral plasma per time point and per patient were denatured with 6 M urea, reduced with 20 mM dithiothreitol for 30 min at 37°C, and alkylated with 50 mM iodoacetamide for 30 min at room temperature in the dark. The urea concentration was diluted to 2 M with 50 mM ammonium bicarbonate and then subjected to Lys-C digestion (Wako, Richmond, VA) at a 1:50 (w:w) enzyme to substrate ratio for 2 h at 30°C with mixing on a shaker at 850 rpm. Urea was further diluted to less than 1 M and then digested overnight with trypsin (Promega, Madison, WI) at a 1:50 (w:w) enzyme to substrate ratio at 37°C with shaking at 850 rpm. Digestion was terminated with formic acid to a final concentration of 1%. The digests were desalted using an Oasis HLB 1cc (30 mg) reverse phase cartridge (Waters, Milford, MA) using a vacuum manifold with 0.1% formic acid and 0.1% formic acid / 80% acetonitrile as buffers A and B, respectively. The cartridges were adapted with 3×500 μl buffer B and then equilibrated with 4×500 μl buffer A. After loading the digests at a reduced flow rate, they were washed with 3×750 μl buffer A and eluted with 3×500 μl buffer B. The eluate was frozen and dried by vacuum centrifugation. The digests were reconstituted in 400 μl 0.1% formic acid and the post-digestion concentration was determined by BCA. Based on the post-digestion concentration, 80 μg aliquots were prepared, frozen, dried to dryness by vacuum centrifugation, and stored at -80°C.

[0354] iTRAQ labeling of plasma samples. According to the manufacturer's instructions for labeling plasma (ABSciex, Framingham, Massachusetts) (it requires the use of twice as many reagents as other samples (cell lysates, tissues, etc.) for plasma), eighty micrograms of dried aliquots of four time points (baseline, 10, 60 and 240min after injury) of each PMI patient were labeled with iTRAQ quadruple reagents. In order to eliminate the bias to any iTRAQ channel, different iTRAQ channel layouts were used for the different time points of four PMI patient samples. After the sample was reconstructed in 30 μl1 m triethylammonium bicarbonate (TEAB), 100 μl of ethanol was added to each sample. The iTRAQ reagents merged from two vials were added to each sample, mixed and incubated at room temperature for 1h. Each sample of three microliters was used to check the labeling by LC-MS / MS, and then the quenching reaction was performed. Once satisfied with labeling efficiency (>95% label incorporation), the reaction was quenched by adding Tris pH 8 to a final concentration of 100 mM and incubated at room temperature for 15 min. Labeled samples representing four different time points of PMI patients were pooled together, dried, and desalted using an Oasis HLB 1 cc (30 mg) reverse phase cartridge as described above. The eluate was frozen, dried to dryness, and stored at -80°C.

[0355] By reverse phase chromatography at high pH (alkaline pHRP) to peptide fractionation. The digested iTRAQ labeled plasma samples of each patient were reconstituted in 540 μl 20 mm ammonium formate / 2% acetonitrile pH 10, loaded onto a Zorbax 300 Extend 2.1×150 mm column (Agilent Technologies, Santa Clara, California), and fractionated by alkaline reverse phase chromatography at a flow rate of 200 μl / min on an Agilent 1100 series HPLC instrument. 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 (300 μg) of sample onto the column, the peptides were separated using the following gradient: isocratic hold at 0% B for 5 min, 0 to 15% solvent B in 8 min; 15% to 28.5% solvent B in 33 min; 28.5% to 34% solvent B in 5.5 min; 34% to 60% solvent B in 13 min, with a total gradient time of 64.5 min. Fractions were collected every 0.6 min through the main elution profile of the separation using a 96×2 ml well plate, for a total of 84 fractions. In addition, the extreme early and late parts of the gradient were collected into two additional larger volume fractions. All fractions were acidified to a final concentration of 1% formic acid, and then the inner 84 fractions were recombined by combining the early, middle and late fractions together, using a concatenation strategy to obtain a total of 28 fractions. These 28 fractions together with 2 additional fractions representing early and late eluting peptides constituted a total of 30 fractions to be analyzed by LC-MS / MS.All fractions were dried to dryness by vacuum centrifugation and stored at -80°C until mass spectrometry analysis.

[0356] NanoLC-MS / MS analysis.For plasma samples from individual patients, each of 30 fractions was reconstructed in 16 μl 5% formic acid / 3% acetonitrile, and 2 μl was equipped with a nano-flow ionization source (James A. Hill Instrument Services, Arlington, Massachusetts) and analyzed on a Q Exactive mass spectrometer connected to an EASY-nLC 1000 UHPLC system (Thermo Fisher Scientific). Chromatography was performed on a 75 μm ID picofrit column (New Objective, Woburn, Massachusetts) filled with Reprosil-Pur C18 AQ 1.9 μm beads (Dr. Maisch, GmbH, Ettlingen, Germany) to 20 cm in length. Column heater sleeve (Phoenix-ST, Chester, Pennsylvania) was used to heat the column to 50 ° C to prevent column overpressure during UHPLC separation. The LC system, column and platinum wire for delivering the electrospray source 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. The peptides were eluted at 200 nL / min with the following gradient: 6% to 35% B in 150 min, 35% to 60% B in 8 min, 60% to 90% B in 3 min, 10 min at 90% B, 90% B to 50% B in 1 min, followed by 10 min at 50% B isocratic conditions. A single Orbitrap MS scan from 300 to 1800 m / z at a resolution of 70,000 was performed (AGC was set to 3e6), followed by up to 12 ms / ms scans at a resolution of 17,500 (AGC was set to 5e4). MS / MS spectra were collected with a normalized collision energy of 27 and a separation width of 2.5 amu. Dynamic exclusion was set to 20 s, and peptide matching was set to on.

[0357] For plasma samples merged from multiple patients and used for iTRAQ / TMT comparison, some of the above parameters were modified. Analyzed with a separation width of 2.0 amu on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). For peptides labeled with TMT, the normalized collision energy was reduced to 26. For peptides labeled with TMT-10, MS / MS spectra were collected with a resolution of 35,000.

[0358] Human protein raw data processing (at ProFound Tx). Raw data from ftp: / / MSV000079033:a@massive.ucsd.edu Download. We used the Spectromine search engine (Biognosys, version 3.2) to search for features detected in the original MS files against the FL69ORF proteome database (updated on November 1, 2022; 800K protein entries). Only peptides with a length of at least seven amino acids and trypsin digestion with up to two missed cleavages were considered. The initial allowed mass tolerance was set to 10ppm at the MS level and to 0.02Da at the MS / MS level. We set N-acetylation (42.010565Da) and oxidation of methionine (15.994915Da) at the N-terminus of the protein as variable modifications, and iTRAQ 4-weight labeling at the N-terminus of the peptide and carbamidomethylation of cysteine ​​as fixed modifications (57.021464 Da). A 1% false discovery rate (FDR) was applied for peptide spectrum matching (PSM) and protein identification using the target-decoy method. iTEAQ 4-plex quantification was performed using the default parameters of the Biognosys method (quantification, iTRAQ 4-plex). Novel ORFs identified in plasma samples from patients with myocardial injury and healthy donors. Example 9.

[0359] This example demonstrates the ability to detect the target protein of the present disclosure in human saliva to verify that it is a circulating factor and a secreted factor. Sample preparation was performed as described in Grassl et al. Genome Medicine [Genome Medicine] Vol. 8, 44 (2016). The following is a detailed description of the method.

[0360] Protein digestion and peptide purification.After collection, the swab was transferred to an Eppendorf tube 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 thoroughly against the inner wall of the Eppendorf tube, and taken out. As estimated by the Bradford protein assay, we can reproducibly recover more than 100 μg of protein in this way. Sample preparation basically follows the in-StageTip protocol. In brief, a total of 20 μg of protein was digested by adding 0.4 μg of trypsin and LysC to our lysis buffer and incubating for 60 min at 37 ° C while shaking. After this short digestion, we acidified the peptides to a final concentration of 1% trifluoroacetic acid (TFA) and loaded them onto the SDB-RPS StageTip. The filter was then washed and the peptides were finally eluted with 60 μl 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.

[0361] Single run and pre-classification liquid chromatography-MS measurement.In order to obtain deep salivary proteome, we use alkaline reverse phase chromatography to classify our eight qualified (waking) samples, and then carry out liquid chromatography (LC)-MS measurement.About 15 μg peptides were separated on a 20cm75 μm inner diameter column filled with ReproSil-Pur C18 beads (Dr. Maisch Co., Ltd., Germany) with 80min gradient.The concentrated fractions were dried in SpeedVac concentrators and resuspended in A buffer to a concentration of 1g / l.Using EASY-nLC 1000 ultra-high pressure system (Thermo Fisher Scientific) and the 40cm column of the internal manufacture of the above type, both the classified samples and the single run samples were subjected to 100min chromatographic gradients.By applying the spray voltage of 2.2kV, the chromatogram was connected online with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The MS scan resolution was set to 120,000 at m / z 200, the scan range was set to 300 to 1650 m / z, and the maximum injection time was set to 55 ms. The 15 most intense ions per MS scan were selected for high energy collision dissociation (HCD) fragmentation with an isolation width of 1.5 m / z and measured at a resolution of 30,000. Dynamic exclusion was used with an exclusion time of 30 s.

[0362] Human protein raw data processing (at BioFang Therapeutics). Raw data from https: / / www.ebi.ac.uk / pride / archive / projects / PXD003028Download. We used the Spectromine search engine (Bio-cognitive Systems, version 3.2) to search for features detected in the original MS file against the FL69ORF proteome database (updated on November 1, 2022; 800K protein entries). Only peptides with a length of at least seven amino acids and a tryptic digestion with at most two missed cleavages were considered. The initial allowed mass tolerance was set to 10ppm at the MS level and to 0.02Da at the MS / MS level. We set N-acetylation (42.010565Da) and oxidation of methionine (15.994915Da) at the N-terminus of the protein as variable modifications, and carbamidomethylation of cysteine ​​as a fixed modification (57.021464Da). A false discovery rate (FDR) of 1% was applied for peptide spectrum matching (PSM) and protein identification using the target-bait method. Relative quantification was performed using the default parameters of the Biognosys method. Novel ORFs identified from saliva samples from four female and four male healthy non-smoking individuals. Example 10. Validation of target proteins that modulate cellular uptake of disease-associated circulating factors.

[0363] This example demonstrates the validation of SEQ ID NO: 26888, SEQ ID NO: 36277, and SEQ ID NO: 75353 as novel peptides capable of increasing 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, lean body mass, fat mass, and BMI; SEQ ID NO: 36277 contains genetic variants associated with HDL cholesterol and triglycerides; and SEQ ID NO: 75353 contains variants associated with HDL cholesterol levels. These peptides may be suitable for both primary prevention (reducing CVD risk in patients without known CVD) and secondary prevention (preventing subsequent heart attacks, strokes, and other CVD events in patients with established CVD).

[0364] The effect of putative cholesterol-lowering peptides was evaluated using an LDL uptake assay in primary human hepatocytes from a pool of 10 healthy donors. Hepatocytes were treated for 4 h with peptides and LDL-labeled BODIPY. The cells were then fixed, permeabilized, and imaged on the Opera Phenix high-content imaging platform. LDL uptake was determined using BODIPY stain intensity.

[0365] Materials and Methods

[0366] Human primary hepatocytes were seeded into 96-well PhenoVue plates at a density of 37K / well and allowed to recover overnight. The next day, hepatocytes were incubated with 5uM peptide or blank control (DMSO) and BODIPY-labeled LDL (Invitrogen TM Image-iT TM Low-density lipoprotein uptake kit, Bodipy FL, I34359). This assay is designed to specifically detect the binding and uptake of LDL through the LDL receptor internalization pathway, allowing maximum control and flexibility in experimental design. Two controls (unlabeled LDL and heparin) are built into the system. Unlabeled LDL provides optional pretreatment to block cell surface receptors before probing with labeled constructs, and heparin is provided as an additional control to chelate LDL in the solution phase outside the cell, thereby preventing the binding and uptake of labeled LDL. Metformin (a type 2 diabetes drug with a cholesterol-lowering effect) is used as a positive control for increased LDL uptake ( Figure 1 ). After peptide, control and LDL-BODIPY treatment, cells were incubated in a 37°C incubator with 5% CO2 for 4 h. After incubation, cells were fixed with 4% paraformaldehyde for 15 min. 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 using the Perkin Elmer Opera Phenix high content imaging platform (fluorescence), with 9 fields per well. BODIPY spot intensity was quantified using Harmony image analysis software.

[0367] like Fig. 9 As shown, peptides SEQ ID NO: 26888, SEQ ID NO: 36277 and SEQ ID NO: 75353 were demonstrated to increase LDL uptake in hepatocytes at a concentration of 5 uM. Example 11. Discovery of secreted proteins associated with systemic lupus erythematosus (SLE) disease.

[0368] This example presents novel findings to identify 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) that exhibit differential expression patterns in plasma samples obtained from individuals with systemic lupus erythematosus (SLE) compared to plasma samples from healthy donors. The ORF proteins identified in this study have significant potential as therapeutic agents or targets for the management of SLE.

[0369] This research aims to use the deep plasma proteomics spectrum analysis of healthy plasma samples and systemic lupus erythematosus (SLE) plasma samples through highly optimized workflow.Low molecular weight proteins (LMWP) are enriched by the combination of protein precipitation and sequential dissolution developed by Broad Treatment Company.LMWP fractions are digested using trypsin / Lys-C mixture.In the initial study, two merged samples were produced, one from 5 healthy donors, and the other from 5 SLE patients.Peptides from these two samples were classified using online reversed phase liquid chromatography (RPLC), and analyzed using the timsTOF Pro 2 MS connected with EvoSepnanoLC using data independent acquisition (DIA) method.Based on the RiboSeq research carried out at Broad Treatment Company, the FL69ORF_DB database containing about 800K ORF entries was searched for MS spectra.Newly discovered ORF proteins were classified by ORF type (including annotated, ncRNA, polycistronic and no transcript). Quantitative analysis was performed using the pooled peptide intensities from each protein, and differentially expressed proteins between SLE samples and healthy samples were identified based on a threshold of at least a two-fold change in plasma samples.

[0370] Materials and Methods

[0371] Sample collection: plasma samples were collected from SLE patients according to standard procedures. In brief, about 2 mL of whole blood samples from each patient were drawn into EDTA-coated collection tubes. The tubes were gently flipped 8-10 times, and the tubes were placed upright on ice for 5 minutes to allow blood cells to settle. The blood samples were centrifuged for 10 min at 1000 x g (RCF). The plasma upper layer (about 1 mL) was separated into a new sample tube containing 10 μL of a protease inhibitor mixture (Therno) to prevent protein degradation. For each SLE patient, healthy donors with matching age, ethnic group and sex were selected. Plasma samples were collected with the same protocol. After collecting samples on site, plasma samples were stored at -80 ° C for proteomic analysis.

[0372] Sample preparation: protein precipitation and differential solubility (PP+DS): An aliquot of 50 μL plasma sample was diluted 1:2 (v / v) with 100 μL denaturing solution (8 M urea) and heat denatured at 70°C for 3 min. Another 50 μL of water was added, then slowly dripped into 1800 μL ice-cold acetone and immediately stirred for 2 h at -20°C, followed by centrifugation at 19,000 g for 15 min at 4°C. The precipitate was absorbed in 300 μL of 80% ACN containing 12 mM HCl and sonicated for 10 s at 40% probe power. The sample was then mixed overnight at 4°C. The sample was centrifuged again at 19000 g for 15 min at 4°C. Low molecular weight proteins (LMWP) were extracted into the supernatant. The sample was dried in a SpeedVac to complete drying, and the sample was stored at 4°C for later use.

[0373] Protein denaturation, reduction and alkylation: Add 1 / 25 volume of 25x TCEP stock and 1 / 10 volume of 10X CAA stock to each sample vial. Heat the samples to 37°C and incubate the samples for 1 hour to reduce and alkylate the proteins.

[0374] In-solution digestion: Dilute the denatured protein sample by adding 8 volumes of μL of digestion buffer, such as 100 mM Tris-HCl (pH 8.0) in water. Add 2 μg of sequencing grade trypsin / Lys-C mixture to each sample at an enzyme:protein ratio of 1:50. Incubate the sample overnight at 37°C in a benchtop thermomixer with mixing at 800 rpm.

[0375] Alkaline reverse phase (HpH) high pressure liquid chromatography (HPLC) fractionation: Equal amounts (about 10 μg) of digestion samples from five healthy donors or five SLE donors were combined into two mixtures (representing healthy plasma digests and SLE plasma digests) for HPLC fractionation. The samples were centrifuged at room temperature for 10 minutes at 16,000 × g. The 50 μg peptide mixture of each combined peptide digest was fractionated into 48 fractions according to the following scheme. In brief, about 50 μg of the peptide mixture was loaded onto a Waters XBridge BEH130 C18 3.5 μm 3mm ID×150 mm length column on an Agilent 1290 HPLC running at 0.6 mL / min. Buffer A consisted of 0.1% ammonium hydroxide in water, and buffer B consisted of 0.1% ammonium hydroxide in 93% ACN and 7% water. All fractionation experiments were performed using the same injection scheme and gradient. Agilent fraction collector was used to collect all fractions at intervals of 0.5min in 96 deep-well plates. Sample was initially loaded onto the post at 0.8mL / min for 10min, and then a step gradient was started at 0.6mL / min, as follows: 3%B to 45%B in 35min, 60%B in 4min, and ramped up to 90%B in 2min. Fraction collection was started 10min after sample loading. Gradient was maintained at 90%B for 5min, then ramped down back to 3%B, and then the post was washed and balanced. In all experiments, the total number of concentrated fractions was set to 96. For optimized results, the first 48 fractions were merged with +49 hole fractions (i.e., merging wells A1+E1, A2+E2, ..., B1+F1, ..., D12+H12), and final 48 fractions were obtained. Before concentrating, ammonium hydroxide was evaporated in a SpeedVac operated at 40°C.

[0376] NanoLC-MS analysis: The dried peptide fractions were reconstituted by 0.1% formic acid in water. For each nanoLC injection, 20% of each fraction (approximately 200 ng digest) was loaded onto the EvoTip. Peptide separation was performed using the standard separation method 40SPD from EvoSep using an IonOpticks column (Aurora ELITE, 75um ID x 150mm L). The EvoSep nanoLC connected to a timTOF pro 2MS (Bruker) was run in DIA mode for data acquisition.

[0377] Database search and data analysis: The Spectronaut search engine (Bio-cognitive Systems, Version 17.1) and the directDIA search engine were used to search for features detected in the original MS files against the FL69ORF proteome database (updated in November 2022; 800K protein entries). Only peptides with a length of at least seven amino acids and complete trypsin digestion with up to two missed cuts were considered. The initial allowed mass tolerance was set to 20ppm at the MS level and to 0.05Da at the MS / MS level. We set the N-acetylation (42.010565Da) and oxidation of methionine (15.994915Da) of the N-terminus of the protein as variable modifications, and the carbamidomethylation of cysteine ​​was set to a fixed modification (57.021464Da). A 1% false discovery rate (FDR) was applied for peptide spectrum matching (PSM) and protein identification using the target-bait method. Label-free quantification was performed using the default parameters of the Biognosys quantitative method (peak integration and merged area based on MS / MS). For each protein, the sum of the peak areas of all identified peptides from all fractions of healthy or SLE samples was combined. Proteins differentially expressed between SLE and healthy samples were identified based on a threshold of at least a two-fold change in plasma samples (i.e., log2 fold change > 1 for upregulation and <-1 for downregulation).

[0378] About 984 secreted proteins (or proteomes) were identified from the LMWP fraction of healthy plasma samples or SLE plasma samples. After removing the proteins for annotation, 23 non-classical proteins were identified, including different types of ORFs (11 polycistronic (2 dORFs and 9 other types), 11 ncRNAs (1 lncRNA and 10 pseudogenes), and one transcriptless protein). Among these novel ORF proteins, 7 ORFs showed upregulated expression and 3 ORF proteins showed downregulated expression in SLE samples compared to healthy controls. For example, SEQ ID NO: 75451 (dORF of SOD2) and SEQ ID NO: 75452 (polycistronic ORF of CISH) and SEQ ID NO: 75453 (overlapping uORF of TMED2) showed higher expression levels in SLE plasma samples. These ORF proteins and upregulated annotated proteins showed significantly enriched GO terms for adaptive immune response and neutrophil degranulation and signaling through interleukins. Inhibition of these ORF targets may lead to a weak immune response in SLE patients. lncRNA (SEQ ID NO: 74932 (NIPBL-DT)) showed downregulation in SLE samples, which is reported to be conserved in protein sequences and associated with immune responses in mammals. SEQ ID NO: 74932 can potentially be used as a therapeutic agent to alleviate SLE symptoms ( Fig.10 , Fig.11A and Fig. 11B ). Table C: Novel ORF proteins (secreted proteins) identified in human plasma from SLE donors and healthy donors and differential expression changes (in log2 scale). Example 12. Validation of two target proteins as novel G protein-coupled receptor (GPCR) ligands.

[0379] This example demonstrates the validation of two novel peptides, SEQ ID NO: 49310 and SEQ ID NO: 42382, that act as GPCR ligands. SEQ ID NO: 49310 blocks CXCR4 and cancer cell migration, making it suitable for treating a variety of cancers. While SEQ ID NO: 42382 agonizes C3AR1, a key regulator of immune responses and inflammation. The target proteins SEQ ID NO: 49310 and SEQ ID NO: 42382 were treated with gpcrMAX, a comprehensive panel covering 168 G protein-coupled receptors (GPCRs) from more than 60 different receptor families. This panel utilizes β-Arrestin Technology (Eurofins DiscoverX). Engineered PathHunter β-arrestin GPCR cell lines to co-express ProLink TM (PK)-tagged GPCR and enzyme acceptor (EA)-tagged β-arrestin. Activation of GPCR-PK induces β-arrestin-EA recruitment, forcing complementation of two β-galactosidase fragments (EA and PK). The resulting functional enzyme hydrolyzes the substrate to generate a chemiluminescent signal.

[0380] Materials and Methods:

[0381] Cell Handling: Expand cell lines from frozen stocks according to standard procedures. Seed the cells into white-walled 384-well microplates at a total volume of 20 μL and incubate at 37 °C for an appropriate time before testing. Cell lines were seeded into white-walled 384-well microplates at a total volume of 20 μL and incubated at 37 °C for an appropriate time before testing.

[0382] Peptide Handling: Target proteins SEQ ID NO: 49310, SEQ ID NO: 42382, and an irrelevant peptide were synthesized using solid-phase peptide synthesis (SPPS) by fluorenylmethoxycarbonyl (Fmoc) protecting group chemistry. For screening: Target proteins SEQ ID NO: 49310, SEQ ID NO: 42382, and 8 irrelevant peptides were treated at a final highest test concentration of 0.12 μM on the gpcrMAX panel. For hit confirmation: Target protein SEQ ID NO: 49310 and the irrelevant peptide were tested at the highest test concentrations of 1 μM and 0.3 μM in the CXCR4 human chemokine GPCR cell-based antagonist arrestin assay.

[0383] Assay Design: Agonist Format: For agonist determination, incubate the cells with the sample to induce a response. Perform an intermediate dilution of the sample stock to produce 5X samples in assay buffer. Add 5 μL of the 5X sample to the cells and incubate at 37 °C or room temperature for 90 to 180 minutes. The vehicle concentration is 1%. Antagonist Format: For antagonist determination, pre-incubate the cells with the antagonist, followed by agonist challenge at the EC80 concentration. Perform an intermediate dilution of the sample stock to produce 5X samples in assay buffer. Add 5 μL of the 5X sample to the cells and incubate at 37 °C or room temperature for 30 minutes. The vehicle concentration is 1%. Add 5 μL of 6X EC80 agonist in assay buffer to the cells and incubate at 37 °C or room temperature for 90 or 180 minutes.

[0384] CXCR4 Human Chemokine GPCR Cell-Based Antagonist Arrestin Assay: For hit confirmation, use the β-Arrestin cell lines, CXCL12 / SDF-1a as activator and plerixafor as inhibitor.

[0385] C3aR Human Complement Peptide GPCR Cell-Based Agonist Inhibitory Protein Assay: For hit confirmation, use the C3AR1 Human Complement Peptide GPCR Agonist Assay β-Arrestin cell line, C3A receptor agonist (short fragment) as control activator.

[0386] Signal detection: by a single addition of 12.5 or 15 μL (50% v / v) The detection reagent mixture is then incubated for one hour at room temperature to generate the assay signal. After signal generation, the microplate is read using a PerkinElmer Envision™ instrument for chemiluminescent signal detection.

[0387] Data analysis: Compound activity was analyzed using the CBIS data analysis suite (ChemInnovation, California). For agonist mode assays, the activity percentage was calculated using the following formula: Activity % = 100% x (average RLU of test samples - average RLU of vehicle controls) / (average RLU of average MAX control ligand - vehicle controls). For antagonist mode assays, the inhibition percentage was calculated using the following formula: Inhibition % = 100% x (1 - (average RLU of test samples - average RLU of vehicle controls) / (average RLU of EC80 controls - average RLU of vehicle controls)).

[0388] Chemotaxis assay: Before the assay, NAMALWA cells (human Burkitt's lymphoma cell line, ATCC) were cultured in serum-free ATCC formulated RPMI for 24h. After starvation, cells were harvested and centrifuged at 1,000xg for 5 minutes to precipitate them. The cells were resuspended in serum-free medium. Using the cell migration / chemotaxis assay kit (96 wells, 8 μm) from Abcam, 150 μL of serum-free medium containing the desired chemoattractant was added to the lower 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 in a CO2 incubator at 37°C for 24 hours. After incubation, 110 μL of cell dye + cell dissociation solution mixture was added to each bottom well and incubated in a CO2 incubator at 37°C for one hour. After incubation, the upper chamber was removed and the plate was read at Ex / Em=530 / 590nm.

[0389] Results: Using the gpcrMAX panel from Eurofins, two target peptides (SEQ ID NO: 49310 and SEQ ID NO: 42382) were identified as novel GPCR ligands. The target peptide SEQ ID NO: 42382 is an agonist of C3AR1, a key anaphylatoxin receptor that plays a key role in inflammation. The target peptide (SEQ ID NO: 49310) blocks CXCR4, a chemokine receptor involved in cell migration and homing. In addition, SEQ ID NO: 49310 was able to significantly inhibit the chemotactic migration of human Burkitt's lymphoma cells, indicating its potential as a chemokine for cancer therapy ( Fig.12 , Fig.13 , Fig.14 , Fig.15 ).

[0390] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0391] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments as encompassed by the appended claims.

Claims

1. A medicament comprising a target protein identified in the sequence list or Table A or a variant thereof and / or regulating the expression or activity of the target protein or a variant thereof.

2. The agent of claim 1, wherein the agent comprises the target protein.

3. The agent of claim 1, wherein the agent modulates the expression of the target protein.

4. The agent of claim 2 or 3, wherein the agent modulates the expression of a gene or gene transcript encoding the target protein.

5. The agent of claim 1, wherein the agent modulates the activity of the target protein.

6. The agent of any one of claims 2-5, comprising a polypeptide, a polynucleotide or a small molecule.

7. The agent of any one of claims 2-6, wherein the agent reduces the expression or activity of the target protein.

8. The agent of claim 7, wherein the agent comprises an inhibitor of the target protein.

9. The agent of claim 8, wherein the inhibitor is a polypeptide.

10. The agent of claim 9, wherein the polypeptide is an antagonist antibody or an antigen-binding fragment thereof that binds to the target protein.

11. The agent of claim 8, wherein the inhibitor is a polynucleotide. 12 . The agent of claim 11 , wherein the polynucleotide comprises a nucleotide sequence complementary to at least a portion of a gene or gene transcript encoding the target protein.

13. The agent of claim 11 or 12, wherein the polynucleotide comprises DNA.

14. The agent of claim 11 or 12, wherein the polynucleotide comprises RNA.

15. The agent of claim 11 or 12, wherein the polynucleotide is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense DNA, an antisense RNA, a micro RNA (miRNA), an antagonist, a guide RNA (gRNA), a locked nucleic acid (LNA), or a morpholino nucleic acid (MNA).

16. The agent of claim 8, wherein the inhibitor is a small molecule. The agent of claim 16 , wherein the small molecule binds to the target protein, thereby reducing the activity of the target protein.

18. The agent of any one of claims 1-6, wherein the agent increases the expression or activity of the target protein.

19. The agent of claim 18, wherein the agent is an isolated polypeptide comprising the amino acid sequence of the target protein, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of the target protein.

20. The agent of claim 19, wherein the isolated polypeptide or variant thereof is a recombinant protein or a synthetic protein.

21. The agent of claim 18, wherein the agent is a polynucleotide encoding the target protein or a variant thereof having at least 80% sequence identity with the amino acid sequence of the target protein.

22. The agent of claim 21, wherein the polynucleotide comprises DNA.

23. The agent of claim 21 or 22, wherein the polynucleotide comprises a vector.

24. The agent of claim 21, wherein the polynucleotide comprises RNA.

25. The agent of claim 24, wherein the RNA is messenger RNA (mRNA) and / or circular RNA (circRNA).

26. The agent of claim 18, wherein the agent comprises an activator of the target protein.

27. The agent of claim 26, wherein the activator is a polypeptide.

28. The agent of claim 27, wherein the polypeptide is an agonist antibody or an antigen-binding fragment thereof that binds to the target protein.

29. The agent of claim 26, wherein the activator is a small molecule.

30. The agent of claim 29, wherein the small molecule binds to the target protein, thereby increasing the activity of the target protein.

31. The agent of any one of claims 1-8, 18 and 26, comprising a gene editing system.

32. The agent of claim 31, wherein the gene editing system is a CRISPR / Cas system, a transposon-based gene editing system, and a transcription activator-like effector nuclease (TALEN) system.

33. The agent of any one of claims 1-32, wherein the target protein is translated from a non-coding RNA.

34. The agent of claim 33, wherein the non-coding RNA is a long intergenic non-coding RNA (lincRNA).

35. The agent of any one of claims 1-32, wherein the target protein is translated from a non-exonic element in an unprocessed precursor mRNA (pre-mRNA).

36. The agent of claim 35, wherein the non-exonic element is an intron in the pre-mRNA.

37. The agent of claim 35, wherein the non-exonic element is a 5' untranslated region (5'UTR) in pre-mRNA.

38. The agent of claim 35, wherein the non-exonic element is a 3' untranslated region (3'UTR) in pre-mRNA.

39. A pharmaceutical composition comprising the agent of any one of claims 1-38 and a pharmaceutically acceptable carrier.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutically acceptable carrier is a carrier.

41. A method for regulating the expression or activity of a target protein identified in the sequence listing, Table A, or a variant thereof in a cell and / or tissue, the method comprising contacting the cell with an agent as described in any one of claims 1 to 38 or a pharmaceutical composition as described in claim 39 or 40.

42. The method of claim 41, wherein the agent reduces the expression or activity of the target protein in the cell and / or tissue.

43. The method of claim 41, wherein the agent increases the expression or activity of the target protein in the cell and / or tissue.

44. The method of any one of claims 41-43, wherein the cells and / or tissues are in a subject, and in: a) the cell is a liver cell (e.g., a hepatocyte (HC), a hepatic stellate cell (HSC), a Kupffer cell (KC) and / or a liver sinusoidal endothelial cell (LSEC)); a pancreatic cell (e.g., an α cell, a β cell, a δ cell and / or a PP cell); a thyroid cell; a glandular cell; In certain embodiments, the target cell is a hepatocyte (HC), a Kupffer cell (KC), a pancreatic β cell, a muscle cell, a heart cell, a brain cell, a kidney cell, a fat cell, or a combination thereof; and / or b) The tissue comprises liver, pancreas, thyroid, ovary, testis, muscle, heart, brain, kidney or adipose tissue or a combination thereof.

45. The method of claim 44, wherein the subject suffers from a disease or condition selected from the group consisting of aging, senescence, fibrosis, metabolic disease, cardiovascular disease, endocrine-related disorders, genetic diseases, cancer, infection, immune disease, indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

46. ​​The method of any one of claims 41-45, wherein the agent modulates the expression or activity of the target protein by at least 10%.

47. The method of any one of claims 41-46, wherein the agent modulates the expression of the gene encoding the target protein by at least 10%, thereby modulating the expression or activity of the target protein.

48. A method for predicting the likelihood of a subject suffering from a disease or condition, the method comprising quantifying the expression or activity of a target protein identified in the sequence listing, Table A, or the aforementioned variant in a sample from the subject, wherein the expression or activity level of the target protein in the sample indicates the likelihood of the subject suffering from cancer, wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer, infection, immune diseases, indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

49. A method for preparing a sample that can be used to detect the likelihood of a subject suffering from a disease or condition, the method include: 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 that can be used to detect the possibility of suffering from cancer; and c) quantifying the expression or activity of the target protein identified in the sequence listing, Table A or the aforementioned variant in the sample prepared in step b), The disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer, infection, immune diseases, indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

50. The method of claim 48 or 49, further comprising administering to the subject an effective amount of the agent of any one of claims 1-38 or the pharmaceutical composition of claim 39 or 40 if the subject is predicted to have a likelihood of developing cancer.

51. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent as described in any one of claims 1-38 or a pharmaceutical composition as described in claim 39 or 40, wherein the disease or condition is selected from aging, senescence, fibrosis, metabolic diseases, cardiovascular diseases, endocrine-related disorders, genetic diseases, cancer, infection, immune diseases, indications for treatment with hormones, growth factors and / or protein replacement, or a combination thereof.

52. A method for identifying an agent that modulates the expression or activity of a target protein identified in a sequence listing, Table A, or the foregoing, the method include: a) contacting the protein identified in the sequence listing or Table A or the aforementioned variants with a pharmaceutical agent; as well as b) determining whether the agent modulates the expression or activity of the target protein, wherein a difference in the expression or activity of the target protein that has been contacted with the agent compared to a reference for the expression or activity of the target protein indicates that the agent modulates the expression or activity of the target protein.

53. The method of claim 52, wherein a difference of at least 10% in the expression or activity of the target protein that has been contacted with the agent compared to the reference indicates that the agent modulates the expression or activity of the target protein.

54. The method of claim 52 or 53, wherein a decrease in the expression or activity of the target protein that has been contacted with the agent compared to the reference indicates that the agent inhibits the expression or activity of the target protein.

55. The method of claim 52 or 53, wherein an increase in the expression or activity of the target protein compared to the reference indicates that the agent activates the expression or activity of the target protein.

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