Compositions and methods for modulating cytokines

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, such as a cytokine. In various embodiments, the disclosure also provides methods of treating inflammation, aging, senescence, fibrosis, autoimmunity, cancer, infection, and / or immunological disease using agents comprising and / or modulating the expression or activity of an immunomodulatory-related protein, 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 / 345,778, 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: 57081057002.xml; created on May 24, 2023, size: 6,780,048 bytes. Background Art

[0003] The main function of the immune system is to mediate protective immunity against pathogens such as viruses, bacteria, fungi, parasites and tumors. During cancer and chronic infection, multiple immune destruction mechanisms are often enhanced. Insufficient immune response may lead to immunodeficiency, prone to infection and cancer (e.g., tumor) progression. However, in the absence of strict regulation, the immune system may also promote acute and chronic inflammation, leading to immune conditions or autoimmunity. As effective signal transduction molecules for regulating inflammation, cytokines are necessary for the function of the immune system in health and disease. Therefore, it is urgent to identify other novel therapeutic targets for regulating immune cells and immune responses, such as targets that act as cytokines or like cytokines and targets that regulate cytokines. Summary of the invention

[0004] The present disclosure provided herein is based, in part, on the identification of non-classical protein targets (eg, proteins encoded by non-classical open reading frames (ORFs)) for modulating immune responses.

[0005] On the one hand, the disclosure relates to a medicament comprising a target protein identified herein (e.g., a target protein in a sequence table) or a variant thereof and / or regulating (e.g., increasing or decreasing) the expression and / or activity of the target protein or its variant. In some embodiments, the medicament comprises a target protein identified herein (e.g., a target protein in a sequence table) 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 in a sequence table or a variant thereof). In some embodiments, the medicament comprises a polypeptide, a polynucleotide, a gene editing system, a small molecule or a cell (e.g., a cell therapy), is essentially composed of it, or is composed of it. 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] On the other hand, the present disclosure provides a method for detecting a disease or condition in a subject or determining the likelihood of a subject suffering 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 of the subject suffering from the disease or condition, wherein the disease or condition is selected from inflammation, aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), immune disease (e.g., disease caused by immune system disorders) or a combination thereof. In certain embodiments, the disease or condition is or relates to inflammation.

[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), Wherein the disease or condition is selected from inflammation, aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), infection, immune disease (e.g., disease caused by immune system disorder) or a combination thereof. In certain embodiments, the disease or condition is or is related to inflammation.

[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] On the other hand, the present disclosure provides a method for selecting a subject suitable for treating 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 treating the disease or condition according to the expression or activity level of the target protein in the sample, wherein the disease or condition is selected from inflammation, aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), infection, immune disease (e.g., disease caused by immune system disorder) or a combination thereof. In certain embodiments, the disease or condition is or involves inflammation.

[0014] In another aspect, the present disclosure provides a method for modulating the expression or activity of a target protein or a variant thereof identified in a sequence list 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 1 Shown is the inhibition of interferon regulatory factor (IRF) pathway by SEQ ID NO:6516 protein treatment in THP-1 double reporter cells. In the presence of lipopolysaccharide (LPS), THP-1 double reporter cells were incubated with increasing concentrations of peptide (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 repeats. Asterisks represent statistical significance (**p<0.01, ****p<0.0001) compared with cells treated with only LPS.

[0018] Figure 2 Shown is the selective inhibition of IRF response by co-treatment of SEQ ID NO:6516 protein and specific TLR agonist in THP-1 double reporter cells.In the presence of SEQ ID NO:6516SEQ ID NO:6516 protein (10 μM) or disordered protein, THP-1 double 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 repetitions of each treatment group from representative experiments.Asterisk represents the statistical significance (*****p<0.0001) compared with the corresponding TLR agonist in the case of cells treated with disordered 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 3BShown is a bar graph demonstrating counts of up- and down-regulated genes when treated with SEQ ID NO: 6516 protein compared to a scrambled protein control.

[0020] Figure 4 Shown is the inhibition of interferon regulatory factor (IRF) pathway by SEQ ID NO:6516 protein treatment in THP-1 double reporter cells. In the presence of lipopolysaccharide (LPS), THP-1 double reporter cells were incubated with increasing concentrations of peptide (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 repeats. Asterisks represent statistical significance (**p<0.01, ****p<0.0001) compared with cells treated with only LPS.

[0021] Figure 5 Shown is the selective inhibition of IRF response by SEQ ID NO:6516 protein and specific TLR agonist co-treatment in THP-1 double reporter cells.In the presence of SEQ ID NO:6516 protein (10 μM) or disordered protein, THP-1 double 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 repetitions of each treatment group from representative experiments.Asterisk represents the statistical significance (*****p<0.0001) compared with the corresponding TLR agonist in the case of cells treated with disordered 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.

[0022] Fig. 6A shows a UMAP visualization of cluster analysis, which revealed 10 different PBMC cell subtypes; Figure 6B Shown is a bar graph demonstrating counts of up- and down-regulated genes when treated with SEQ ID NO: 6516 protein compared to a scrambled protein control.

[0023] Figure 7 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.

[0024] Figure 8 Shown 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.

[0025] Fig. 9 Shown is the relative activation of the IL-4 signaling pathway, determined via SEAP expression by measuring the optical density at 600 nm.

[0026] Fig.10 Shown is the relative activation of the IL-15 signaling pathway, determined via SEAP expression by measuring the optical density at 600 nm.

[0027] Fig.11 Shown is the relative activation of the IFN-α signaling pathway, determined via SEAP expression by measuring the optical density at 600 nm.

[0028] Figure 12A-12C It is shown that SEQ ID NO: 4524 protein treatment increases T cell activation markers in CD8+ T cells. Isolated CD8+ T cells were incubated with 10 μM scrambled protein or 10 μM SEQ ID NO: 4524 protein for 24 hours in the presence of T cell stimulators (CD3 / CD28 antibody cocktail). Fig. 12A Shown are the concentrations of TNFα measured by standard curve analysis. Fig. 12B Shown are the concentrations of IL-2 measured by standard curve analysis. Fig. 12C The percentage of CD69 positive cells assessed by flow cytometry analysis using CD69 antibody staining is shown, using no stained cells as negative gating control. Data are presented as the mean of triplicate replicates of each healthy donor of PBMC samples. Data were analyzed using a ratio paired t-test.

[0029] Fig.13The effect of synthetic protein (SEQ ID NO:4524) on CD8+T cell activation and IFN-γ production is shown. The separated CD8+T cells are cultured with or without CD3 / CD28 stimulants, and treated with wild-type or point mutation (C to A) peptides of three different concentrations (10 μM, 5 μM and 1 μM). Scrambled protein and untreated are used as controls. Five technical repetitions are performed for each condition. After 3 days of cultivation, IFN-γ production is measured using ELISA, and concentration is calculated using standard curve analysis. Error bars represent the standard deviation (SD) of mean value. Statistical analysis (* p<0.05, **** p<0.0001) is performed using common two-way ANOVA and Sidak (Sidak) multiple comparison test and single merged variance.

[0030] Fig.14 A schematic diagram showing the cloning of target protein genes into pFL69-TRE-rtTA-Blast (Cellecta #SVRTTATB-P) using the Bst XI restriction enzyme site. DETAILED DESCRIPTION

[0031] Following is a description of an exemplary embodiment. Target protein

[0032] 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., comprising a polypeptide selected from the group consisting of SEQ ID NO: 6520, SEQ ID NO: 4227, SEQ ID NO: 6091, SEQ ID NO: 3702, SEQ ID NO: 3585, SEQ ID NO: 6516, SEQ ID NO: 1184, SEQ ID NO: 3915, SEQ ID NO: 1238_L8V, SEQ ID NO: 4524, SEQ ID NO: 3953, SEQ ID NO: 6519, SEQ ID NO: 5862, SEQ ID NO: 2758, SEQ ID NO: 6518, SEQ ID NO: 5843, SEQ ID NO: 4615, SEQ ID NO: 4174, SEQ ID NO: 5627, SEQ ID NO: 5631, SEQ ID NO: 6295, SEQ ID NO: 5848, SEQ ID NO: 5931, SEQ Target proteins having amino acid sequences of SEQ ID NO: 6517, SEQ ID NO: 3494, SEQ ID NO: 3462, SEQ ID NO: 4566, SEQ ID NO: 5537, SEQ ID NO: 5730). The target protein can be produced recombinantly (e.g., via DNA or mRNA) or synthetically.

[0033] In some embodiments, the target protein is an extracellular (secreted) protein.

[0034] In various embodiments, the target protein is a protein comprising the amino acid sequence shown in the sequence table. In certain embodiments, the target protein consists of the amino acid sequence shown in the sequence table. In certain embodiments, the target protein comprises an amino acid sequence having an amino acid substitution relative to the amino acid sequence shown in the sequence table, wherein the substitution is to replace the N-terminal residue in the amino acid sequence in the sequence table with a methionine (Met) residue. In certain 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 table, wherein the substitution is to replace the N-terminal residue in the amino acid sequence in the sequence table with a methionine (Met) residue. In certain embodiments, the target protein comprises the amino acid sequence shown in the sequence table, and is further included in a methionine (Met) residue at its N-terminus. In certain embodiments, the target protein consists of the amino acid sequence shown in the sequence table and a methionine (Met) residue at its N-terminus.

[0035] Certain target proteins in the sequence listing have been identified as being differentially expressed (e.g., upregulated or downregulated) in a disease and / or condition state selected from the following compared to a reference state (e.g., a normal state): aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumors), infection, immune diseases (e.g., inflammation and / or autoimmune diseases) 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.

[0036] Without wishing to be bound by theory, it is believed that certain target proteins disclosed herein act as cytokines or like cytokines, or regulate the expression or activity of cytokines. In some embodiments, the target proteins disclosed herein reduce inflammation (e.g., by inhibiting interferon activity, such as type I interferon activity; by activating NFκB). In some embodiments, the target proteins disclosed herein induce immune cell activation (e.g., by inducing IL-2 or TNF expression, activity and / or secretion). In some embodiments, the target proteins disclosed herein induce immune cell proliferation.

[0037] 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).

[0038] 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, the reference for a disease state can be a normal healthy state; the reference for a mutated protein can be a protein that has not been mutated; the 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).

[0039] In some embodiments, the proteins are used as targets for immune and / or disease states (e.g., exhausted T cells, CD4 + T cells (e.g., Th1, Th2, Th17), CD8 + T cells or a combination thereof).

[0040] In certain embodiments, the target protein has a higher expression level in activated immune cells. In certain embodiments, the expression level of the target protein in activated immune cells is at least about 0.5 times higher than the expression level of the target protein in a reference (e.g., an unactivated immune cell of the same type), 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 higher, 100 times higher).

[0041] In certain embodiments, the target protein has a lower expression level in activated immune cells. In certain embodiments, the expression level of the target protein in activated immune cells is at least about 0.5 times lower than the expression level of the target protein in a reference (e.g., an unactivated immune cell of the same type), 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).

[0042] 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).

[0043] 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).

[0044] 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.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] 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).

[0049] In some embodiments, the target protein is translated from non-exon elements in unprocessed precursor mRNA (pre-mRNA). In some embodiments, the non-exon element is an intron in the pre-mRNA. In some embodiments, the non-exon element is 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.

[0050] 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.

[0051] In some embodiments, the target protein of the present disclosure is a modulator of one or more GPCRs. In some embodiments, the target protein is an agonist of one or more GPCRs. In some embodiments, the target protein is an antagonist of one or more GPCRs. In some embodiments, the target protein is a direct modulator of one or more GPCRs, such as a ligand of one or more GPCRs. In some embodiments, the target protein is an indirect modulator of one or more GPCRs.

[0052] The expression and / or activity of various GPCRs have been linked to different diseases / disorders, conditions and indications, including those shown in Table A (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. 55:1-13. 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 A that are known to be associated with GPCR expression and / or activity.

[0053] Table A. Disease-related GPCRs

[0054] Agents that modulate target proteins

[0055] Provided herein are agents for regulating the expression of a target protein disclosed herein (such as a target protein in a sequence list), a variant thereof, or a fragment thereof (e.g., a biologically active fragment of a target protein). The expression of a target protein or a variant thereof or a 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 number of copies 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 a protein, protein stability, protein degradation (e.g., cleavage, such as protease cleavage), or a combination of the foregoing.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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 FADH2), 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).

[0060] 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.

[0061] 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).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] In some embodiments, the agent induces downregulation of the target protein (e.g., increases target protein degradation); prevents multimerization (e.g., dimerization) of the target protein; sequesters the target protein (e.g., secreted target protein); 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 phagocytosis 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 participates 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 participate in the binding of the target protein to the binding partner.

[0066] Non-limiting examples of binding partners include cytokine receptors and immune signaling receptors.

[0067] In some embodiments, the binding partner comprises: a) type I cytokine receptors (e.g., interleukin 1 receptor, erythropoietin receptor, GM-CSF receptor, G-CSF receptor, growth hormone receptor, prolactin receptor, oncostatin M receptor, leukemia inhibitory factor receptor); b) type II cytokine receptors (e.g., type II interleukin receptor, interferon-α / β receptor, interferon-γ receptor); c) members of the immunoglobulin superfamily (e.g., interleukin-1 receptor, colony stimulating factor 1 receptor (CSF1R), C-kit receptor, interleukin-18 receptor); d) members of the tumor necrosis factor receptor family (e.g., CD27, CD30, CD40, CD120, lymphotoxin beta receptor); e) chemokine receptors (e.g., interleukin-8 receptor, CC chemokine receptor type 1 (CCR1), CXC chemokine receptor type 4 (CXCR-4), MCAF receptor, NAP-2 receptor); f) members of the transforming growth factor (TGF) beta receptor family (e.g., TGF beta receptor 1, TGF beta receptor 2), or a combination of the foregoing.

[0068] In some embodiments, the binding partner comprises: a) Pattern recognition receptors (PRRs) (e.g., Toll-like receptors (TLRs), NOD-like receptors (NLRs)); b) Killer cell activating receptor (KAR); c) Killer cell inhibitory receptors (KIR); d) complement receptors; e) Fc receptors; f) B cell receptor; g) T cell receptor; h) cytokine receptors, or a combination of the foregoing.

[0069] 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 the multimerization (e.g., dimerization) of the binding partner of the target protein; 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, 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 some embodiments, the agent binds to the target binding site of the binding partner. In specific embodiments, the agent further binds to at least one residue of the binding partner of the target protein, and the at least one residue participates in the binding between the target protein and the binding partner. In more specific embodiments, the agent further binds to one or more binding sites and / or domains of a binding partner of the target protein, said one or more binding sites and / or domains being involved in the binding between the target protein and the binding partner.

[0070] In some embodiments, the agent modulates (eg, activates or inhibits) immune signaling, cytokine signaling, inflammatory signaling, or a combination of the foregoing.

[0071] 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.

[0072] 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.

[0073] In certain embodiments, the agent reduces the function of (eg, a blocking antibody) directed against TNFα.

[0074] 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.

[0075] 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.

[0076] 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).

[0077] 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 a 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 Library of Medicine) of the United States. (Altschul et al., 1990).

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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

[0083] 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.

[0084] 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).

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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 by 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 following: 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.

[0090] 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.

[0091] "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.

[0092] 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.

[0093] 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%.

[0094] 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%.

[0095] 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).

[0096] 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 Edward 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).

[0097] 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.

[0098] 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.

[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 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.

[0100] 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.

[0101] 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.

[0102] In some embodiments, polypeptide is a cell penetrating peptide. In certain embodiments, polypeptide is connected to cell penetrating peptide. Suitable cell penetrating peptide sequence 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 [cell penetrating peptides for tumor targeting and future cancer therapy applications], Pharmaceuticals [Pharmaceuticals] 5 (9): 991-1007 (2012). Non-limiting examples of cell penetrating peptides include TAT (48-60), transmembrane peptides, pVEC, MPG8, transporter (Transportan), transporter 10, PepFect3, PepFect 6, PepFect 14, polyarginine, stearoyl-polyarginine, Pep-1, Pep-3, CADY, YTA2, YTA4, SynB1, SynB3, Maurocalcine and PTD4.

[0103] In some embodiments, the polypeptide is a circulating factor (eg, a cytokine).

[0104] 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).

[0105] 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 [Therapeutic Proteins: Methods and Protocols] (Mark C. Smales and David C James, ed., 2005); Pharmaceutical Biotechnology: Fundamentals and Applications [Drug Biotechnology: Basics 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.

[0106] 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.

[0107] 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)).

[0108] In some embodiments, the polypeptide comprises one or more neoantigens or variants thereof selected from the sequence list. As used herein, the term "neoantigen" refers to a tumor antigen produced by a target protein as described herein. In some embodiments, the neoantigen is a neoantigen specific to cancer. 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.

[0109] 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

[0110] 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.

[0111] 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 includes 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.

[0112] 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).

[0113] 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.

[0114] 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 a portion thereof (e.g., a biologically active portion or fragment thereof).

[0115] In some embodiments, the polynucleotide comprises a nucleotide sequence complementary to at least a portion of a gene or gene transcript encoding a target protein disclosed herein (e.g., fully complementary or partially complementary), 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 complementary to at least a portion of a gene or gene transcript encoding a protein capable of regulating the expression or activity of a target protein disclosed herein.

[0116] In some embodiments, the polynucleotide encodes a target protein disclosed herein, or a variant thereof (e.g., a biologically active variant thereof), or a portion thereof (e.g., a biologically active portion or fragment thereof).

[0117] In some embodiments, the nucleic acid encoding the target protein or its variant or part (e.g., fragment) is a gene sequence or part thereof. In some embodiments, the coding nucleic acid is an unprocessed RNA transcript (e.g., pre-mRNA) or part thereof (e.g., 5'UTR, 3'UTR, intron). In some embodiments, the coding nucleic acid is an mRNA molecule or part thereof. In some embodiments, the coding nucleic acid is present in a non-coding RNA (e.g., long intergenic non-coding RNA (lincRNA), long non-coding RNA (lncRNA) or miRNA).

[0118] The encoding nucleic acid may comprise a classical open reading frame (ORF) or a non-classical ORF. In certain embodiments, the encoding nucleic acid comprises a non-classical ORF.

[0119] The polynucleotide may be single-stranded (ss) or double-stranded (ds). In some embodiments, the polynucleotide is double-stranded (ds). In some embodiments, the double-stranded polynucleotide is about 15-50 base pairs in length, 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 length. In some embodiments, the polynucleotide is about 19-23 base pairs in length. In some embodiments, the polynucleotide is about 21 base pairs in length.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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).

[0125] 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.

[0126] 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.

[0127] 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%.

[0128] 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.

[0129] 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 acts as miRNA; in other embodiments, miRNA acts 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, miRNA reduces protein output by translational inhibition 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 sites are within the mRNA 3'UTR; miRNAs appear to target sites that have almost complete complementarity with 2-8 nucleotides at the 5' end of the miRNA (see, e.g., Rajewsky, Nat Genet [Natural Genetics] 38 Suppl: S8-13 (2006) and Lim et al., Nature [Natural] 433: 769-73 (2005)). This region is referred to as the seed region. Because siRNA and miRNA are interchangeable, exogenous siRNAs downregulate mRNAs that have seed complementarity with the siRNA (see, e.g., Birmingham et al., Nat Methods [Natural Methods] 3: 199-204 (2006)). Multiple target sites within the 3'UTR produce stronger downregulation (see, e.g., Doench et al., Genes Dev [Genes and Development] 17: 438-42 (2003)).

[0130] 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)).

[0131] 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).

[0132] In some embodiments, mRNA is produced by in vitro transcription. In some embodiments, mRNA is modified to optimize its activity. In some embodiments, mRNA comprises modified bases, 5' caps, 5' cap analogs, anti-reverse cap analogs (ARCA) or a combination thereof.

[0133] 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)).

[0134] 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)).

[0135] In some embodiments, the mRNA comprises a modified 3' untranslated region (UTR), a 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, the 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)).

[0136] In some embodiments, the mRNA comprises RNA base modifications. 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 [Control Release Magazine] 217: 337-44 (2015) and Svitkin et al., Nucleic Acids Res. [Nucleic Acids Research] 45: 6023-36 (2017)).

[0137] In some embodiments, the RNA (e.g., mRNA) is a circular RNA.

[0138] 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, e.g., 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)).

[0139] 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.

[0140] 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.

[0141] 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).

[0142] One of ordinary skill in the art can readily prepare suitable polynucleotide preparations for use in the compositions, kits, and methods described herein using the locus information (such as chromosomal location, start and end nucleotide positions, and polymorphism identification) of the protein sequences contained in the sequence listing incorporated herein.

[0143] C. Agents containing gene editing systems

[0144] In some embodiments, the medicament comprises a gene editing system. In some embodiments, the gene editing system produces a nucleotide deletion, a nucleotide substitution, a nucleotide addition, or a combination thereof in a gene encoding a target protein.

[0145] In some embodiments, the gene editing system is a CRISPR / Cas system, a transposon-based gene editing system, or a transcription activator-like effector nuclease (TALEN) system. In some embodiments, the gene editing system is a CRISPR / Cas system. In some embodiments, the gene editing system is a Class II CRISPR / Cas system.

[0146] In some embodiments, the gene editing system (e.g., CRISPR / Cas system) reduces (e.g., reduces, inhibits) or eliminates (e.g., via gene knockout) the expression of the target protein. In some embodiments, the gene editing system (e.g., CRISPR / Cas system) reduces (e.g., reduces, inhibits) or eliminates (e.g., via gene knockout) the expression of a protein that can regulate the expression or activity of the target protein. In some embodiments, the gene editing system (e.g., CRISPR / Cas system) increases (e.g., via gene knock-in or gene replacement) the expression of the target protein. In some embodiments, the gene editing system (e.g., CRISPR / Cas system) increases (e.g., via gene knock-in or gene replacement) the expression of a protein that can regulate the expression or activity of the target protein.

[0147] In certain embodiments, the CRISPR system specifically catalyzes the cutting in the gene encoding the target protein, thereby inactivating the gene. Repairing nucleic acid chain breakage by non-homologous end joining (NHEJ) often results in the change of DNA sequence at the cleavage site, resulting in a small insertion or deletion (insertion and deletion (Indel)). In certain embodiments, NHEJ is used to knock out the gene encoding the target protein. In certain embodiments, homology directed repair (HDR) is used to simultaneously inactivate the gene encoding the target protein and insert a heterologous sequence into the inactivated locus. The cell in which knockout and / or knock-in events have occurred can be identified and / or selected by methods well known in the art.

[0148] 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.

[0149] In some embodiments, the type II Cas endonuclease is Cas9 (e.g., 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 / ).

[0150] 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").

[0151] 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.

[0152] 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 Cpf1 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 (for example, 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)).

[0153] 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.

[0154] 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.

[0155] 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.

[0156] Cpf1 related CRISPR arrays are processed into mature crRNA without the need for tracrRNA. Cpf1 endonucleases associate with T-rich PAM sites (e.g., 5'-TTN). Cpf1 can also recognize 5'-CTA PAM motifs. Cpf1 cuts target DNA by introducing misplaced or staggered double-strand breaks with 5' overhangs of 4 or 5 nucleotides, such as cutting such target DNA, where the misplaced or staggered cutting of 5 nucleotides is located at 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 overhangs of 5 nucleotides produced by such misplaced cutting allow DNA insertion by homologous recombination to be more accurately edited than the insertion at the DNA of blunt end cutting. See, for example, Zetsche et al., Cell [cell] 163: 759-71 (2015).

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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). .

[0161] 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).

[0162] 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.

[0163] 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.

[0164] 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).

[0165] 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.

[0166] 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.

[0167] 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 from, for example, Advantgene.

[0168] 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).

[0169] 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. c) Small molecule drugs

[0170] 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.

[0171] 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.

[0172] In certain embodiments, the agent comprises a proteolysis targeting chimera (PROTAC).

[0173] 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. d) Therapeutic cells and cell-based therapies

[0174] 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 or variant thereof in a sequence table), 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., recombinant DNA, RNA (such as mRNA or siRNA)) and / or a gene editing system (e.g., CRISPR / Cas system).

[0175] 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.

[0176] 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

[0177] In another aspect, the present disclosure provides an expression vector comprising a polynucleotide described herein.

[0178] 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.

[0179] In certain 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 certain embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In certain 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 that is suitable for artificial selection. The selectable marker element can be a negative or positive selection marker. Non-limiting examples of expression vectors used 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 edited, 4th edition 2012).

[0180] In another aspect, the present disclosure provides an expression host cell comprising any one or more of the polynucleotides or expression vectors described herein.

[0181] The term "expression host cell" refers to a cell that can be used to receive, maintain, replicate and / or amplify a vector.

[0182] 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).

[0183] 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

[0184] 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.

[0185] 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).

[0186] 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.

[0187] 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.

[0188] In some embodiments, the heterologous part is polyethylene glycol (PEG), hexadecanoic 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).

[0189] 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).

[0190] In some embodiments, the composition is formulated to be administered as a combination therapy with one or more additional therapeutic agents (e.g., with a second therapeutic agent). As used herein, "combination therapy" or "combined administration" means that two (or more) different agents 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 agents or treatments include biological agents (e.g., antibodies, peptides), cell therapy, gene therapy, immunotherapy, and small molecules that affect immune-mediated diseases or conditions.

[0191] 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.

[0192] 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).

[0193] 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 virus, lentivirus, alpharetrovirus, gammaretrovirus, foamy virus (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.

[0194] 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).

[0195] In certain 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 around 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 the degradation of plasma enzymes, and are loaded and transported across biomembranes and blood-brain barriers (BBB) ​​(see, e.g., Spuch and Navarro, J Drug Deliv. [Drug Delivery Magazine] 2011: 469679 (2011)).

[0196] Vesicles can be made of 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. Patent 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 Deliv. [Drug Delivery Magazine] 2011: 469679 (2011)). Extruded lipids can be prepared by extrusion through a filter with a gradually decreasing size, such as Templeton et al., Nature Biotech [Natural Biotechnology], 15: 647-52 (1997), which is incorporated herein by reference for its teachings on the preparation of extruded lipids.

[0197] 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.

[0198] 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 important components of drug delivery. These nanoparticles can effectively guide drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), i.e., novel carriers that combine 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 [Nanomaterials] 7 (6): 122 (2017).

[0199] 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.

[0200] 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 (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and globulin.

[0201] 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, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-( The following are some examples of the compounds: 1,2-diisopropylaminoethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol hydrochloride), diheptadecylamido glycylidene spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC).

[0202] 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).

[0203] 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.

[0204] 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).

[0205] 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.

[0206] In some 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, which is incorporated herein by reference for its teachings on anellosome design, preparation and use. Methods for regulating target proteins

[0207] On the other hand, the present disclosure provides a method for regulating the expression or activity of a target protein or a variant thereof identified in a sequence list 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.

[0208] In some embodiments, the target cell is a cancer cell (e.g., a metastatic cancer cell), a cell in a tumor microenvironment (e.g., a stromal cell), or a combination thereof. In specific embodiments, the target cell is a metastatic cancer cell.

[0209] 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.

[0210] In some embodiments, the target cell is involved and / or participates in fibrosis, aging and / or senescence. In certain embodiments, the target cell is an epithelial cell, an endothelial cell, a stem cell, a non-immune cell, or a combination thereof.

[0211] 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.

[0212] 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.

[0213] The target tissue can be any tissue in the body.

[0214] In certain embodiments, the target tissue comprises a tumor, a tumor microenvironment, a metastatic site, or a combination thereof.

[0215] 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.

[0216] 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%.

[0217] 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.

[0218] 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

[0219] 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 developing 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 developing the disease or condition, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumors), immune diseases (e.g., inflammation and / or autoimmune diseases), or a combination thereof.

[0220] On the other hand, the present disclosure provides a method for classifying a subject based on the predicted likelihood of suffering from a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from a subject; predicting the likelihood of suffering from a disease or condition based on the expression or activity of the target protein in the sample; and classifying the patient based on the predicted likelihood, wherein the disease or condition is selected from inflammation, aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), immune disease (e.g., disease caused by immune system disorder) or a combination thereof. In certain embodiments, the disease or condition is or relates to inflammation.

[0221] On the other hand, the present disclosure provides a method for stratifying a group of subjects with 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 subject group for treatment according to the expression and / or activity level of the individual subject of the target protein in the sample, wherein the disease or condition is selected from inflammation, aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), immune disease (e.g., disease caused by immune system disorder) or a combination thereof. In certain embodiments, the disease or condition is or involves inflammation.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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; c) quantifying the expression or activity of the target protein in the sample prepared in step b); and Wherein the disease or disorder is selected from inflammation, aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), immune disease (e.g., disease caused by immune system disorder) or a combination thereof. In certain embodiments, the disease or disorder is or involves inflammation.

[0226] 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.

[0227] 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.

[0228] As used herein, "treatment" and "treating" refer to 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 and / or undesirable clinical manifestations of the disease, disorder, or disorder are reduced and / or the time course of progression is 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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).

[0233] 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.

[0234] In another aspect, the disclosure provides a method for regulating the expression or activity of a target protein or variant thereof identified in a sequence listing 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.

[0235] 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), 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.

[0236] 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.

[0237] 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 e) Cancer

[0238] 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.

[0239] 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; 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 carcinoma (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. ;

[0240] Metastasis of the above cancers can also be treated according to the methods described herein. In some embodiments, the cancer is a metastatic cancer.

[0241] 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.

[0242] 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.

[0243] 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, or a combination of the foregoing.

[0244] 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%.

[0245] 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%.

[0246] 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.

[0247] 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%.

[0248] 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%.

[0249] 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%.

[0250] In some embodiments, the effective amount is sufficient to modulate (eg, increase or decrease) expression of the target protein in cancer (eg, tumor) cells.

[0251] In some embodiments, the effective amount is sufficient to modulate the body's response to cancer, such as by inhibiting the growth of the cancer, reducing the malignancy of the cancer, inhibiting the metastasis of the cancer, promoting remission, modulating (increasing and / or decreasing) immune-mediated responses associated with the cancer, or a combination of the foregoing. f) Immuno-oncology

[0252] 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.

[0253] In some embodiments, treatment modulates (eg, increases) the subject's immune system against cancer.

[0254] In certain embodiments, treatment modulates (e.g., increases or decreases): a) Lymph node innervation; b) development of high endothelial venules (HEVs) and / or tertiary lymphoid organs (TLOs); c) immune cell: migration, proliferation, recruitment, lymph node homing, lymph node egress, tumor homing, tumor egress, differentiation, activation, polarization, cytokine production, degranulation, maturation, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antigen presentation, target (e.g., protein or transcript) expression, or a combination thereof, or a combination of the foregoing.

[0255] In some embodiments, the effective amount is sufficient to modulate (eg, increase) the subject's immune system against cancer.

[0256] In certain embodiments, an effective amount is sufficient to modulate (e.g., increase or decrease): a) Lymph node innervation; b) development of HEV and / or TLO; c) immune cell: migration, proliferation, recruitment, lymph node homing, lymph node egress, tumor homing, tumor egress, differentiation, activation, polarization, cytokine production, degranulation, maturation, ADCC, ADCP, antigen presentation, target (e.g., protein or transcript) expression, or a combination thereof, or a combination of the foregoing.

[0257] 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%.

[0258] 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%.

[0259] 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 innervation, lymph node homing of immune cells (e.g., dendritic cells and / or T cells), immune cell (e.g., dendritic cells) and / or T cells) , cells and / or T cells), lymph node outflow of immune cells, differentiation of immune cells, activation of immune cells, polarization of immune cells, cytokine production (e.g., increasing pro-inflammatory cytokine production, decreasing pro-inflammatory cytokine production, increasing anti-inflammatory cytokine production, decreasing anti-inflammatory cytokine production), 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 immune cells (e.g., CD8 + T cells), tumor outflow, target protein expression, or a combination of the foregoing. g) Immunity / Inflammation

[0260] 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.

[0261] 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.

[0262] 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).

[0263] 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.

[0264] 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).

[0265] 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).

[0266] In certain embodiments, the target protein is an agonist against IL-2.

[0267] 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.

[0268] In some embodiments, treatment modulates (eg, increases) the subject's immune system.

[0269] In certain embodiments, treatment modulates (e.g., increases or decreases): a) Lymph node innervation; b) development of HEV and / or TLO; c) immune cell: migration, proliferation, recruitment, lymph node homing, lymph node egress, tumor homing, tumor egress, differentiation, activation, polarization, cytokine production, degranulation, maturation, ADCC, ADCP, antigen presentation, target (e.g., protein or transcript) expression, or a combination thereof, or a combination of the foregoing.

[0270] In some embodiments, the effective amount is sufficient to modulate (eg, increase or decrease) the immune system of a subject.

[0271] In certain embodiments, an effective amount is sufficient to modulate (e.g., increase or decrease): a) Lymph node innervation; b) development of HEV and / or TLO; c) immune cell: migration, proliferation, recruitment, lymph node homing, lymph node egress, tumor homing, tumor egress, differentiation, activation, polarization, cytokine production, degranulation, maturation, ADCC, ADCP, antigen presentation, target (e.g., protein or transcript) expression, or a combination thereof, or a combination of the foregoing.

[0272] In some embodiments, an effective amount is sufficient to modulate (e.g., increase or decrease): a) Development of HEV and / or TLO; b) immune cell activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment; 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.

[0273] 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; 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.

[0274] 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%.

[0275] 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; 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.

[0276] 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%.

[0277] In some embodiments, the 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 number of relapses and / or outbreaks; e) reduce viral load; or f) reduce (e.g., control) infection, or a combination of the foregoing.

[0278] In some embodiments, the effective amount is sufficient to increase organ function, inflammation, or a combination thereof. In certain embodiments, the increase is at least about 10%, such as at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, the increase is 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%.

[0279] 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%.

[0280] In some embodiments, the target protein activates immune cells. In some embodiments, the target protein inhibits immune cells. Immune cells are cells that play a role in immune response. Immune cells have hematopoietic origin, including lymphocytes (e.g., B cells and T cells), natural killer cells and myeloid cells (e.g., basophils, eosinophils, granulocytes, macrophages, mast cells and monocytes). The target protein can be expressed on cancer cells (e.g., metastatic cancer cells), in tumor microenvironment (e.g., on stromal cells) or on non-malignant cells (e.g., immune cells).

[0281] 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%.

[0282] 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%.

[0283] 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%.

[0284] 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%.

[0285] In certain embodiments, the effective amount is sufficient to modulate expression of the target protein in an immune cell.

[0286] 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. h) Aging

[0287] 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.

[0288] 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%. i) Aging

[0289] 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.

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

[0291] 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.

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

[0293] 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.

[0294] 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.

[0295] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0296] As used herein, the indefinite articles “a,” “an,” and “the” should be understood to include plural referents unless the context clearly indicates otherwise.

[0297] 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".

[0298] 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.

[0299] 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".

[0300] 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 that the target protein acts as a cytokine on T cells.

[0301] This example demonstrates the ability of the target protein of the present disclosure to function as a cytokine on mammalian T cells. In this example, human T cell line (Jurkat, ATCC) cells were treated with supernatants from transduced HEK293T cells, each of which contained an overexpressed target protein of interest. HEK293T cells (1 x 10 5 / well) were seeded in 96-well plates (200 μl / well) to reach about 70%-90% confluence on the next day (eg, 12-36 h).

[0302] Two V-bottom 96-well plates were prepared and a mix of each construct was prepared as indicated in Table B below.

[0303] Table B The constructs used were designed as follows:

[0304] Doxycycline-inducible expression of target proteins was generated using the InDOXible Tet-activated lentiviral cDNA expression system (Celecota). All target protein genes were cloned into pFL69-TRE-rtTA-Blast (Celecota #SVRTTATB-P) using the Bst XI restriction enzyme site ( Fig.14 ).

[0305] In order to produce a lentivirus containing the target protein, the cloned lentiviral vector was transfected into HEK293T cells with a lentiviral packaging mixture (Celecota #CPCP-K2A), as described in detail below. 24h after cell inoculation, the plate A mixture was transferred to plate B and mixed by gently pipetting. The cells were incubated and DNA-lipofectamine was added to the cell mixture. The transfected cells were incubated in a CO2 incubator at 37°C for 24h.

[0306] To harvest the virus, the transfected cells were centrifuged at 1650 rpm for 5 min in a 96-well plate. 150 μl of the supernatant was transferred to a new V-bottom 96-well plate and centrifuged at 1650 rpm for 5 min. 150 μl of the supernatant was carefully transferred to the cells already placed in the 96-well plate (U-bottom).

[0307] To transduce Jurkat cells, 150 μl of virus was transferred into Jurkat cells. The cells were centrifuged at 1650 rpm for 1 h (32° C.) in a 96-well plate using the spinoculation method. The supernatant was discarded and the cells were resuspended in 200 μl of complete medium. The cells were incubated overnight at 37° C.

[0308] 24h after spinoculation, cells were centrifuged and resuspended in complete medium (with penicillin / streptomycin) plus blasticidin (final concentration 10μg / ml, 1:1000 dilution from original tube ThermoFisher #A1113903). After 48h, cells were processed for flow cytometry and stained for expression of CD69 (Invitrogen, #MA5-18150). 5μl CD3 / CD28 antibody complex (StemCell Technologies, ImmunoCult TM Human CD3 / CD28 T cell activator) was used as a positive control to stimulate T cells. The cells were incubated at 37°C for 24 h. The upregulation of CD69 expression in Jurkat cells indicated that the target protein was a cytokine that acted on T cells. Example 2: Validation of target proteins as cytokines (ie, scRNAseq hashing).

[0309] This example demonstrates the ability of the target protein of the present disclosure to act as a cytokine by single cell RNA sequencing to look for changes in the cell population after treatment with the target protein. In this example, human primary blood mononuclear cells were cultured at 1 x 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. Target proteins (e.g., listed in the sequence table) were ordered by GenScript Biotech (Piscataway, New Jersey). Peptides were synthesized using solid phase peptide synthesis (SPPS) by fluorenylmethoxycarbonyl (Fmoc) protecting group chemistry.

[0310] 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 for 10 min at 4 ° C. 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).

[0311] Single cells were processed by the 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.

[0312] After sequencing, the reads were aligned to the human reference genome (GENCODE34 / GRCH38). The reads were 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 contained more than 500 genes). The raw data was normalized. Principal component analysis was performed, and the cell clusters were annotated. Differential gene expression analysis was performed between the control and the target protein treatment. Inflammatory cell populations (e.g., Th1 CD4 + T cells, CD8 + In addition, in cells where the target protein is immunosuppressive, an increase in immunosuppressive cell populations (e.g., T regulatory cells) following treatment with the target protein is observed. Example 3. Validation of target proteins as cytokines (ie, scRNAseq hashing).

[0313] On the one hand, this demonstrates the validation of the target protein (SEQ ID NO: 6516) as a cytokine regulator. SEQ ID NO: 6516 is a novel secreted peptide that inhibits the innate immune response caused by other circulating factors (toll-like receptor agonists). Therefore, SEQ ID NO: 6516 is a monocyte and dendritic cell regulatory cytokine and will be suitable for treating diseases caused by pathological innate immune responses, such as lupus, multiple sclerosis and rheumatoid arthritis.

[0314] 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 primary human immune cells were most responsive to the target proteins, target proteins were applied to peripheral blood mononuclear cells (PBMCs), which were then subjected to single-cell RNA-seq 24 hours later.

[0315] Materials and Methods

[0316] Dual reporter assay: NF-kB-SEAP and IRF-Lucia luciferase reporter monocytic (THP1) cells (InvivoGen) 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.

[0317] 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.

[0318] scRNA-seq: From the example: 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 1x10 PBMC. 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 (Bai Jin Company). 5 μl of human TruStain FcX (Bai Jin 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).

[0319] 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 Inc. 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.

[0320] 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.

[0321] SEQ ID NO: 6516 significantly blocks the IRF signaling pathway responses from several TLRs. Upregulation of the IRF pathway is associated with many autoimmune diseases (especially lupus). Consistent with the association with innate immunity-related diseases (like lupus), SEQ ID NO: 6516 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 3A and Figure 3B ). Other signs: Target protein Relative activity SEQ ID NO:6520 +++ Example 4: Validation of target protein as cytokine on monocytes.

[0322] This example demonstrates the ability of the target protein disclosed herein to act as a cytokine on mammalian monocytes. In this example, the target protein (e.g., listed in the sequence table) was ordered by GenScript Biotech (Piscataway, New Jersey). Peptides were synthesized using solid phase peptide synthesis (SPPS) by fluorenylmethoxycarbonyl (Fmoc) protecting group chemistry. NF-kB-SEAP and IRF-Lucia luciferase reporter monocytic cells (THP1) cells (Anvivogen, #thpd-nfis) were cultured in RPMI 1640 culture medium supplemented with HEPES buffer (10mM), sodium pyruvate (1mM), glucose (4.5g / L), fetal bovine serum (10%), penicillin (100U / mL), streptomycin (100μg / mL) and 2-mercaptoethanol (0.05mM). THP1 cells were treated with 1 μg / mL LPS (Sigma-Aldrich) for 24 h, treated with target protein (final concentration 10 μM), or left untreated.

[0323] After treatment, THP1 cells were cultured for 12 h and then the reporter activity was measured. TM (Anvivogen, #rep-qbs) (SEAP test kit) and QUANTI-Luc TM(Anvivogen, #rep-qlc1) (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 a biological sample (such as a cell culture supernatant). QUANTI-Luc is a lyophilized assay reagent containing all the components required for quantitatively measuring the activity of Lucia luciferase and other luciferases that utilize coelenterazine. An increase in reporter activity after treatment with the target protein was observed in cells where the target protein was immunostimulatory. In addition, a decrease in reporter activity after treatment with the target protein was observed in cells where the target protein was immunosuppressive. Example 5. Validation of target protein as cytokine on monocytes.

[0324] On the one hand, this example demonstrates the validation of a target protein (SEQ ID NO: 6516) as a cytokine that affects monocytes. SEQ ID NO: 6516 is a novel secreted peptide that inhibits the activity of toll-like receptors on monocytes and dendritic cells. Therefore, SEQ ID NO: 6516 is an innate immune cell regulatory cytokine and will be suitable for treating diseases caused by pathological innate immune responses, such as lupus, multiple sclerosis, and rheumatoid arthritis.

[0325] 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 primary human immune cells were most responsive to the target proteins, target proteins were applied to peripheral blood mononuclear cells (PBMCs), which were then subjected to single-cell RNA-seq 24 hours later.

[0326] Materials and Methods

[0327] Dual reporter assay: NF-kB-SEAP and IRF-Lucia luciferase reporter monocytic (THP1) cells (Anvivogen) were cultured in RPMI 1640 medium supplemented with HEPES buffer (10 mM), sodium pyruvate (1 mM), glucose (4.5 g / L), fetal bovine serum (10%), penicillin (100 U / mL), streptomycin (100 μg / mL) and 2-mercaptoethanol (0.05 mM). THP1 cells were treated with 100 ng / mL LPS (Sigma-Aldrich) for 24 h, treated with target protein (final concentration 10 μM), or left untreated.

[0328] 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.

[0329] scRNA-seq: From the example: 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 1x10 PBMC. 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 (Bai Jin Company). 5 μl of human TruStain FcX (Bai Jin Company) was added to the cells and incubated at 4 ° C for 10 min. 1 μg of single cell hash antibody (TotalSeq, Biogen) was added to the cells along with approximately 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 (approximately 1000 cells / μl).

[0330] 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 Inc. 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.

[0331] 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.

[0332] SEQ ID NO: 6516 significantly blocks the IRF signaling pathway responses from several TLRs. Upregulation of the IRF pathway is associated with many autoimmune diseases (especially lupus). Consistent with the association with innate immunity-related diseases (like lupus), SEQ ID NO: 6516 strongly affects activated monocytes and dendritic cells, while having relatively little effect on T cells, B cells, and NK cells ( Figure 4 , Figure 5 , Fig. 6A and Figure 6B ). Other signs: Target protein Relative activity SEQ ID NO:1184 +++ Example 6: Validation of the target protein as a cytokine through activation and differentiation of immune cells.

[0333] This example shows the ability of target protein to act as cytokine by activating and / or differentiating immune cells (such as monocytes to macrophages). In this example, one or more target proteins (e.g., listed in the sequence table) of this disclosure are ordered by GenScript Biotech (Piscataway, New Jersey). Peptides are synthesized by fluorenylmethoxycarbonyl (Fmoc) protecting group chemistry using solid phase peptide synthesis (SPPS). Human mononuclear cells (THP-1, ATCC) are cultured in RPMI 1640 culture medium supplemented with HEPES buffer (10mM), sodium pyruvate (1mM), glucose (4.5g / L), fetal bovine serum (10%), penicillin (100U / mL), streptomycin (100μg / mL) and 2-mercaptoethanol (0.05mM). Monocytes were treated with 1 μg / mL LPS (Sigma-Aldrich) and M-CSF (50 ng / ml) for 24 h, 50 nM PMA (Sigma-Aldrich) for 72 h, treated with target protein (final concentration 10 μM), or left untreated.

[0334] Untreated cells, LPS-treated cells or target protein-treated cells were resuspended in 2 mL fresh culture medium at a cell concentration of 500,000 cells / mL and inoculated into 24-well TCPS plates. The plates were cultured for 24 h. After 24 h, cells were mechanically dissociated and released from the material and collected. Nikon Diaphot optical microscope (Nikon, Melville, New York) was used to count cells on a hemocytometer. Viable cells were determined via trypan blue dye exclusion. The supernatant collected at 24 h was stored at -20 °C until cytokine concentrations were analyzed.

[0335] The cells treated with PMA were rinsed with PBS and separated with 4mL trypsin / EDTA (Clonetics, Walkersville, Maryland) per T-75 flask. The flask was incubated for 4min and then 8mL trypsin neutralization solution (TNS; Clonetics) was added. The cells were then resuspended and inoculated onto the material in the same manner as untreated cells and LPS-treated cells. 24h after inoculation, the supernatant was collected and stored at -20°C. Then 260 μL trypsin / well was used, incubated for 4min, and then 520 μL TNS / well was added to loosen the cells from the material. The viable cells determined by trypan blue dye exclusion were counted on a hemocytometer via an optical microscope.

[0336] In order to evaluate the level of monocyte differentiation, the expression of cell surface marker CD11b (marker of macrophage differentiation) was measured by flow cytometry. Untreated cells, LPS-treated cells, PMA-treated cells or target protein-treated cells were separated as described above and fixed with 3.7% paraformaldehyde for 15 min. The cells were then rinsed in PBS, incubated with 10% goat serum (blocking buffer; Sigma), and incubated with 10 μg / mL mouse anti-human CD11b (Becton Dickinson). The cells were rinsed again, then incubated with Alexa fluor488 goat anti-mouse secondary antibody (Invitrogen), rinsed, and post-fixed in 10% formalin. The fluorescence intensity of the labeled cells was measured using a FACSCalibur flow cytometer (Becton Dickinson). At least 10,000 events were obtained for each sample to determine the geometric mean fluorescence intensity. Cytokines including TNF-α, MCP-1, MIP-1α, IL-8, IL-1β, IL-6, VEGF, IL-1ra, IL-10, IL-2, IL-4, IL-5, IL-7, IL-9, IL-12 (p70), IL-13, IL-15, IL-17, IFN-γ, IP-10, G-CSF and GM-CSF were measured in the supernatant collected from monocytes / macrophages seeded on the surface of the biomaterial. Increased expression of these cytokines indicates that the target protein is pro-inflammatory, and decreased expression of these cytokines indicates that the target protein is anti-inflammatory. Example 7: Validation of a target protein as a cytokine mutated in a disease.

[0337] This example shows the target protein of the present disclosure as the ability of cytokine-driven immune diseases. In this example, the target protein is mutated to contain non-synonymous amino acid mutations (e.g., functional gain variants) in the genome and subsequent transcripts. In order to test whether the target protein variant causes disease, immune signaling and downstream NF-kB activation are determined in human monocytes (Anvivogen, #thp1-nfkb) containing NF-κB Lucia reporters. Chemical synthesis contains variants and wild-type peptides. Human monocytes are cultured in RPMI1640 culture medium supplemented with HEPES buffer (10mM), sodium pyruvate (1mM), glucose (4.5g / L), fetal bovine serum (10%), penicillin (100U / mL), streptomycin (100μg / mL) and 2-mercaptoethanol (0.05mM). The monocytes were treated with 1 μg / mL LPS (Sigma-Aldrich) for 24 h, treated with variants of the target protein (final concentration 10 μM), treated with wild-type target protein (final concentration 10 μM), or left untreated. The level of NF-κB-induced Lucia in the cell culture supernatant was easily assessed using QUANTI-Luc (luciferase assay reagent) as described above. An increase in reporter activity after treatment with a target protein containing a variant was observed in cells where the target protein is immunostimulatory, which contributes to excessive immune activity in the disease. Example 8. 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.

[0338] This example demonstrates the validation of the target protein (SEQ ID NO: 3585) as an anti-inflammatory cytokine modulator suitable for the treatment of autoimmune diseases such as psoriasis, arthritis and multiple sclerosis.

[0339] 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 cytometry.

[0340] Materials and Methods

[0341] 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).

[0342] PBMC plating: Frozen PBMCs from normal healthy human volunteers (Stem Cell Technology) 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.

[0343] 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 the final concentration, for example, a final concentration of 1 uM peptide would need to be prepared at 20 uM.

[0344] 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).

[0345] 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.

[0346] *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 used for activation. Only TransAct is required in the control wells. The purpose of this procedure is to determine the possible response from the peptide alone.

[0347] 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.

[0348] SEQ ID NO: 3585 significantly blocks the release of 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 7 , Figure 8 ).

[0349] 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 9. Validation of target proteins acting as inhibitors of proinflammatory cytokine activity.

[0350] This example demonstrates the validation of SEQ ID NO: 1238_L8V, SEQ ID NO: 4227 and SEQ ID NO: 6091 as novel peptides capable of inhibiting the proinflammatory cytokine pathways of IL-4 and IL-15, and SEQ ID NO: 3915 as a novel peptide capable of activating the signaling pathway of IFN-α. These peptides may be suitable for treating autoimmune diseases such as psoriasis, arthritis and multiple sclerosis.

[0351] The effects of putative inhibitors on proinflammatory cytokine pathways were assessed using HEK-Blue cells engineered for detection of bioactive cytokines via activation of the JAK / STAT pathway and subsequent expression of the SEAP reporter gene. Cells were treated with their relevant cytokines + / - peptides or control antibodies, and 20 hours after treatment, activation of the relevant cytokine receptors via SEAP expression was determined by mixing the cell supernatant with the Quanti-Blue colorimetric reagent and measuring the optical density at 600 nm.

[0352] Methods and Materials:

[0353] The reporter system HEK-Blue IL-4 / IL-15 / IFN-α was cultured in DMEM (Gibco) with GlutaMAX and 10% HI FBS. The cells were seeded into a black 96-well optically clear flat-bottom PhenoPlate at a density of 25,000 cells / well and 100uL / well and allowed to stand overnight in a 37°C incubator with 5% CO2. After 24 hours, cells were treated with 10uL of their relevant cytokines (Stem Cell Technologies) with or without 10uL of peptide (5uM final concentration) or control antibody (1ug / well) at a previously determined EC75 for activation (1.164ng / mL for HEK-Blue IL-4; 2.26ng / mL for HEK-Blue IL-15; and 0.456ng / mL for HEK-Blue IFN-α). The cells were maintained overnight in a 37°C incubator with 5% CO2.

[0354] 20-22 hours after treatment, 20uL of cell supernatant was collected and transferred to a 96-well white flat bottom opaque plate (Costar), and each sample was mixed with 180uL Quanti-Blue reagent. The colorimetric reaction was allowed to develop in a 37°C incubator with 5% CO2 for a certain duration (3 hours for HEK-Blue IL-4; 2 hours for HEK-Blue IL-15; and 1 hour for IFN-α). The assay plate was then removed from the incubator and the optical density of each well was measured at 600nm on a GloMax plate reader. The relative activation of the cytokine signaling pathway in question was determined by calculating the ratio of the optical density of each sample to the average optical density of cells from the same plate treated with cytokines alone.

[0355] like Fig. 9 As shown in Figure 1, peptides SEQ ID NO: 1238_L8V and SEQ ID NO: 4227 were confirmed as strong inhibitors and moderate inhibitors of the IL-4 signaling pathway, respectively. Peptide SEQ ID NO: 6091 was confirmed as a strong inhibitor of the IL-15 signaling pathway, as shown in Figure 1. Fig.10 As shown; and peptide SEQ ID NO: 3915 was confirmed to be a moderate activator of the IFN-α signaling pathway, as Fig.11 shown. Example 10. Validation that the target protein acts as a cytokine on T cells.

[0356] This example shows that the target protein (SEQ ID NO:4524) acts as a validation of a cytokine on T cells. SEQ ID NO:4524 is a novel secretory peptide that enhances T cell activation. Therefore, SEQ ID NO:4524 is a T cell regulatory cytokine, and will be suitable as a drug target for treating diseases (such as psoriasis, multiple sclerosis and rheumatoid arthritis) caused by pathological T cell responses.

[0357] To determine the effect of target proteins on T cell activity, target proteins, irrelevant proteins, single amino acid mutant target proteins, and controls + / -CD3 / CD28 were applied to primary human T cells isolated from peripheral blood mononuclear cells (PBMCs). Extracellular CD69, IL-2, and TNFα levels were evaluated 24 hours later (72 hours for IFN-γ).

[0358] Materials and Methods

[0359] Isolation of PBMCs from Leukopak: Using EasySep TMDirect Human PBMC Isolation Kit (Cat. No. 19654, Stem Cell Technologies, Vancouver, British Columbia, Canada) was used to isolate peripheral blood mononuclear cells (PBMCs) from leukopak samples according to the manufacturer's instructions. Leukopak samples were prepared in a volume range of 125 mL and added to a T-75 cm2 cell culture flask. EDTA was added to the sample to a final concentration of 6 mM. The separation mixture provided in the kit was added to the sample without vortexing, and the sample was mixed and incubated at room temperature (RT) for 5 minutes. EasySep buffer was added to double the original sample volume, followed by the addition of vortexed RapidSpheres TM Place the flask in a magnet and incubate at room temperature for 5 minutes. Carefully pipette the cell suspension into a new T-75 cm2 flask and place the RapidSpheres TM Add to the enriched cells. Place the flask back in the magnet and separate for a second time at room temperature for 5 minutes. Finally, carefully pipette the cell suspension into a new tube or centrifuge bottle and prepare the separated PBMCs for use.

[0360] Isolation of primary human T cells: After isolating PBMCs, use EasySep TM Human CD8+T Cell Isolation Kit (Cat. No. 17953, Stem Cell Technologies, Vancouver, BC, Canada) was used to isolate CD8+T cells according to the manufacturer's instructions. PBMC samples were prepared at a concentration of 5 x 10^7 cells / mL in a 2 mL volume range and added to a 5 mL polystyrene round-bottom tube. The isolation mixture (50 μL / mL sample) provided in the kit was added to the sample without vortexing, mixed, and incubated at room temperature for 5 minutes. RapidSpheres TM Vortex for 30 seconds to ensure even dispersion, then add to the sample at a concentration of 50 μL / mL sample. Add EasySep buffer to replenish the sample to a total volume of 2.5 mL, and mix the sample by gently pipetting up and down 2-3 times. Place the tube (without cap) in a magnet and incubate at room temperature for 3 minutes. After incubation, pick up the magnet, and in one continuous motion, invert the magnet and tube, and pour the enriched cell suspension into a new tube. The isolated CD8+T cells are now ready for use.

[0361] Peptide treatment: Isolated CD8+ T cells were plated at a density of 1 x 10^5 cells / well in a 96-well format. Each well was initially filled with 100 μl of ImmunoCult-XF T Cell Expansion Medium (Stem Cell Technologies). For T cell activation and peptide treatment, a second solution was prepared. This solution contained a CD3 / CD28 antibody cocktail (Stem Cell Technologies) at a concentration of 25 μl / ml and a synthetic peptide (SEQ ID NO: 4524) from a 1 mM stock solution or a scrambled control to a final concentration of 10 μM. 100 μl of this second solution was added to each well, making a final volume of 200 μl in each well. Subsequent incubations were performed under appropriate conditions.

[0362] ELISA: All reagents, working standards and samples were prepared as indicated in the Abcam ELISA kit manual. For each assay, 50 μL of undiluted sample or standard was added to the appropriate wells, followed by 50 μL of antibody mixture. The plate was sealed and incubated for 60 minutes at room temperature on a plate shaker set at 400 rpm. After incubation, each well was washed three times with 350 μL 1X wash buffer. Washing was performed by aspirating or decanting from the wells, and then 350 μL 1X wash buffer was distributed to each well. After the final wash, the plate was inverted and patted on a clean paper towel to remove excess liquid. Subsequently, 100 μL TMB colorimetric solutions were added to each well. The plate was incubated for 10 minutes on a plate shaker set at 400 rpm in the dark. Finally, 100 μL of stop solution was added to each well. The plate was vibrated on a plate shaker for 1 minute to ensure mixing. End point readout was performed by recording optical density at 450 nm using a GloMax microplate reader (Promega).

[0363] Antibody staining for flow cytometry analysis: After sample collection, cells were centrifuged at 600 x g for 5 minutes. The supernatant was carefully discarded and the cells were washed with 200 μl of Dulbecco's phosphate buffered saline (DPBS). This was followed by another round of centrifugation at 600 x g for 5 minutes and the supernatant was discarded again.

[0364] For antibody staining, 100 μl of prepared antibody (anti-CD69, Invitrogen) staining solution (1:100 dilution in flow cytometry staining solution, e Bioscience (eBioscience)) was added to each well. The cells were then incubated at 4 °C for 30 minutes, and the plate was covered with foil to avoid light. After incubation, another 100 μl DPBS was added to each well, and the sample was centrifuged at 600 x g for 5 minutes. The supernatant was discarded, and the cells were washed once again with 200 μl DPBS, and then centrifuged at 600 x g for 5 minutes. After discarding the supernatant, the cells were fixed by adding 100 μl of fixed buffer (Becton Dickinson and Company) to each well, and then incubated at 4 °C for 20 minutes. Then, 100 μl DPBS was added to each well, and the cells were finally centrifuged at 600 x g for 5 minutes. After discarding the supernatant, the cells were resuspended in 110 μl of flow cytometry staining solution. The prepared samples were stored at 4°C, covered with aluminum foil until further flow cytometry analysis. Flow cytometry was performed using a BD symphony A1 analyzer (BD Dickinson).

[0365] Monitoring of T cell activation markers TNFα, IL-2, CD69 and IFN-γ: SEQ ID NO: 4524 significantly upregulated T cell activation markers CD69, IL-2 and TNFα. Interestingly, a single point mutation targeting cysteine ​​XXX completely abolished its activity. Therefore, SEQ ID NO: 4524 acts as a cytokine to activate T cells (Figure 12, Fig.13 ). ORF ID Relative activity SEQ ID NO:4524 +++ SEQ ID NO:3935 + SEQ ID NO:6519 ++ SEQ ID NO:5862 ++++ SEQ ID NO:2758 ++

[0366] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0367] 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 pharmaceutical agent comprising a target protein or a variant thereof identified in a sequence list 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. 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 or variant thereof identified in a sequence listing in a cell, 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 expression or activity of the target protein in the cell.

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

44. The method of any one of claims 41-43, wherein the cell is in a subject, and wherein the cell is: a) cancer cells, cells in a tumor microenvironment, or a combination thereof; b) epithelial cells, endothelial cells, stem cells, non-immune cells or a combination thereof; c) immune cells (e.g., macrophages, lymphocytes such as helper T cells), or A combination of the preceding.

45. The method of claim 44, wherein the subject has a disease or condition selected from the group consisting of aging, senescence, fibrosis, autoimmunity, cancer, infection, immune disease, 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 developing a disease or condition, the method comprising quantifying the expression or activity of a target protein identified in a sequence listing 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 developing cancer, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmunity, cancer, infection, immune disease, or a combination thereof.

49. A method of 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 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 in the sample prepared in step b), The disease or disorder is selected from inflammation, aging, senescence, fibrosis, autoimmunity, cancer, infection, immune disease 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 of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent 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 inflammation, aging, senescence, fibrosis, autoimmunity, cancer, infection, immune disease, or a combination thereof.

52. A method for identifying an agent that modulates the expression or activity of a target protein or variant thereof identified in a sequence listing, the method comprising: a) contacting a protein identified in the sequence listing or a variant thereof 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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