Compositions and methods for modulating immune response

Abstract

In various embodiments, the disclosure provides compositions, such as polypeptides, polynucleotides, gene editing systems, small molecules, vectors, or host cells, comprising and / or modulating the expression or activity of an immunomodulatory-related protein. In various embodiments, the disclosure also provides methods of treating aging, senescence, fibrosis, autoimmunity, cancer, infection, and / or immune 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

By citing and incorporating materials

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 345,765, filed May 25, 2022. The entire teachings of the foregoing application are incorporated herein by reference. By referencing materials incorporated into XML

[0002] This application incorporates the sequence list contained in the following accompanying eXtensible Markup Language (XML) file: a) Filename: 57081055002.xml; created on May 23, 2023, with a size of 40,525,913 bytes. Background Technology

[0003] The primary function of the immune system is to mediate protective immunity against pathogens such as viruses, bacteria, fungi, parasites, and tumors. During cancer and chronic infections, multiple immune-destructive mechanisms are often enhanced. Inadequate immune responses can lead to immunodeficiency, susceptibility to infection, and cancer (e.g., tumor) progression. However, without strict regulation, the immune system can also promote acute and chronic inflammation, leading to immunopathologies or autoimmunity. Therefore, there is an urgent need to identify additional novel therapeutic targets for modulating immune cells. Summary of the Invention

[0004] This disclosure is based in part on the identification of non-classical protein targets (e.g., proteins encoded by non-classical open reading frames (ORFs)) for regulating immune responses.

[0005] In one aspect, this disclosure relates to a pharmaceutical agent comprising a target protein identified herein (e.g., a target protein listed in the sequence listing) or a variant thereof and / or regulating (e.g., increasing or decreasing) the expression and / or activity of the target protein or a variant thereof. In some embodiments, the pharmaceutical agent comprises a target protein identified herein (e.g., a target protein listed in the sequence listing) or a variant thereof. In some embodiments, the pharmaceutical agent regulates (e.g., increasing or decreasing) the expression and / or activity of the target protein identified herein (e.g., a target protein listed in the sequence listing or a variant thereof). In some embodiments, the pharmaceutical agent comprises, substantially comprises, or comprises of a polypeptide, polynucleotide, gene editing system, small molecule, or cell (e.g., cell therapy). The pharmaceutical agent may be an inhibitor or activator of a target protein identified herein. In some embodiments, the pharmaceutical agent regulates the expression of a target protein identified herein. In some embodiments, the pharmaceutical agent regulates the activity of a target protein identified herein.

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

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

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

[0009] On the other hand, this disclosure provides a method for detecting a disease or condition in a subject or determining the likelihood of a subject having said disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, wherein the level of expression or activity of the target protein in the sample indicates the likelihood of the subject having said disease or condition, wherein said disease or condition is selected from aging, senescence, fibrosis, autoimmune disease, cancer (e.g., tumor), infection, immune disease (e.g., inflammation and / or autoimmune disease) or a combination thereof.

[0010] On the other hand, this disclosure provides a method for preparing a sample that can be used to determine the likelihood of a subject having a disease or condition, the method comprising: a) Samples obtained or already obtained 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 likelihood of developing cancer; and c) Quantify the expression or activity of the target protein in the sample prepared in step b). The disease or condition mentioned therein is selected from aging, senescence, fibrosis, autoimmune disease, cancer (e.g., tumor), infection, immune disease (e.g., inflammation and / or autoimmune disease) or a combination thereof.

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

[0012] On the other hand, this disclosure provides a method for treating a disease or condition in a subject in need (e.g., a human subject with cancer), the method comprising administering to the subject an effective amount of a pharmaceutical agent or a pharmaceutical composition comprising the pharmaceutical agent, the pharmaceutical agent comprising a target protein identified herein and / or regulating the expression or activity of the target protein.

[0013] In another aspect, this 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 based on the expression or activity level of the target protein in the sample, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmune diseases, cancer (e.g., tumors), infections, immune diseases (e.g., inflammation and / or autoimmune diseases) or combinations thereof.

[0014] In another aspect, this disclosure provides a method for regulating the expression or activity of a target protein or a variant thereof identified in a sequence listing in cells (e.g., cancer cells, such as cancer cells of a subject), the method comprising contacting the cells (e.g., in vitro, ex vivo, or in vivo) with a pharmaceutical agent or a pharmaceutical composition comprising the pharmaceutical agent containing the target protein identified herein and / or regulating the expression or activity of the target protein.

[0015] On the other hand, this disclosure provides a method for identifying an agent that regulates the expression or activity of the target protein identified herein, the method comprising: a) bringing the target protein into contact with the drug; and b) Determine whether the drug modulates the expression or activity of the target protein. The 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 regulates the expression or activity of the target protein. Attached Figure Description

[0016] The foregoing will become clear from the following more specific description of exemplary embodiments, as illustrated in the accompanying drawings, in which the same reference numerals refer to the same parts in different views. The drawings are not necessarily to scale, but rather focus on illustrating the embodiments.

[0017] Figure 1 The dose-dependent reduction of hTNFa on CD3 / CD28 (TransAct)-activated PBMCs was shown compared to the control (TA_H2O). *** = p < 0.0002 and **** = p < 0.0001, compared to the corresponding controls, by two-way ANOVA.

[0018] Figure 2 This study showed a lack of IL-1b inhibition on CD3 / CD28 (TransAct)-activated PBMCs compared to the control (TA_H2O). The positive control showed significant inhibition. **** = p < 0.0001, compared to the corresponding control, by two-way ANOVA.

[0019] Figure 3 The inhibition of the interferon regulatory factor (IRF) pathway by treatment with the protein SEQ ID NO:37413 in THP-1 dual reporter cells is illustrated. THP-1 dual reporter cells were incubated for 24 h with progressively increasing concentrations of the peptide (0, 2.5, 5, 7.5, and 10 μM) in the presence of lipopolysaccharide (LPS). Relative IRF activity was assessed by measuring reporter gene expression. Data are presented as mean ± SD of 10 technical replicates. An asterisk indicates statistical significance compared to cells treated with LPS only (**p < 0.01, ****p < 0.0001).

[0020] Figure 4 This study demonstrates the selective inhibition of IRF responses in THP-1 dual-reporter cells by co-treatment with a specific TLR agonist using SEQ ID NO:37413 protein. THP-1 dual-reporter cells were treated for 24 hours with 10 different TLR agonists (TLR1 / 2, TLR2, TLR3, TLR4, TLR5, TLR6 / 2, TLR7, TLR8, and TLR9) in the presence of SEQ ID NO:37413 protein (10 μM) or the out-of-order peptide. Relative IRF activity was assessed by measuring reporter gene expression. Data are presented as mean ± SD of 10 technical replicates from each treatment group in representative experiments. An asterisk indicates statistical significance compared to the corresponding TLR agonist in cells treated with the out-of-order peptide (*****p<0.0001). When co-treated with TLR1 / 2, TLR4, and TLR6 / 2 agonists, the protein inhibits the IRF response, while no significant effect was observed with TLR2, TLR3, TLR5, TLR7, TLR8, or TLR9 agonists.

[0021] Figure 5A The UMAP visualization of cluster analysis is shown, revealing 10 distinct PBMC cell subtypes; while Figure 5B A bar graph is shown, which displays the counts of genes upregulated and downregulated when treated with the protein SEQ ID NO:37413, compared to the disordered protein control.

[0022] Figure 6 The screening design for validating the function of the novel protein in primary NK cells activating Raji cell killing is shown.

[0023] Figure 7 The study shows the enrichment of CRISPR-mediated KO of known controls ICAM1 and FAS in post-NK Raji cells, supporting their role in enhancing NK cell cytotoxicity. Similar enrichment was observed for three exemplary novel proteins: SEQ ID NO:33482, SEQ ID NO:33482, SEQ ID NO:37776, and SEQ ID NO:35050. As expected, no enrichment was observed in the intergenetic (neutral) control guide RNA. Box plots summarize the effects of numerous guide RNAs targeting each target (n=4); the effects of four biological replicates from pre-NK and post-NK samples are shown.

[0024] Figure 8 The screening design for verifying the function of novel proteins in inhibiting Raji cell killing by primary NK cells is shown.

[0025] Figure 9 The study demonstrates the depletion of known controls B2M and TAP1's CRISPR-mediated KO in post-NK Raji cells, supporting their role in protecting cells from NK cell cytotoxicity. Similar depletion was observed in three exemplary novel proteins, SEQ ID NO:2013, SEQ ID NO:35586, and SEQ ID NO:36545. As expected, no depletion was observed in the intergenetic (neutral) control guide RNA. Box plots summarize the effects of numerous guide RNAs targeting each target (n=4); the effects of four biological replicates from pre-NK and post-NK samples are shown. Detailed Implementation

[0026] The following is a description of an exemplary embodiment. target protein

[0027] In one respect, this disclosure provides target proteins identified herein (e.g., target proteins comprising amino acid sequences selected from SEQ ID NO:33885, SEQ ID NO:37413, SEQ ID NO:33482, SEQ ID NO:36545, SEQ ID NO:2013, SEQ ID NO:35586, SEQ ID NO:35050). As used herein, the expressions “target proteins identified herein” and “target proteins of this disclosure” include the polypeptides disclosed in the sequence listing. Target proteins can be generated by recombinant synthesis (e.g., via DNA or mRNA) or by synthesis.

[0028] In some embodiments, the target protein is an extracellular protein (e.g., a secreted protein, such as a ligand). In some embodiments, the target protein is a transmembrane protein (e.g., a receptor). In certain embodiments, the target protein is membrane-bound and extracellular, but not transmembrane. In more specific embodiments, the target protein is embedded in a membrane, but not transmembrane.

[0029] In various embodiments, the target protein is a protein comprising the amino acid sequence shown in the sequence listing. In some embodiments, the target protein consists of the amino acid sequence shown in the sequence listing. In some embodiments, the target protein comprises an amino acid sequence having one amino acid substitution relative to the amino acid sequence shown in the sequence listing, wherein the substitution is replacing an N-terminal residue in the amino acid sequence shown in the sequence listing with a methionine (Met) residue. In some embodiments, the target protein comprises the amino acid sequence shown in the sequence listing and further comprises a methionine (Met) residue at its N-terminus. In some embodiments, the target protein consists of the amino acid sequence shown in the sequence listing and a methionine (Met) residue at its N-terminus.

[0030] Certain target proteins in the sequence listing have been identified as differentially expressed (e.g., upregulated or downregulated) in a range of diseases and / or conditions, selected from: aging, senescence, fibrosis, autoimmune diseases, cancer (e.g., tumors), infection, immune diseases (e.g., inflammation and / or autoimmune diseases), or combinations thereof, such that regulation of the level and / or activity of the target proteins plays a role in treating, alleviating, and / or preventing the development of diseases or conditions.

[0031] 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. Thus, a differentially expressed protein, or “DEP,” may have a higher expression level in a reference state (e.g., normal state) compared to a disease state in which DEP is expressed at a lower level or not expressed at all. Conversely, DEP may have a higher expression level in a disease state compared to a reference state (e.g., normal state) in which DEP is expressed at a lower level or not expressed at all. Furthermore, expression can be considered differential if DEP is identifiable altered between the two states under comparison (e.g., mutation). Identifiable alterations can include amino acid substitutions, insertions and / or deletions (including N-terminal and C-terminal truncation), and modifications (e.g., post-translational modifications).

[0032] As used herein, the term "reference" refers to a standard used for one or more comparative purposes. Those skilled in the art can select an appropriate reference for one or more specific comparative purposes. Thus, for example, a reference for a disease state may be a normal state of health; a reference for a mutated protein may be an unmutated protein; a reference for disease treatment may be no treatment or standard care. In some embodiments, particularly those 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 the 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 single cell, sample, or subject (e.g., cells or samples from a healthy subject or a subject without a specific disease; healthy subject, subject without 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., cells or samples from a healthy subject or a subject without a specific disease; healthy subject, subject without 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 statistics (e.g., mean or median).

[0033] In some embodiments, the protein is used to represent immune and / or disease states (e.g., exhausted T cells, CD4+). + T cells (e.g., Th1, Th2, Th17), CD8 + Markers of T cells (or combinations thereof).

[0034] In some embodiments, the target protein is expressed at a higher level in activated immune cells. In some embodiments, the expression level of the target protein in activated immune cells is at least about 0.5 times higher than that in a reference (e.g., unactivated immune cells 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 higher (e.g., 50 times higher, 100 times higher).

[0035] In some embodiments, the target protein is expressed at a lower level in activated immune cells. In some embodiments, the expression level of the target protein in activated immune cells is at least about 0.5 times lower than the target protein expression level in a reference (e.g., unactivated immune cells 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 lower (e.g., 50 times lower, 100 times lower).

[0036] In some embodiments, the expression level of the target protein in the disease (e.g., as determined from a sample of cells or tissues from a subject without the disease) is at least about 0.5 times higher than the target protein expression level in a reference (e.g., a sample from cells or tissues from a subject with the disease), 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 higher (e.g., 50 times higher, 100 times higher).

[0037] In some embodiments, the expression level of the target protein in the disease (e.g., as determined from samples containing or obtained from cells or tissues of a subject without the disease) is at least about 0.5 times lower than the target protein expression level in a reference (e.g., a sample from cells or tissues of a subject without the disease), 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 lower (e.g., 50 times lower, 100 times lower). In some embodiments, the target protein is not expressed in the disease or is expressed at an undetectable level (e.g., as determined from samples containing cells or tissues of a subject with the disease).

[0038] In some embodiments, the target protein transcript level in a disease (e.g., as determined from a sample of cells or tissues from a subject without the disease) is at least about 0.5 times higher than the target protein transcript level in a reference (e.g., a sample from cells or tissues from a subject with the disease), 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 higher (e.g., 50 times higher, 100 times higher). In certain embodiments, the increase in the target protein transcript level contributes to (e.g., causes) the disease or condition described herein.

[0039] In some embodiments, the target protein transcript level in a disease (e.g., as determined from a sample containing or obtained from a subject with the disease) is at least about 0.5 times lower than the target protein transcript level in a reference (e.g., a sample from cells or tissues of a subject without the disease), 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 lower (e.g., 50 times lower, 100 times lower). In some embodiments, the target protein transcript 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 with the disease). In certain embodiments, the reduction in the target protein transcript level contributes to (e.g., causes) the disease or condition described herein.

[0040] In a particular embodiment, the gene encoding the target protein in the disease described herein contains at least one mutation (e.g., fusion, deletion, insertion, point mutation, and / or amplification of amino acid duplication).

[0041] 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, and cells isolated from a subject.

[0042] 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 some 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).

[0043] In some embodiments, the target protein is translated from a non-exon element in unprocessed precursor mRNA (premRNA). In some embodiments, the non-exon element is an intron in the premRNA. In some embodiments, the non-exon element is the 5' untranslated region (5'UTR) in the premRNA. In some embodiments, the non-exon element is the 3' untranslated region (3'UTR) in the premRNA.

[0044] In some embodiments, the target protein is 2,000 or fewer amino acids long, such as 1,000 or fewer, 750 or fewer, 500 or fewer, 250 or fewer, 150 or fewer, or 100 or fewer. In some embodiments, the target protein is 7 or more amino acids long, such as 8, 9, 10, 15, 18, 25, 50, 75, or 100 or more. In some embodiments, the target protein is from about 50 to about 200 amino acids long, for example from about 100 to about 150 amino acids long. In a particular embodiment, the target protein is 7 or more amino acids long. In a more specific embodiment, the target protein is about 18 amino acids long.

[0045] In some embodiments, the target protein disclosed herein is a regulator 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 regulator of one or more GPCRs, such as a ligand of one or more GPCRs. In some embodiments, the target protein is an indirect regulator of one or more GPCRs.

[0046] The expression and / or activity of various GPCRs have been associated with different diseases / disorders, symptoms, and indications (including those shown in Table A) (see, for example, 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). DrugDiscov. [Nature Reviews: Drug Discovery] 2020 June 19(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 regulators of GPCRs) may be used to treat and / or diagnose one or more diseases / disorders, conditions and / or indications, such as cancer or precancerous conditions, or any diseases / disorders, conditions and indications known to be associated with GPCR expression and / or activity as listed in Table A.

[0047] Table A. Disease-related GPCRs Drugs that regulate target proteins

[0048] This document provides agents for regulating the expression of target proteins disclosed herein (such as those listed in the sequence listing), their variants, or fragments thereof (e.g., biologically active fragments of the target protein). The expression of the target protein, its variants, or fragments can be regulated directly or indirectly through a wide range of processes, resulting in an increase or decrease in target protein levels. Non-limiting examples include alterations to: the copy number of the gene encoding the target protein, transcription initiation, elongation, or termination, RNA processing, RNA stability (e.g., mRNA stability), RNA degradation, translation initiation, post-translational modifications of the protein, protein stability, protein degradation (e.g., cleavage, such as protease cleavage), or combinations thereof.

[0049] 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 the target protein. In some embodiments, the agent decreases (e.g., inhibits, reduces, or neutralizes) the activity of the target protein. In some embodiments, the agent increases (e.g., activates) the activity of the target protein. In some embodiments, the agent decreases (e.g., inhibits or downregulates) the expression of the target protein. In other embodiments, the agent increases (e.g., activates or upregulates) the expression of the target protein.

[0050] As used herein, the terms "increasing" or "increase" refer to a regulation that results in a higher level of expression, activity, function, or a combination thereof, or a measure of a target protein (e.g., cancer cell death or DNA methylation at a target site), relative to a reference (e.g., the level before drug intervention or in the absence of drug intervention). In some embodiments, the drug increases the expression or activity or measure of the target protein by at least about 5% relative to a reference, for example, 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.

[0051] As used herein, the terms "decreasing" or "decrease" refer to a regulation that results in a lower level of expression, activity, function, or a combination thereof, or a measure of a target protein (e.g., cancer cell death or DNA methylation at a target site), relative to a reference (e.g., the level before drug intervention or in the absence of drug intervention). 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, for example, 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.

[0052] Non-limiting examples of measurement include energy production or conversion in the liver (e.g., regulation of ATP synthesis, β-oxidation, oxidation of metabolites derived from glycolysis, oxidation of metabolites derived from amino acids), mitochondrial transcription, mitochondrial ribosome assembly, mitochondrial translation, mitochondrial thermogenesis, 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, and signaling processes (e.g., calcium signaling).

[0053] In some embodiments, such as after a treatment regimen has begun, after the agent has been exposed to (e.g., cells) or administered (e.g., to a subject) for at least about 1 day, such as 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, the level of expression, activity, function, or a combination thereof or a measure thereof of the target protein is measured.

[0054] In some embodiments, the agent comprises, or is substantially composed of, peptides, polynucleotides, gene editing systems, small molecules, or cells (e.g., cell therapy).

[0055] In some embodiments, the target protein activates immune cells, and the agent modulates (e.g., increases or decreases) the level of the target protein's expression, activity, function, or a combination thereof. In some embodiments, the target protein inhibits immune cells (e.g., inhibits immune cell activation, induces immune cell death (e.g., apoptosis), or a combination thereof), and the agent modulates (e.g., increases or decreases) the level of the target protein's expression, activity, function, or a combination thereof.

[0056] In some embodiments, the agent modulates (e.g., increases or decreases) the levels of expression, activity, function, or combinations thereof of target proteins in the following cells: cancer cells (e.g., metastatic cancer cells), cells in the tumor microenvironment (e.g., stromal cells), target cells of the 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 combinations thereof. In some embodiments, the agent modulates the levels of expression, activity, function, or combinations thereof of target proteins in tumors, tumor microenvironments, metastatic sites, lymph nodes, spleen, secondary lymphoid organs, tertiary lymphoid organs, barrier tissues, skin, intestines, airways, wounds, other immune tissues, non-immune tissues, or combinations thereof.

[0057] In some embodiments, the agent modulates (e.g., increases or decreases) inflammation, reduces the level of autoantibodies, increases organ function, reduces the rate or number of relapses or outbreaks, reduces viral load, controls infection, or a combination of the foregoing.

[0058] In some embodiments, the agent induces downregulation of the target protein (e.g., increased target protein degradation); prevents target protein polymerization (e.g., dimerization); chelates the target protein (e.g., secreted target protein); modulates (e.g., agonizes, antagonizes, or disrupts) the known function of the target protein; reduces the binding between the target protein and its binding partner (e.g., via steric hindrance); modulates (e.g., increases or decreases) downstream cell signaling; induces phagocytosis of cells expressing the target protein; or a combination of the foregoing. In some embodiments, the agent lacks agonistic activity against the target protein. In some 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 a particular embodiment, the agent binds to at least one residue of the target protein, said at least one residue participating 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, said one or more binding sites and / or domains participating in binding of the target protein to the binding partner.

[0059] Non-limiting examples of binding couples include cytokine receptors and immune signaling receptors.

[0060] In some embodiments, the agent induces downregulation of the binding partner of the target protein; chelates the binding partner of the target protein (e.g., a secretory binding partner); prevents the polymerization (e.g., dimerization) of the binding partner of the target protein; chelates the binding partner of the target protein (e.g., a secretory binding partner); modulates (e.g., activates, antagonizes, or disrupts) 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); modulates (e.g., increases or decreases) downstream cell signaling; or a combination of the foregoing. In some embodiments, the agent lacks agonistic activity against the binding partner of the target protein. In some embodiments, the agent has agonistic activity against the binding partner of the target protein. In some embodiments, the agent lacks antagonistic activity against the binding partner of the target protein. In some embodiments, the agent has antagonistic activity against the binding partner of the target protein. In a particular embodiment, the agent further binds to at least one residue of the binding partner of the target protein, said at least one residue being involved in the binding between the target protein and the binding partner. In more specific embodiments, the agent further binds to one or more binding sites and / or domains of the binding partner of the target protein, said one or more binding sites and / or domains participating in the binding between the target protein and the binding partner.

[0061] In some embodiments, the agent modulates (e.g., activates or inhibits) immune signaling, cytokine signaling, inflammatory signaling, or a combination thereof.

[0062] In some embodiments, the agent enhances signals involved in T cell activation and / or survival. In some embodiments, the agent activates stimulating checkpoint molecules. Non-limiting examples of stimulating checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, GITR, and inducible T cell costimulators (ICOS). In a particular embodiment, the agent is an agonist targeting CD28.

[0063] In some embodiments, the agent reduces signals involved in T cell impotence and / or exhaustion. In some embodiments, the agent inhibits inhibitory checkpoint molecules. Non-limiting examples of inhibitory checkpoint molecules include PD-1, PD-L1, PD-L2, TIM-3, LAG-3, CTLA-4, A2AR, CD276, B7-H4, BTLA, IDO, KIR, NOX2, VISTA, SIGLEC 7, and SIGLEC9. In specific embodiments, the agent is an inhibitor of PD-1 (e.g., a blocking antibody).

[0064] In some embodiments, the agent further modulates (e.g., increases or decreases) the expression, activity, function, or combinations thereof of checkpoint proteins other than the target protein.

[0065] In some embodiments, the agent modulates (e.g., increases or decreases) the expression, activity, function, or combinations thereof of a variant of the target protein disclosed herein. In some embodiments, the variant comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the target protein disclosed herein. For example, sequence identity with the variant can be at least about: 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, sequence identity is about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, 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.

[0066] As used herein, the term "sequence identity" refers to the degree to which two nucleotide sequences or two amino acid sequences have identical residues at the same positions when sequences are aligned to achieve the maximum level of identity (expressed as a percentage). For sequence alignment and comparison, typically a sequence is designated as the reference sequence to which the test sequence is compared. Sequence identity between the reference and test sequences is expressed as a percentage of positions along the entire length of the reference sequence where the reference and test sequences have the same nucleotides or amino acids when aligned to achieve the maximum level of identity. As an example, two sequences are considered to have 70% sequence identity when the test sequence has the same nucleotide or amino acid residues at 70% of the same positions along the entire length of the reference sequence when aligned to achieve the maximum level of identity.

[0067] Sequence alignments used to achieve the maximum level of identity can be readily performed by those skilled in the art using appropriate alignment methods or algorithms. In some cases, alignment may include introducing gaps to provide the maximum level of identity. 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), the 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 Ausubel et al., Current Protocols in Molecular Biology).

[0068] When using sequence comparison algorithms, the test and reference sequences are input into the computer, with subsequent coordinates specified if necessary, and the sequence algorithm program parameters are also specified. The sequence comparison algorithm then calculates the percentage of sequence identity between one or more test sequences and the reference sequence based on the specified program parameters. A commonly used tool for determining the percentage of sequence identity is the Basic Local Alignment Search Tool for Proteins (BLASTP), which is available from the National Center for Biotechnology Information at the National Library of Medicine, National Institutes of Health (Altschul et al., 1990).

[0069] In some embodiments, the amino acid sequence of a variant of the target polypeptide disclosed herein contains at least one amino acid substitution relative to the amino acid sequence of the target protein. In some embodiments, the number of amino acid substitutions in the variant relative to the amino acid sequence of the target protein disclosed herein is at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, the number of amino acid substitutions is at least about 5, 6, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60. In some embodiments, the number of amino acid substitutions is at most about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 6, or 5. In some embodiments, the number of amino acid substitutions is about: 1-60, 1-55, 2-55, 2-50, 3-50, 3-45, 4-45, 4-40, 5-40, 5-35, 6-35, 6-30, 7-30, 7-25, 8-25, 8-20, 9-20, 9-15, 10-15, 5-60, 10-60, 10-55, 15-55, 15-50, 20-50, 20-45, 25-45, 25-40, or 30-40. In some embodiments, the number of amino acid substitutions is about: 10-35, 10-33, 11-33, 11-31, 12-31, 12-29, 13-29, 13-27, 14-27, or 14-25.

[0070] One or more amino acid substitutions in the variant can be made with classical or non-classical amino acids. Non-classical amino acids include, but are not limited to, D amino acids, such as the D form of classical L-amino acids.

[0071] In some embodiments, amino acid substitution is a conservative substitution. The terms "one or more conservative amino acid substitutions" or "one or more conservative substitutions" refer to amino acid substitutions that have a value of 0 or greater in BLOSUM62.

[0072] In some embodiments, the amino acid substitution is a highly conserved substitution. The terms "one or more highly conserved amino acid substitutions" or "one or more highly conserved substitutions" refer to amino acid substitutions having a value of at least 1 (e.g., at least 2) in BLOSUM62.

[0073] In some embodiments, variants of the target protein disclosed herein contain about 5-60 amino acid substitutions relative to the amino acid sequence of the target protein disclosed herein. In some embodiments, the amino acid substitutions include at least one conserved substitution. In some embodiments, the amino acid substitutions include at least one highly conserved substitution. A. Polypeptide drugs

[0074] The terms “polypeptide,” “peptide,” or “protein” refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). Proteins, peptides, or polypeptides may 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-aminohexanoic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitrulline, homoserine, leucine, valine, ornithine). The amino, carboxyl, and / or other functional groups on a peptide may 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 disclosed, for example, in Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. The functional groups of proteins, peptides, or polypeptides may also be derivatized (e.g., alkylated) or labeled (e.g., with detectable labels such as fluorophores or haptens) using methods known in the art. If desired, proteins, peptides, or polypeptides may contain one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclization), N-methyl-α-amino group substitution). Additionally, proteins, peptides, or polypeptides may be analogs of known and / or naturally occurring peptides, such as peptide analogs with one or more conserved amino acid residue substitutions.

[0075] In some embodiments, the pharmaceutical preparation 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 the expression and / or activity of the target protein disclosed herein (e.g., a direct or indirect inhibitor). In some embodiments, the polypeptide is an activator of the expression and / or activity of the target protein disclosed herein (e.g., a direct or indirect activator). In some embodiments, the polypeptide reduces the expression or activity of the target protein disclosed herein. In other embodiments, the polypeptide increases the expression or activity of the target protein disclosed herein. In some embodiments, the polypeptide is the target protein disclosed herein or a portion thereof (e.g., its biologically active portion, such as a biologically active fragment of the target protein).

[0076] 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 capable of modulating the expression or activity of the target protein.

[0077] In some embodiments, the polypeptide is an antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule 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 an immunoglobulin molecule. As used herein, the term "antibody" refers to a full-length antibody or a polymer thereof (e.g., IgM) comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (V... H ) and heavy chain constant region (containing structural domains CH1, hinge, CH2, and CH3). Each light chain contains light chain variable region (V L ) and the light chain constant region (CL). V H and V L The region can be further subdivided into highly variable areas called Complementary Determinant Regions (CDRs), which are scattered within the Frame Regions (FRs). H and V L Each antibody contains three CDRs and four FR segments arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies can belong to any species, such as rodent (e.g., mouse, rat, guinea pig) antibodies, human antibodies, or can be humanized or chimeric antibodies.

[0078] In some embodiments, the antibody comprises an IgA (e.g., IgA1 or IgA2) heavy chain constant region, an IgD heavy chain constant region, an IgE heavy chain constant region, an IgG (e.g., IgG1, IgG2 (e.g., IgG2a, IgG2b or IgG2c), IgG3 or IgG4) heavy chain constant region, or an IgM heavy chain constant region. In some embodiments, the antibody comprises an IgG heavy chain constant region. In some embodiments, the antibody comprises a κ light chain constant region. In some embodiments, the antibody comprises a λ light chain constant region.

[0079] 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 primate-derived (e.g., humanized). In some embodiments, the antibody is multispecific, such as bispecific, trispecific, or tetraspecific. In some embodiments, the antibody is a heteroconjugated antibody.

[0080] 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 full-length parental antibody. Non-limiting examples of antigen-binding fragments include V H District, V L Region, Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment and a V H A structural domain or a V L Domain-specific antibodies (dAbs) composed of structural domains, etc. V 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 pair intramolecularly or intermolecularly; in the latter case, V H and V L The domain is expressed by a separate chain to form a monovalent antigen-binding site, such as a single-chain Fv (scFv) or a biantibody. In some embodiments, the polypeptide disclosed herein is an antigen-binding fragment selected from: Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv (sdFv, such as a biantibody, triantibody, or tetraantibody), scFv, SMIP, or rlgG. In some embodiments, the polypeptide is scFv. The antigen-binding fragment can be generated by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art.

[0081] Peptide agents (e.g., monoclonal antibodies) can be monovalent, bivalent, or multivalent. Monoclonal antibodies can be monospecific or multispecific (e.g., bispecific). A monospecific antibody binds to one antigenic epitope. Multispecific antibodies (such as bispecific or trispecific antibodies) are included in the term monoclonal antibody.

[0082] "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.

[0083] "Isolated antibody" refers to an antibody or its antigen-binding fragment that is substantially free of other antibodies with different antigen specificities (e.g., isolated anti-target protein antibody is substantially free of antibodies that specifically bind to antigens other than the target protein). In the case of bispecific antibodies, the bispecific antibody specifically binds to two target antigens and is substantially free of antibodies that specifically bind to antigens other than the two target antigens. In some embodiments, the polypeptide agent (e.g., monoclonal antibody) is at least 80% pure, for example, about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0084] In some embodiments, the polypeptide is an antagonist antibody that binds to a target protein (e.g., a target protein whose expression or activity is increased in a cancer state relative to a reference state). In some embodiments, the antibody described herein is an antagonist antibody that binds to a protein capable of modulating 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 (e.g., a target protein or a protein capable of modulating the expression or activity of a target protein) upon binding. 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 signal transduction molecule, and the antagonist antibody reduces the activity of the enzyme or attenuates signal transduction pathways mediated by the signal transduction molecule. In some embodiments, the antagonist antibody reduces 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%, 98%, or 99%.

[0085] 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 capable of modulating the expression or activity of the target protein. As used herein, the term "agonist antibody" refers to an antibody that enhances the function of an antigen (e.g., a target protein or a protein capable of modulating the expression or activity of the target protein) upon binding. 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 signal transduction molecule, and the agonist antibody increases the activity of the enzyme or activates a signal transduction pathway mediated by a signal transduction 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%.

[0086] In some embodiments, the agonist antibody does not perform at least one of the following functional properties: reducing (e.g., inhibiting) the activity of the antigen; inducing antibody-dependent cell killing of antigen-expressing cells (e.g., via natural killer (NK) cells, monocytes, macrophages, neutrophils, dendritic cells, or eosinophils); inducing phagocytosis of antigen-expressing cells (e.g., via macrophages); inducing opsonization of antigen-expressing cells; and inducing downregulation of antigens on the cell surface (e.g., via hypercrosslinking or clustering antigens to induce internalization and degradation).

[0087] Suitable techniques, assays, and reagents for preparing and using therapeutic antibodies against antigens are known in the art. Methods for preparing 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, for example, Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by Zhiqiang An, 1st edition 2009); Antibodies: A Laboratory Manual (edited by Edward A. Greenfield, 2nd 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).

[0088] In some embodiments, the polypeptide is an antibody mimic that binds to the target protein disclosed herein. The term "antibody mimic" refers to a polypeptide that mimics the ability of an antibody to bind to an antigen but is structurally different from that of a natural antibody. Non-limiting examples of antibody mimics include adnectin, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, fynomer, Kunitz domain peptide, monobody, nanobody, nanoCLAMP, and versabody.

[0089] 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 at least 70% of the same amino acid sequence as at least a portion (e.g., a biologically active portion or fragment) of the target protein. For example, the percentage of identity with the full-length target protein or its biologically active portion or fragment can be at least about: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. 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.

[0090] 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 conserved substitution. In some embodiments, the amino acid substitution is a highly conserved substitution.

[0091] In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises at least 70% of the same amino acid sequence as at least a portion of a protein capable of regulating the expression or activity of a target protein. For example, the identity percentage can be at least about: 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the polypeptide comprises the amino acid sequence of a protein capable of regulating the expression or activity of a target protein.

[0092] 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 conserved substitution. In some embodiments, the amino acid substitution is a highly conserved substitution.

[0093] In some embodiments, the polypeptide is a cell-penetrating peptide. In some embodiments, the polypeptide is linked to a cell-penetrating peptide. Suitable cell-penetrating peptide sequences may be protein-derived, engineered, or chimeric (modified). See, for example, Regberg et al., Applications of cell-penetrating peptides for tumor targeting and future cancer therapies, Pharmaceuticals 5(9):991-1007 (2012). Non-limiting examples of cell-penetrating peptides include TAT (48-60), membrane-penetrating peptides, pVEC, MPG8, Transportan, Transportan 10, PepFect3, PepFect6, PepFect14, polyarginine, stearoyl-polyarginine, Pep-1, Pep-3, CATY, YTA2, YTA4, SynB1, SynB3, Maurocalcine, and PTD4.

[0094] In some embodiments, the polypeptide is a circulating factor (e.g., a cytokine).

[0095] In some embodiments, the biological properties (e.g., biological activity or half-life) of the polypeptide (e.g., isolated polypeptide) and the target protein are similar. Non-limiting examples of biological activity include, in particular, enzyme activity or properties (e.g., selectivity, homeostasis, or kinetics), binding activity (e.g., nucleic acid (DNA, RNA) binding to proteins) or properties (e.g., specificity, affinity, or kinetics), cell signaling activity, immune activity, and structural activity (e.g., cell adhesion). Non-limiting examples of enzyme activity include transferase activity (e.g., transferring a functional group from one molecule to another), oxidoreductase activity (e.g., catalyzing redox reactions), hydrolase activity (e.g., cleaving chemical bonds via hydrolysis), lyase activity (e.g., generating double bonds), ligase activity (e.g., linking two molecules via covalent bonds), and isomerase activity (e.g., catalyzing structural changes within a molecule from one isomer to another).

[0096] 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 generating therapeutic polypeptides are known in the art. See, for example, Therapeutic Proteins: Methods and Protocols (edited by Mark C. Smales and David C. James, 2005); Pharmaceutical Biotechnology: Fundamentals and Applications (edited by Daan J. A. Commelin, Robert D. Sindelar, and Bernd Meibohm, 2013). The polypeptide can be recombinantly expressed using mammalian cells, insect cells, yeast, or bacteria, under the control of a suitable promoter.

[0097] In some embodiments, the polypeptides described herein (e.g., target proteins or portions thereof, polypeptide agents that regulate target proteins) are modified, for example by cleavage (e.g., protease cleavage) or post-translational modification. In some embodiments, one or more modifications will affect the activity of the polypeptide, for example by making an inactive polypeptide active or by altering (e.g., increasing, decreasing) the activity level of the polypeptide. In certain embodiments, the polypeptides described herein are provided as prodrugs, for example, which can be converted in vivo (e.g., by proteolytic cleavage, post-translational modification) into an active polypeptide. In some embodiments, the polypeptide comprises post-translational modifications or other chemical modifications. Non-limiting examples of post-translational modifications include acetylation, amidation, formylation, glycosylation, hydroxylation, methylation, myristylation, phosphorylation, deamidation, isopreneation (e.g., farnesylation, geranylation, etc.), ubiquitination, ribosylation, and sulfation. Phosphorylation can occur on amino acids such as tyrosine, serine, threonine, or histidine.

[0098] In some embodiments, the polypeptide is linked to a heteropeptide 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 contains a tag (e.g., a detectable tag (such as a fluorophore or enzyme) or a purification tag (such as an epitope tag)).

[0099] In some embodiments, the polypeptide comprises one or more neoantigens selected from the sequence listing or variants thereof. As used herein, the term "neoantigen" refers to a tumor antigen generated by the target protein described herein. In some embodiments, the neoantigen is a cancer-specific neoantigen. There are various ways to generate neoantigens. For example, a neoantigen may be generated in vitro as a polypeptide prior to being formulated into a tumor vaccine or 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.

[0100] Alternatively, neoantigens can be generated in vivo by introducing a polynucleotide encoding a neoantigen or an expression vector (e.g., a viral expression vector) into cells or tissues (e.g., of a subject in need). In some embodiments, the polypeptide comprises at least two neoantigens. In some embodiments, the polypeptide comprises a T-cell enhancer amino acid sequence. In some embodiments, the T-cell enhancer is selected from the group consisting of: a constant chain, a leader sequence of tissue plasminogen activator, a PEST sequence, a cyclin disruption cassette, a ubiquitination signal, and a SUMOylation signal. B. Polynucleotide drugs

[0101] In some embodiments, the agent comprises a polynucleotide or its analogue or derivative. In some embodiments, the polynucleotide or its analogue or derivative is an inhibitor of the target protein. In some embodiments, the polynucleotide or its analogue or derivative is an activator of the target protein. In some embodiments, the polynucleotide or its analogue or derivative reduces (e.g., decreases or neutralizes) the expression or activity of the target protein. In other embodiments, the polynucleotide or its analogue or derivative increases the expression or activity of the target protein.

[0102] Polynucleotides can have sequences containing naturally occurring ribonucleotide or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or combinations thereof. Therefore, polynucleotides can comprise, for example, nucleotides containing naturally occurring bases (e.g., A, G, C, or T) and nucleotides containing modified bases (e.g., 7-dezoguanosine, inosine, or methylated nucleotides such as 5-methyldCTP and 5-hydroxymethylcytosine). In some embodiments, the polynucleotide comprises at least one modified nucleotide. Non-limiting examples of modified nucleotides include 2'-fluoro, 2'-o-methyl, 2'-deoxy, non-locked nucleic acids, 2'-hydroxy, thiophosphate, 2'-thiouridine, 4'-thiouridine, and 2'-deoxyuridine. In some embodiments, the modification increases nuclease resistance, increases serum stability, reduces immunogenicity, or a combination thereof.

[0103] 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).

[0104] In some embodiments, the polynucleotide includes analogs or derivatives of the polynucleotide. In some embodiments, the analog or derivative is a peptide nucleic acid (PNA). In some embodiments, the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative comprises one or more phosphate thioester links. In some embodiments, the agent comprises a deoxyribonucleic acid guanidine (DNG) nucleotide. In some embodiments, the agent comprises a ribonucleic acid guanidine (RNG) nucleotide.

[0105] In some embodiments, polynucleotides regulate the expression and / or activity of nucleic acids encoding target proteins disclosed herein (e.g., target proteins in the sequence listing) or portions thereof (e.g., their biologically active portions or fragments).

[0106] In some embodiments, the polynucleotide comprises a nucleotide sequence complementary (e.g., fully complementary or partially complementary) to at least a portion of a gene or gene transcript encoding a target protein disclosed herein, such that the polynucleotide sequence is capable of hybridizing or annealing to the gene or gene transcript (e.g., under physiological conditions). In other embodiments, the polynucleotide comprises a nucleotide sequence 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.

[0107] In some embodiments, the polynucleotide encodes the target protein disclosed herein or a variant thereof (e.g., its biologically active variant) or a portion thereof (e.g., its biologically active portion or fragment).

[0108] In some embodiments, the nucleic acid encoding a target protein or a variant thereof or a portion thereof (e.g., a fragment) is a gene sequence or a portion thereof. In some embodiments, the encoding nucleic acid is an unprocessed RNA transcript (e.g., pre-mRNA) or a portion thereof (e.g., 5'UTR, 3'UTR, intron). In some embodiments, the encoding nucleic acid is an mRNA molecule or a portion thereof. In some embodiments, the encoding nucleic acid is present in non-coding RNA (e.g., long intergenic non-coding RNA (lincRNA), long non-coding RNA (lncRNA), or miRNA).

[0109] The encoding nucleic acid may contain either a classic open reading frame (ORF) or a non-classical ORF. In some embodiments, the encoding nucleic acid contains a non-classical ORF.

[0110] Polynucleotides can be single-stranded (ss) or double-stranded (ds). In some embodiments, the polynucleotide is double-stranded (ds). In some embodiments, the length of the double-stranded polynucleotide is about 15-50 base pairs, for example, about: 15-45, 15-40, 15-35, 15-30, 15-25, 18-50, 18-45, 18-40, 18-35, 18-30, 18-25, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40, or 40-50 base pairs. In some embodiments, the polynucleotide is about 19-23 base pairs in length. In some embodiments, the polynucleotide is about 21 base pairs in length.

[0111] In some embodiments, the polynucleotide is single-stranded (ss). In some embodiments, the length of the single-stranded polynucleotide is about 15-50 nucleotides, for example 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.

[0112] In some embodiments, the polynucleotide prevents newly generated nuclear RNA transcripts from maturing into mRNA for transcription. In some embodiments, the polynucleotide comprises a nucleotide sequence complementary to the sequence at the intron and exon boundaries.

[0113] In some embodiments, the polynucleotide (e.g., an antisense oligonucleotide) may hybridize with the mRNA encoding the target protein (e.g., under physiological conditions). In some embodiments, the polynucleotide is at least about 10 nucleotides in length, for example 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, or 20-25 nucleotides. In some embodiments, the polynucleotide is at least 75% identical to the antisense sequence of the target transcript, for example at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0114] In some embodiments, the polynucleotide further includes an overhang sequence (e.g., an unpaired overhang nucleotide that does not directly participate in the formation of the double helix structure of the core sequence). In some embodiments, the polynucleotide includes 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, phosphorothioate, or a deoxynucleotide reversed (3' to 3' linked) nucleotide.

[0115] Non-limiting examples of polynucleotide agents suitable for use in the compositions, kits and methods described herein include small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antagomir, antisense DNA, antisense RNA, morpholinonucleotide (MNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), aptamers and guide RNA (gRNA).

[0116] In some embodiments, polynucleotides suppress gene expression (e.g., through the biological process of RNA interference (RNAi)). Polynucleotides suitable for RNA interference can be readily designed and generated by those skilled in the art using techniques, assays, and reagents (including computational tools) known in the art. See, for example, Pei et al. 2006, Reynolds et al. 2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005, Chalk et al. 2004, and Amarzguioui et al. 2004.

[0117] In some embodiments, the polynucleotide is a miRNA. In some embodiments, the miRNA is about 22 nucleotides in length. The miRNA binds to a target site on the mRNA molecule and silences the mRNA, for example by inducing mRNA cleavage, mRNA destabilization, or inhibition of mRNA translation.

[0118] 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 a 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%.

[0119] In some embodiments, the polynucleotide is shRNA. shRNA is an RNA molecule containing a hairpin turn that reduces the expression of a target gene via RNAi. shRNA can be delivered to cells in the form of plasmids (e.g., viral or bacterial vectors), for example, by transfection, electroporation, or transduction.

[0120] siRNA and shRNA are analogous to intermediates in the processing pathway of endogenous microRNA (miRNA) genes (see, for example, Bartel, Cell 116:281-97 (2004)). In some embodiments, siRNA functions as miRNA; in others, miRNA functions as siRNA (see, for example, Zeng et al., Mol Cell 9:1327-33 (2002); Doench et al., Genes Dev 17:438-42 (2003)). Like siRNA, microRNAs use RISC to downregulate target genes, but unlike siRNA, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein output through translational repression or poly-A removal and mRNA degradation (see, for example, Wu et al., Proc Natl Acad SciUSA 103:4034-39 (2006)). Known miRNA binding sites are located within the 3'UTR of mRNA; miRNAs appear to target sites that are almost perfectly complementary to the 2–8 nucleotides at the 5' end of the miRNA (see, for example, Rajewsky, Nat Genet, Nature Genetics 38 Supplement: S8-13 (2006) and Lim et al., Nature 433: 769-73 (2005)). This region is called the seed region. Because siRNAs and miRNAs are interchangeable, exogenous siRNAs downregulate mRNAs that are seed-complementary to the siRNA (see, for example, Birmingham et al., Nat Methods 3: 199-204 (2006)). Multiple target sites within the 3'UTR produce stronger downregulation (see, for example, Doench et al., GenesDev 17: 438-42 (2003)).

[0121] In some embodiments, the polynucleotide is a messenger RNA (mRNA) or circular RNA (circRNA) encoding the 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 to limit mRNA destabilization caused by rare codons (see, for example, Presnyak et al., Cell. 160(6):1111-24 (2015) and Thess et al., Mol Ther. 23(9):1456-64 (2015)).

[0122] 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 (edited by Mouldy Sioud, 2010) and Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Medicine 9:60 (2017).

[0123] In some embodiments, mRNA is generated by in vitro transcription. In some embodiments, the mRNA is modified to optimize its activity. In some embodiments, the mRNA comprises modified bases, a 5' cap, a 5' cap analog, an anti-reverse cap analog (ARCA), or a combination thereof.

[0124] In some embodiments, the mRNA contains a poly(A) tail. In some embodiments, the poly(A) tail is about 100-200 nucleotides long. In some embodiments, the poly(A) tail improves mRNA expression and / or stability (see, for example, Kaczmarek et al., Genome Medicine 9:60 (2017)).

[0125] In some embodiments, the mRNA contains a 5' cap. In some embodiments, the mRNA contains a 5' cap analog. In some embodiments, the 5' cap analog is a cap modified with 1,2-dithiobisphosphate (see, for example, Strenkowska et al., Nucleic Acids Res. 44:9578-90 (2016)).

[0126] In some embodiments, the mRNA comprises a modified 3' untranslated region (UTR), 5' UTR, or both. In some embodiments, the modified UTR comprises a sequence responsible for recruiting RNA-binding proteins (RBPs) and miRNAs (e.g., to enhance the level of protein products (see, for example, Kaczmarek et al., Genome Medicine 9:60 (2017))). In some embodiments, the 3' UTR, 5' UTR, or both are modified to encode regulatory elements. In some embodiments, the regulatory elements comprise a K-turn motif, a miRNA binding site, or a combination thereof to control RNA expression in a cell-specific manner (see, for example, Wroblewska et al., Nat Biotechnol. 33:839-41 (2015)).

[0127] In some embodiments, the mRNA contains RNA base modifications. In some embodiments, the mRNA contains pseudouridine. In some embodiments, the mRNA contains N1-methyl-pseudouridine (e.g., to mask immunostimulatory activity and enhance translation initiation (see, for example, Andries et al., J Control Release 217:337-44 (2015) and Svitkin et al., Nucleic Acids Res. 45:6023-36 (2017)).

[0128] In some embodiments, the RNA (e.g., mRNA) is circular RNA.

[0129] Compositions and methods for generating mRNA are disclosed in, for example, WO 2016011306, WO 2016014846, WO2016022914, WO 2016077123, WO 2016164762, WO 2016201377, WO 2017049275, US9937233, US 8710200, US10022425, US 9878056, US 9572897, WO 2010084371, US 9353153, WO 2015034925 and WO 2019236673. See also, for example, Jemielity et al., RNA 9(9):1108-22 (2003); Mockey et al., Biochem Biophys Res Commun. 340:1062-88 (2006); Strenkowska et al., Nucleic Acids Res. 44:9578-90 (2016); Presnyak et al., Cell 160:1111-24 (2015); and Kaczmarek et al., Genome Medicine 9:60 (2017). In some embodiments, mRNA is prepared in lipid nanoparticle (LNP) formulations (e.g., for in vivo delivery, see, for example, U.S. Patent 9,764,036, U.S. Patent 9,682,139, Kauffman et al., Nano Lett. 15:7300-6 (2015), and Fenton et al., Adv Mater. 28:2939-43 (2016)).

[0130] In some embodiments, the polynucleotide is an aptamer. In some embodiments, the aptamer binds to the target protein disclosed herein. In particular embodiments, the aptamer binds to a binding pair of the target protein disclosed herein.

[0131] In some embodiments, the polynucleotide is linked to the delivery polymer (e.g., covalently). In some embodiments, the link between the polynucleotide and the delivery polymer is reversible. In some embodiments, the polynucleotide is linked to the delivery polymer via a physiologically unstable linker. In some embodiments, the physiologically unstable linker is a disulfide bond.

[0132] 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 (e.g., to cells or a subject).

[0133] Those skilled in the art can readily prepare suitable polynucleotide agents for use in the compositions, kits, and methods described herein using locus information (such as chromosomal location, start and stop nucleotide positions, and polymorphism identification) of protein sequences contained in the sequence listings incorporated herein.

[0134] C. Drugs containing gene-editing systems

[0135] In some embodiments, the agent comprises a gene editing system. In some embodiments, the gene editing system produces a nucleotide deletion, nucleotide substitution, nucleotide addition, or a combination thereof in a gene encoding a target protein.

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

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

[0138] In some embodiments, the CRISPR system specifically catalyzes a cleavage in the gene encoding the target protein, thereby inactivating the gene. Repairing nucleic acid strand breaks via non-homologous end joining (NHEJ) often results in alterations to the DNA sequence at the cleavage site, leading to small insertions or deletions (indels). In some embodiments, NHEJ is used to knock out the gene encoding the target protein. In some 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. Cells that have undergone knockout and / or knock-in events can be identified and / or selected using methods well known in the art.

[0139] In some embodiments, the gene editing system comprises a single Cas endonuclease or a polynucleotide encoding a single Cas endonuclease. In some embodiments, the single Cas endonuclease is Cas9, Cpf1, C2C1, or C2C3. In some embodiments, the single Cas endonuclease is Cas9 (e.g., Streptococcus Pyogenes). In some embodiments, the single Cas endonuclease is Cpf1. In some embodiments, Cpf1 is AsCpf1 (from a species of the genus Acidaminococcus) or LbCpf1 (from a species of the family Lachnospiraceae). The selection of the nuclease and one or more gRNAs will typically be determined based on whether the deletion, substitution, or addition of one or more nucleotides of the target sequence is required.

[0140] In some embodiments, the type II Cas endonuclease is Cas9 (e.g., of Streptococcus pyogenes). In some embodiments, the modified Cas9 is a nickase Cas9, an inactive Cas9 (dCas9), or 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 dual-nickase Cas9 (e.g., for more accurate genome editing, see, for example, Ran et al., Cell 154:1380-89 (2013)). Wild-type Cas9 produces double-strand breaks (DSBs) at specific DNA sequences targeted by gRNAs. Nickase Cas9 produces only single-strand breaks. dCas9 is non-catalytically active. In some embodiments, dCas9 is fused with a nuclease (e.g., FokI to produce DSBs at target sequences homologous to both gRNAs). Various CRISPR / Cas9 plasmids are publicly available from the Addgene repository (Addgene, Cambridge, MA: addgene.org / crispr / ).

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

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

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

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

[0145] In some embodiments, the target DNA sequence is adjacent to a "prototype spacer neighbor motif" ("PAM") that is specific to Cas endonucleases. PAM sequences appear to be distributed throughout a given genome. CRISPR endonucleases of various prokaryotic species have unique PAM sequence requirements. Non-restrictive 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 endonuclease) associate with G-rich PAM sites (e.g., 5'-NGG) and cleave the target DNA at a position (5') 3 nucleotides upstream of the PAM site.

[0146] In some embodiments, the gene editing system includes: a) Wild-type or modified type II Cas endonucleases or polynucleotides encoding wild-type or modified type II Cas endonucleases; and b)crRNA.

[0147] Cpf1-associated CRISPR arrays are processed into mature crRNA without the need for tracrRNA. The Cpf1 endonuclease associates with T-rich PAM sites (e.g., 5'-TTN). Cpf1 can also recognize 5'-CTA PAM motifs. Cpf1 cleaves target DNA by introducing misaligned or staggered double-strand breaks with 4 or 5 nucleotides of 5' overhang, for example, cleaving target DNA where the 5-nucleotide misaligned or staggered cut is located 18 nucleotides downstream (3') of the PAM site on the coding strand and 23 nucleotides downstream of the PAM site on the complementary strand. The 5-nucleotide overhang resulting from such misaligned cuts allows for more precise genome editing via DNA insertion through homologous recombination than insertion at blunt-end cuts. See, for example, Zetsche et al., Cell[Cell] 163:759-71 (2015).

[0148] In some embodiments, the gene editing system activates or inhibits the transcription of a target gene. In some embodiments, the gene editing system comprises: a) Chimeric proteins containing dCas9 and one or more effector domains; and b) One or more sgRNAs.

[0149] 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 a DNA sequence recognized by the sgRNA. In some embodiments, the chimeric protein demethylates a DNA sequence recognized by the sgRNA.

[0150] The effector domain contains the biologically active portion of an effector protein (e.g., a transcription activator or repressor). In some embodiments, the gene editing system contains one effector domain. In some embodiments, the gene editing system contains at least two effector domains, such as two, three, or four effector domains. In some embodiments, the effector domain contains KRAB. In some embodiments, the effector domain contains VP64. In some embodiments, the effector domain contains VP64, p65, and Rta. In some embodiments, dCas9 is D10A. In some embodiments, dCas9 is H840A.

[0151] Because dCas9 is non-catalytically active, it does not cleave target DNA but instead interferes with transcription through steric hindrance. dCas9 chimeric proteins (e.g., dCas9-VPR) (guided by one or more gRNAs to the upstream sequence of the transcription start site (TSS) of the target gene) regulate the transcription of the target gene. See, for example, Gilbert et al., CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes, Cell 154, 442-51 (2013); Cheng et al., Multiplexed Activation of Endogenous Genes by CRISPR-on, An RNA-Guided Transcriptional Activator System, Cell Res. 23:1163-71 (2013); Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation, Cell 159:647-61 (2014); Tanenbaum et al., A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging, Cell 159:635-46 (2014); Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Nature 517:583-88 (2015); Chavez et al., Highly efficient Cas9-mediated transcriptional programming, Nat. Methods.[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, Nature Methods 7:563-67 (2016).

[0152] CRISPR technology for editing genes in eukaryotes is disclosed in U.S. Patent Application Publication 2016 / 0138008A1 and US2015 / 0344912 A1, as well as U.S. Patents 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. The Cpf1 endonuclease, along with the corresponding guide RNA and PAM site, is disclosed in U.S. Patent Application Publication 2016 / 0208243A1. CRISPR technology for generating mtDNA dysfunction in the mitochondrial genome was disclosed in Jo et al., BioMed Res. Int. 2015:305716 (2015). Co-delivery of Cas9 and sgRNA using nanoparticles was disclosed in Mou et al., ACS Nano 11(3):2452-58 (2017).

[0153] In some embodiments, the agent comprises a transposon-based gene editing system. An example of a suitable transposon-based gene editing system used in this disclosure is the 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.

[0154] In some embodiments, the agent comprises a transcription activator-like effector nuclease (TALEN) system. TALEN-based systems comprise proteins 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-cutting domain (a nuclease that cuts the DNA strand). The aforementioned FokI restriction enzyme is an exemplary enzyme domain suitable for use in TALEN-based gene regulation systems.

[0155] TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when plants are infected. The DNA-binding domain contains a highly conserved repeating sequence of 33-34 amino acids, with the 12th and 13th amino acids differing. These two positions (called repeat variable double residues (RVDs)) are highly variable and strongly correlated with specific nucleotide recognition. Therefore, the TAL effector domain can be engineered to bind specific target DNA sequences by selecting combinations of repeat segments containing appropriate RVDs. The nucleic acid specificity of RVD combinations is as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (although, in some embodiments, NN may also bind adenine with lower specificity).

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

[0157] In some embodiments, the gene regulatory system comprises two or more TAL effector fusion proteins (each containing 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, or 100% identical, to a target DNA sequence defined by a set of genomic coordinates.

[0158] Methods and compositions for assembling TAL effector repetitive 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 used to construct TAL effector repetitive sequences are commercially available from, for example, AdGene.

[0159] In some embodiments, the reagent comprises a zinc finger nuclease (ZFN) system. Commercially available plasmids can be used, such as the plasmid pair (CSTZFN-1KT) from Sigma Aldrich (St. Louis, Missouri). A custom zinc finger nuclease (ZFN) R-3257609 was used to generate ZFN domains. Plasmids can be prepared using commercial systems according to the manufacturer's protocol (e.g., NEB Monarch Miniprep (catalog number T1010), New England Biolabs, Ipswich, MA).

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

[0161] 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 capable of regulating the expression or activity of the target protein. In some embodiments, the small molecule is an inhibitor of the target protein (e.g., a direct inhibitor, an indirect inhibitor). In some embodiments, the small molecule is an activator of the target protein (e.g., a direct activator and an indirect activator).

[0162] Examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic, or inorganic compounds. The atoms in a small molecule are typically linked together by covalent and / or ionic bonds. In some embodiments, the small molecule is a small organic molecule. The arrangement of atoms in a small organic molecule can represent a chain (e.g., a carbon-carbon chain or a carbon-heteroatom chain), or it can represent a ring containing carbon atoms (e.g., benzene or a polycyclic system) or a ring of combinations of carbon and heteroatoms (i.e., heterocycles, such as pyrimidines or quinazolines). Although small molecules can have a wide range of molecular weights, they typically include molecules with a molecular weight of less than about 5,000 Daltons. For example, such small molecules can be less than about 1,000 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., identified, isolated, purified) and / or synthesized (e.g., by conventional organic synthesis, bio-mediated synthesis, or a combination thereof). See, for example, Ganesan, Drug Discov. Today, 7(1):47-55 (January 2002); Lou, Drug Discov. Today, 6(24):1288-1294 (December 2001). Examples of naturally occurring small molecules include, but are not limited to, hormones, neurotransmitters, nucleotides, amino acids, sugars, lipids and their derivatives.

[0163] In a particular embodiment, the agent comprises a protein hydrolysis-targeting chimera (PROTAC).

[0164] Small molecules suitable for use in the compositions, kits and methods disclosed herein can be identified by a person skilled in the art using any of the screening methods disclosed herein. E. Therapeutic cells and cell-based therapies

[0165] In some embodiments, the agent comprises therapeutic cells. In some embodiments, the therapeutic cells express and / or are engineered to express the target protein described herein (e.g., the target protein in the sequence listing or a variant thereof), peptide (e.g., an antibody, an antigen-binding fragment, or a peptide comprising at least 70% of the same amino acid sequence as at least a portion of the target protein), polynucleotide (e.g., recombinant DNA, RNA (such as mRNA or siRNA)), and / or gene editing systems (e.g., a CRISPR / Cas system).

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

[0167] Therapeutic cells suitable for use in the compositions, kits, and methods disclosed herein can be generated, identified, and / or enriched using methods known to those skilled in the art. Non-limiting examples of the methods include purifying, propagating, and / or differentiating cells from a subject (e.g., a human) into specific cell products; engineered somatic cells for gene therapy; cell immortalization; in vitro genetic modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technologies); in vivo genetic modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technologies); genome editing; cell plasticity techniques; gene modification; and flow cytometry. In some embodiments, the therapeutic cells are autologous or syngeneic. In other embodiments, the therapeutic cells are allogeneic. Expression vector and host

[0168] On the other hand, this disclosure provides expression vectors containing the polynucleotides described herein.

[0169] The term "expression vector" refers to a reproducible nucleic acid that can express one or more proteins when it is transformed into a suitable expression host cell.

[0170] In some embodiments, the expression vector comprises an expression control polynucleotide sequence operatively linked to a polynucleotide, a polynucleotide sequence encoding an optional marker, or both. In some embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In some embodiments, the expression control polynucleotide sequence comprises an inducible promoter sequence. The term “promoter” refers to a region of DNA to which an RNA polymerase binds and initiates gene transcription. The term “operatively linked” means that a nucleic acid is located in a recombinant polynucleotide (e.g., a vector) in such a way that the nucleic acid can be expressed under the control of an element (e.g., a promoter) to which it is linked. The term “optional marker element” is an element that confers traits suitable for artificial selection. Optional marker elements can be negative or positive selection markers. Non-limiting examples of expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Molecular Cloning: A Laboratory Manual (edited by Michael R. Green and Joseph Sambrook, 4th edition, 2012).

[0171] On the other hand, this disclosure provides expression host cells comprising any one or more of the polynucleotides or expression vectors described herein.

[0172] The term "expression host cell" refers to a cell that can be used to receive, maintain, replicate, and / or amplify a vector.

[0173] Non-limiting examples of host cells that express expression include mammalian cells, such as hybridoma cells, young 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. For methods of host cell culture used to produce protein therapeutics, see, for example, *Mammalian Cell Cultures for Biologics Manufacturing* (edited by Weichang Zhou and Anne Kantardjieff, 2014); for purification of protein therapeutics, see, for example, *Protein Biotechnology: Isolation, Characterization, and Stabilization* (edited by Felix Franks, 2013) and *Protein Purification Protocols* (edited by Paul Cutler, 2010); and for formulation of protein therapeutics, see, for example, *Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic* (edited by Brian K Meyer, 2012).

[0174] The polynucleotides or expression vectors described herein can be introduced into suitable or desired host cells using techniques known in the art, including transformation, electroporation, and transduction. The introduced nucleic acids may be extrachromosomal in the host cell or may be integrated into the host cell's genome. Pharmaceutical Composition

[0175] On the other hand, this disclosure provides pharmaceutical compositions comprising the pharmaceutical agents and pharmaceutically acceptable carriers disclosed herein. As used herein, the term "pharmaceutical composition" means a composition or its final dosage form or formulation that has pharmacological activity or other direct effects in alleviating, treating or preventing cancer.

[0176] In some embodiments, the composition (e.g., a pharmaceutical composition) comprises a pharmaceutically acceptable carrier, excipient, stabilizer, diluent, or tonifier (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor (1980)). A suitable pharmaceutically acceptable carrier, excipient, or stabilizer is non-toxic to the receptor at the dose and concentration employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents, or supplements include buffers (e.g., phosphates, citrates, histidines), 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), and PLURONICS. TM And polyethylene glycol (PEG).

[0177] In some embodiments, the pharmaceutical agent (e.g., peptide, polynucleotide, or small molecule) of the pharmaceutical composition is modified, for example, by conjugation to a heterologous moiety. The term "conjugation" refers to attachment via covalent or non-covalent interactions. Conjugation can be performed using any suitable linker; non-limiting examples include peptide linkers, compound linkers, and chemical crosslinking agents.

[0178] In some embodiments, the heterologous portion is a molecule of a marker (e.g., a fluorescent marker or a radioactive marker), a molecule of a stabilizing agent, a molecule of a targeting agent (e.g., targeting specific cells or tissues to facilitate or prevent crossing the blood-brain barrier), or a combination thereof.

[0179] In some embodiments, the heterosource portion is polyethylene glycol (PEG), hexadecanoic acid, hydrogel, nanoparticles, polymerized domains, and carrier peptides. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are polymer nanoparticles. In some embodiments, the polymer is an amphiphilic polymer. In other embodiments, the polymer is a hydrophobic or hydrophilic polymer. Non-limiting examples of polymers include poly(lactic acid)-poly(ethylene glycol), poly(lactic acid-co-glycolic acid)-poly(ethylene glycol), poly(lactic acid-co-glycolic acid) (PLGA), poly(lactic acid-co-glycolic acid)-d-α-tocopherol 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 nanoparticles comprise poly(lactic acid-co-glycolic acid) (PLGA).

[0180] In some embodiments, the composition (e.g., a pharmaceutical composition) is formulated for a suitable administration regimen and route. Non-limiting examples of routes of administration include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical administration. In some embodiments, the composition (e.g., a pharmaceutical composition) is stored as an aqueous solution or a dried formulation (e.g., lyophilized). In some embodiments, the composition is formulated for administration by infusion (e.g., intravenous infusion).

[0181] In some embodiments, the composition is formulated for administration in combination with one or more additional therapeutic agents (e.g., with a second therapeutic agent) as a combination therapy. As used herein, “combination therapy” or “combination administration” means administering two (or more) different agents or treatments 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 biologics (e.g., antibodies, peptides), cell therapies, gene therapies, immunotherapies, and small molecules that affect immune-mediated diseases or conditions.

[0182] The treatment regimen defines the dosage and duration of administration of each agent such that the individual effects of the agent overlap with those of the subject. In some embodiments, two or more agents are administered sequentially as part of a prescription regimen. In other embodiments, the delivery of two or more agents is simultaneous or concurrent. In some embodiments, two or more agents are co-formulated. In some embodiments, the combined administration of two or more agents or treatments results in a reduction in symptoms or other parameters related to the disorder that would be observed with the delivery of a single agent or treatment alone or in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Each of the two or more therapeutic agents may be administered via any suitable route, including but not limited to oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The two or more therapeutic agents may be administered via the same route or via different routes.

[0183] In some embodiments, the agents or pharmaceutical compositions disclosed herein are delivered via a viral vector (e.g., by contacting cells with a viral vector), administered topically (e.g., by injection) to a tumor, or administered systemically (e.g., intravenously or orally) to a subject (e.g., a human patient).

[0184] Viral genomes provide a rich source of vectors that can be used to efficiently deliver exogenous genes into 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 via general 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 viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses (such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses)), positive-strand RNA viruses (such as piconemaviruses and alphaviruses), and double-stranded DNA viruses (including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, EB virus, cytomegalovirus, replication-defective herpesviruses), and poxviruses (e.g., cowpox, modified Ankara cowpox (MVA), fowlpox, and canarypox virus)). Further non-limiting examples include, for example, norovirus, capsular virus, flavivirus, reovirus, papillomavirus, hepatotropic DNA virus, human papillomavirus, human foamy virus, and hepatitis virus. Non-limiting examples of retroviruses include: avian leukosis-sarcoma virus, avian type C virus, mammalian type C, type B, and type D viruses, carcinogenic retroviruses, HTLV-BLV group viruses, lentiviruses, alpha retroviruses, gamma retroviruses, and foam viruses (see, for example, Coffin JM. Retroviridae: The viruses and their replication. [Retroviridae: 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). Other 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 leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Further non-limiting examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the teachings of which are incorporated herein by reference.

[0185] In some embodiments, the pharmaceutical agents or compositions disclosed herein are formulated for delivery in vivo, in vitro, ex vivo, or in situ via membrane-based carriers. In some embodiments, the membrane-based carriers are cell-based carriers (e.g., mammalian (such as human) cells). In some embodiments, the membrane-based carriers are vesicle-based carriers. In some embodiments, the membrane-based carriers comprise one or more of the vectors described herein (e.g., plasmids, viruses, virus-like particles, or virions).

[0186] In some embodiments, the pharmaceutical agents or pharmaceutical compositions disclosed herein are formulated for delivery via one or more liposomes. Liposomes are spherical vesicle structures consisting of a single or multiple lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible, non-toxic, capable of delivering both hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes, and loading and transporting them across biological membranes and the blood-brain barrier (BBB) ​​(see, for example, Spuch and Navarro, J Drug Deliv. [Journal of Drug Delivery] 2011:469679(2011)).

[0187] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilayer vesicular lipids are known in the art (see, for example, U.S. Patent No. 6,693,086, whose teachings on the preparation of multilayer vesicular lipids are incorporated herein by reference). Although vesicle formation can be spontaneous when the lipid membrane is mixed with an aqueous solution, it can also be accelerated by applying force in an oscillatory manner using a homogenizer, sonicator, or extrusion device (see, for example, Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)). Extruded lipids can be prepared by extruding through a filter with a gradually decreasing size, as described in Templeton et al., Nature Biotech, 15:647-52 (1997), whose teachings on the preparation of extruded lipids are incorporated herein by reference.

[0188] In some embodiments, the pharmaceutical agents or pharmaceutical compositions disclosed herein are formulated for delivery via lipid nanoparticles (LNPs). In one embodiment, an LNP formulation comprising the pharmaceutical agents or pharmaceutical compositions disclosed herein has one or more of the following characteristics: (a) the LNP formulation comprises cationic lipids, neutral lipids, cholesterol, and PEG lipids; and (b) the average particle size of the LNP formulation is between 80 nm and 160 nm.

[0189] Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component 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), novel carriers combining liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs consist of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, the two components increase drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs. See, for example, Li et al., Nanomaterials 7(6):122 (2017).

[0190] In some embodiments, the pharmaceutical agents or compositions disclosed herein are formulated for delivery via a carbohydrate carrier (e.g., anhydride-modified plant glycogen or glycogenotype material). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin.

[0191] In some embodiments, the pharmaceutical agents or pharmaceutical compositions disclosed herein are formulated for delivery via a protein carrier (e.g., a protein covalently linked to a cyclic polynucleotide). Non-limiting examples of protein carriers include human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and globulins.

[0192] In some embodiments, the pharmaceutical agents or compositions disclosed herein are formulated for delivery via a cationic carrier (e.g., a cationic lipid polymer or a transfection reagent). Non-limiting examples of cationic carriers include lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-β-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationic gelatin, dendritic polymers, chitosan, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleylenyl-3-( 2-Hydroxyethyl)imidazoline trichloroethylene (DOTIM), 2,3-diolenoyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propaneammonium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol hydrochloride), bis(heptadecylamidoglycylspermine) (DOGS), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-diolenoyl-N,N-dimethylammonium chloride (DODAC).

[0193] In some embodiments, the pharmaceutical agents or pharmaceutical compositions disclosed herein are formulated for delivery via exosomes, adipocytes, and / or erythrocytes. See, for example, Ha et al., Acta Pharm Sin B. 6(4):287-96 (2016).

[0194] In some embodiments, the pharmaceutical agents or compositions disclosed herein are formulated for delivery via one or more fusosomes. The fusosomes have been engineered to impart target cell specificity for fusion and payload delivery, allowing for the creation of delivery media with programmable cell specificity. See, for example, patent application WO 2020014209, whose teachings on the design, preparation, and use of fusosomes are incorporated herein by reference.

[0195] In some embodiments, the agents or pharmaceutical compositions disclosed herein are formulated for delivery via in vitro differentiated erythrocytes. See, for example, WO 2015073587; WO 2017123646; WO 2017123644; WO 2018102740; WO2016183482; WO 2015153102; WO 2018151829; WO 2018009838; Shi et al., PNAS, 111(28):10131-36 (2014); U.S. Patent 9,644,180; Huang et al., Nature Communications 8:423 (2017).

[0196] In some embodiments, the pharmaceutical agents or pharmaceutical compositions disclosed herein are formulated for delivery via one or more microsomes, virus-like particles (VLPs), or plant nanovesicles and plant messenger packets (PMPs). See, for example, WO 2011097480, WO2013070324, WO 2017004526 and WO 2020041784.

[0197] In some embodiments, the pharmaceutical agents or pharmaceutical compositions disclosed herein are formulated for delivery via one or more anellosomes. The preparation and use of anellosomes for delivering therapeutic products are described in U.S. Patent 11,166,996, the teachings of which regarding the design, preparation, and use of anellosomes are incorporated herein by reference. Methods of regulating target proteins

[0198] On the other hand, this disclosure provides a method for regulating the expression or activity of a target protein or a variant thereof identified in a sequence listing in cells (target cells, cells of target tissues), the method comprising contacting cells (e.g., in vitro, ex vivo, or in vivo) with a pharmaceutical agent or a pharmaceutical composition containing the pharmaceutical agent, the pharmaceutical agent containing the target protein identified herein and / or regulating the expression or activity of the target protein.

[0199] In some embodiments, the target cells are cancer cells (e.g., metastatic cancer cells), cells in the tumor microenvironment (e.g., stromal cells), or a combination thereof. In a particular embodiment, the target cells are metastatic cancer cells.

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

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

[0202] In some embodiments, the target cells are immune cells. In some embodiments, the target cells are 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 combinations thereof.

[0203] In some embodiments, the target proteins disclosed herein are used to mediate the depletion of cell populations (e.g., cancer cell (such as tumor cell) populations; immune cell populations). In some embodiments, the target proteins disclosed herein, for example, act as conjugates of surface markers to facilitate cell targeting (e.g., delivery of therapeutic agents in a cell type-specific manner).

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

[0205] In some embodiments, the target tissue includes a tumor, the tumor microenvironment, a metastatic site, or a combination thereof.

[0206] In some embodiments, the target tissue is immune tissue. In some embodiments, the target tissue is non-immune tissue. In some embodiments, the target tissue includes lymph nodes, spleen, secondary lymphoid organs, tertiary lymphoid organs, barrier tissue, skin, intestines, airways, wounds, immune tissue, non-immune tissue, or a combination of the foregoing.

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

[0208] In some embodiments, the effective amount is sufficient to increase the expression of the target protein in target cells and / or target tissues. 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 approximately 10%-99%, for example, approximately: 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 times, for example 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 times.

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

[0210] On the other hand, this disclosure provides a method for detecting a disease or condition in a subject or predicting the likelihood (or risk level) of a subject having a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, wherein the level of expression or activity of the target protein in the sample indicates the likelihood of the subject having a disease or condition, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmune disease, cancer (e.g., tumor), infection, immune disease (e.g., inflammation and / or autoimmune disease) or a combination thereof.

[0211] On the other hand, this disclosure provides a method for classifying subjects based on the predictive likelihood of having a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject; predicting the likelihood of having a disease or condition based on the expression or activity of the target protein in the sample; and classifying the patient based on the predictive likelihood, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmune disease, cancer (e.g., tumor), infection, immune disease (e.g., inflammation and / or autoimmune disease) or a combination thereof.

[0212] On the other hand, this disclosure provides a method for stratifying a group of subjects suffering from a disease or condition, the method comprising: quantifying the expression and / or activity of a target protein in samples from individual subjects in the group; and stratifying the group of subjects receiving treatment based on the expression and / or activity levels of the target protein in the samples from individual subjects, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmune diseases, cancer (e.g., tumors), infections, immune diseases (e.g., inflammation and / or autoimmune diseases) or combinations thereof.

[0213] In some embodiments, higher levels of target protein expression or activity in a subject's sample compared to a suitable control (e.g., a reference standard) indicate a disease or condition or the likelihood of having a disease or condition. In some embodiments, lower levels of target protein expression or activity in a subject's sample compared to a suitable control (e.g., a reference standard) indicate a disease or condition or the likelihood of having a disease or condition.

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

[0215] In some embodiments, the method further includes administering an effective amount of the agent or pharmaceutical composition disclosed herein to a subject who is identified as having or is predicted to have a disease or condition.

[0216] On the other hand, this disclosure provides a method for preparing a sample that can be used to detect the likelihood that a subject has a disease or condition, the method comprising: a) Samples obtained or already obtained from the subject; b) Add 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 likelihood of developing cancer; c) Quantify the expression or activity of the target protein in the sample prepared in step b); and d) Wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), infection, immune disease (e.g., inflammation and / or autoimmune disease) or a combination thereof.

[0217] On the other hand, this disclosure provides a method for treating a disease or condition in a subject in need, the method comprising administering to the subject an effective amount of the agent or pharmaceutical composition disclosed herein.

[0218] On the other hand, this disclosure provides a method for treating a disease or condition in a subject in need, the method comprising administering to the subject an effective amount of the agent or pharmaceutical composition disclosed herein, wherein the expression and / or activity level of the target protein disclosed herein in the subject is altered.

[0219] As used herein, “treatment” and “treating” refer to medical management of a subject aimed at improving, alleviating, stabilizing (i.e., not worsening), preventing, or curing a disease, pathological condition, or disorder. “Treatment” includes active treatment (treatment aimed at improving a disease, pathological condition, or disorder), etiological treatment (treatment addressing the cause of the related disease, pathological condition, or disorder), palliative treatment (treatment aimed at relieving symptoms), preventive treatment (treatment aimed at minimizing or partially or completely suppressing the development of the related disease, pathological condition, or disorder), and supportive treatment (treatment used to supplement another therapy). Treatment also includes reducing the severity of a disease or condition; preventing the spread of a disease or condition; delaying or slowing the progression of a disease or condition; alleviating or mitigating a disease or condition; and remission (whether partial or complete), whether detectable or undetectable. “Alleviating” or “mitigating” a disease or condition means that the severity and / or undesirable clinical manifestations of a disease, disorder, or condition are reduced and / or the time course of progression is slowed or prolonged compared to the severity or time course without treatment. "Treatment" also includes extending survival compared to the expected survival without treatment. Those who need treatment include those who already have a condition or disability, those who are susceptible to a condition or disability, or those who need to prevent a condition or disability.

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

[0221] As used herein, the terms “effective amount,” “therapeutic effective amount,” or “sufficient amount” refer to an amount sufficient to achieve a therapeutic effect (e.g., produce a beneficial or desired outcome) (including effects at the cellular, tissue, or clinical level) 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 a dose of medicine sufficient to achieve a response compared to a response obtained without the administration of the medicine. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors such as the given medicine, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight), or the host receiving treatment, but can still be routinely determined by those skilled in the art. In some embodiments, the “therapeutic effective amount” of the compositions disclosed herein is the amount that produces a beneficial or desired outcome (e.g., compared to a control) in a subject. Therapeutic effective amounts of the compositions disclosed herein can be readily determined by those skilled in the art using conventional methods known in the art. Dosing regimens can be adjusted to provide an optimal therapeutic response.

[0222] 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., intra-arterial, intravenous, intramuscular, subcutaneous, intradermal injection), intravenous infusion, and inhalation (e.g., intrabronchial, intranasal, or oral inhalation, nasal drops), depending on the compound and the specific disease or condition to be treated. Administration may be local or systemic, as directed. Preferred methods of administration may vary depending on the specific compound selected.

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

[0224] Administration of two or more therapeutic agents encompasses the co-administration of the therapeutic agents in a substantially simultaneous manner (such as in a combination of drugs). Alternatively, such administration encompasses the co-administration of each therapeutic agent in multiple containers or single containers (e.g., capsules, powders, and liquids). Such administration also encompasses the sequential use of the therapeutic agents at substantially 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.

[0225] On the other hand, this disclosure provides methods for regulating the expression or activity of target proteins or variants thereof identified in the sequence listing in cells, said methods comprising contacting cells with the pharmaceutical agents or pharmaceutical compositions disclosed herein. In some embodiments, the cells are in a subject.

[0226] On the other hand, this disclosure provides a method for identifying agents that regulate the expression and / or activity of target proteins (e.g., target proteins in the sequence listing), said method comprising: a) Contacting a sample containing the target protein (e.g., a biological sample, such as cells or tissues) with an agent (e.g., a candidate agent for testing its ability to regulate the expression and / or activity of the target); and b) Determine whether the drug modulates the expression or activity of the target protein. The difference in the expression or activity of target proteins that have been exposed to the drug compared to the reference indicates that the drug regulates the expression or activity of the target proteins.

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

[0228] In some embodiments, a decrease in the expression or activity of a target protein that has been contacted with the 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 cancer

[0229] 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, rectal tumors, testicular tumors, head and / or neck tumors, pancreatic tumors, brain tumors, and skin tumors). Thus, in some embodiments, the cancer is a solid tumor cancer. Solid tumor cancers treatable 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, cancers are blood cancers (e.g., leukemia, lymphoma, and myeloma). Blood cancers treatable 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.

[0230] Examples of cancers treatable according to the methods described herein include acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; adrenocortical carcinoma, children; HIV-related cancers (e.g., Kaposi's sarcoma, HIV-related lymphoma, primary CNS lymphoma); anal cancer; appendiceal 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 sarcoma, osteosarcoma, and malignant fibrous histiocytoma); brain tumors / cancer; breast cancer; Burkitt lymphoma; carcinoid tumors (gastrointestinal); carcinoid tumors, children; cardiac / heart tumors, children; embryonic tumors. Gestational tumors, children; germ cell tumors, children; primary CNS lymphomas; cervical cancer; cervical cancer in children; cholangiocarcinoma; chordoma, children; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); chronic myeloproliferative neoplasms; colorectal cancer; colorectal cancer in children; craniopharyngioma, children; cutaneous T-cell lymphomas (e.g., mycosis fungoides and Sezari syndrome); ductal carcinoma in situ (DCIS); embryonal tumors, central nervous system, children; endometrial cancer (uterine cancer); ependymoma, children; esophageal cancer; esophageal cancer in children; nasal glioma; Ewing sarcoma; extracranial germ cell tumors, children; gonadal germ cell tumors; eye / ocular cancer; intraocular melanoma in children; intraocular melanoma Retinoblastoma; Fallopian tube cancer; Malignant fibrous histiocytoma and osteosarcoma; Gallbladder cancer; Gastric / stomach cancer; Pediatric gastric / stomach cancer; Gastrointestinal carcinoid tumors; Gastrointestinal stromal tumors (GIST); Pediatric gastrointestinal stromal tumors; Germ cell tumors; Pediatric central nervous system germ cell tumors (e.g., pediatric extracranial germ cell tumors, gonadal germ cell tumors, ovarian germ cell tumors, testicular cancer); Gestational trophoblastic disease; Hairy cell leukemia; Head and neck cancer; Cardiac tumors, pediatric; Hepatocellular carcinoma; Histiocytosis, Langerhans cell; Hodgkin's lymphoma; Hypopharyngeal carcinoma; Intraocular melanoma; Pediatric intraocular melanoma; Islet cell tumors, pancreatic neuroendocrine tumors. Tumors; Kaposi's sarcoma; Renal (renal cell) carcinoma; Langerhans cell histiocytosis; Laryngeal cancer; Leukemia; Lip and oral cancer; Liver cancer; Lung cancer (non-small cell and small cell); Pediatric lung cancer; Lymphoma; Male breast cancer; Malignant fibrous histiocytoma and osteosarcoma of bone; Melanoma; Pediatric melanoma; Melanoma, intraocular (eye); Pediatric intraocular melanoma; Merkel cell carcinoma; Mesothelioma, malignant; Pediatric mesothelioma; Metastatic cancer; Metastatic squamous neck cancer with occult primary lesions; Mid-trimester carcinoma with NUT gene alterations; Oral cancer; Multiple endocrine neoplasia syndrome; Multiple myeloma / plasma cell tumor; Mycosis fungoides; Myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm; Myeloid leukemia, chronic (CML);Myeloid leukemia, acute (AML); myeloproliferative neoplasms, chronic; nasal cavity and sinus carcinoma; nasopharyngeal carcinoma; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung cancer; oral cavity cancer, lip and oral cavity cancer, and oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; pediatric ovarian cancer; pancreatic cancer; pediatric pancreatic cancer; pancreatic neuroendocrine tumors; papillomatosis (children, larynx); paraganglioma; pediatric paraganglioma; sinus and nasal cavity carcinoma; parathyroid carcinoma; penile cancer; pharyngeal carcinoma; pheochromocytoma; pediatric pheochromocytoma; pituitary adenoma; plasma cell tumor / multiple myeloma; pleural pulmonary blastoma; gestational breast cancer; primary central nervous system (CNS) lymphoma; primary peritoneal cancer; prostate cancer; rectal cancer; recurrent cancer; renal cell (renal) carcinoma; retinoblastoma; rhabdomyosarcoma, children; salivary gland cancer; sarcoma (e.g., pediatric rhabdomyosarcoma). Rhabdomyosarcoma, pediatric angiomyolipoma, Ewing sarcoma, Kaposi's sarcoma, osteosarcoma (bone cancer), soft tissue sarcoma, uterine sarcoma); Cézari syndrome; skin cancer; pediatric skin cancer; small cell lung cancer; small bowel cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; occult squamous neck carcinoma, metastatic; stomach (gastric) cancer; pediatric stomach (gastric) cancer; T-cell lymphoma, skin (e.g., mycosis fungoides and Cézari syndrome); testicular cancer; pediatric testicular cancer; laryngeal cancer (e.g., nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma); thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine cancer, endometrial cancer; uterine sarcoma; vaginal cancer; pediatric vaginal cancer; angiomyolipoma; vulvar cancer; and nephroblastoma and other pediatric kidney tumors.

[0231] Metastasis of the aforementioned cancers can also be treated according to the methods described herein. In some embodiments, the cancer is metastatic cancer.

[0232] In some embodiments, the cancer is selected from lung cancer, breast cancer, Hodgkin's 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.

[0233] In some embodiments, treatment: a) Inhibit the growth, proliferation, metastasis, invasion, or migration of cancer cells, or a combination thereof; b) Promotes cancer cell death; c) Inducing autophagy in cancer cells. Or a combination thereof.

[0234] In some embodiments, the effective amount is sufficient: a) Inhibit the growth, proliferation, metastasis, invasion, or migration of cancer cells, or a combination thereof; b) Promotes cancer cell death; c) Inducing autophagy in cancer cells. d) or a combination thereof.

[0235] In some embodiments, the effective amount is sufficient to reduce cancer (e.g., tumor) growth, proliferation, metastasis, invasion, migration, autophagy, or a combination of the foregoing. In 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 approximately 10%-99%, for example, approximately: 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%.

[0236] 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%, 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 some embodiments, the reduction in cancer cell proliferation or tumor growth is approximately 10%-99%, for example, approximately: 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%.

[0237] In some embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease) tumor autophagy, for example by increasing at least one tumor-suppressive function of autophagy and / or decreasing at least one tumor-promoting function of autophagy.

[0238] In some embodiments, the effective amount is sufficient to increase cancer autophagy. 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 approximately 10%-99%, for example, approximately: 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%.

[0239] In some embodiments, the effective amount is sufficient to reduce cancer autophagy. 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 approximately 10%-99%, for example, approximately: 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%.

[0240] In some embodiments, the effective amount is sufficient to prevent the death of the subject, thereby reducing cancer (e.g., tumor) mortality. In some embodiments, the reduction in cancer (e.g., tumor) mortality 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 in cancer (e.g., tumor) mortality is approximately 10%-99%, such as approximately: 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%.

[0241] In some embodiments, the effective amount is sufficient to regulate (e.g., increase or decrease) the expression of the target protein in cancer (e.g., tumor) cells.

[0243] In some embodiments, the effective amount is sufficient to modulate the body’s response to cancer, for example by inhibiting cancer growth, reducing cancer malignancy, inhibiting cancer metastasis, promoting remission, modulating (increasing and / or decreasing) cancer-related immune-mediated responses, or a combination of the foregoing. Immuno-oncology

[0244] In some embodiments, this disclosure provides treatments utilizing the immune system. In some embodiments, the methods relate to immuno-oncology (e.g., cancer immunotherapy). In still other embodiments, the immune system is the innate immune system. In some embodiments, the immune system is the adaptive immune system. In still other embodiments, the treatment involves humoral immunity or antibody-mediated immunity. In some embodiments, the treatment involves cell-mediated immunity, such as in cancer. In some embodiments, the methods relate to treatment at or before early disease progression, while in other embodiments, the methods relate to treatment or prevention of late disease progression. In still other embodiments, stimulation of the immune system induces immuno-oncological effects.

[0245] In some embodiments, the effective amount is sufficient to modulate (e.g., increase) the subject's immune system against cancer. In some embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease): a) Immune cell-related readouts, immune cell activation, degranulation, maturation, migration, polarization, proliferation, and recruitment of immune cells (e.g., macrophages, monocytes, or dendritic cells); b) Lymph node activation, innervation, differentiation, egress, and homing; c) Cytokine production; d) Antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent cytophagy (ADCP); e) Antigen presentation; f) Target protein expression, g) or a combination thereof.

[0246] In some embodiments, the adjustment is an increase. 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 approximately 10%-99%, for example, approximately: 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%.

[0247] In some embodiments, the adjustment is a reduction. 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 approximately 10%-99%, for example, approximately: 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 (e.g., immuno-oncology-specific treatments), the effective amount is sufficient to modulate (e.g., increase or decrease) immune cell-related readout, 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), and lymph node homing of immune cells (e.g., dendritic cells and / or T cells). Tumor efflux, differentiation of immune cells, activation of immune cells, polarization of immune cells, cytokine production (e.g., increasing pro-inflammatory cytokines, decreasing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, decreasing anti-inflammatory cytokines), degranulation of immune cells, maturation of immune cells, ADCC of immune cells, ADCP of immune cells, antigen presentation, tumor homing of immune cells (e.g., T cells); increased tumor efflux of immune cells (e.g., regulatory T cells), decreased tumor efflux of immune cells (e.g., CD8+ T cells), target protein expression, or a combination thereof. Immune / Inflammation

[0249] In some embodiments, the methods disclosed herein relate to immunity. In some embodiments, immunity involves 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.

[0250] In some embodiments disclosed herein, the methods relate to the treatment of inflammation. In some embodiments, the inflammation is acute or has a relatively short duration, lasting from minutes to hours. In other embodiments, the inflammation is chronic or has a longer duration, lasting from 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 some embodiments, such conditions include, but are not limited to, skin flushing, pain or tenderness, swelling, fever, fatigue, fever, joint pain or stiffness, canker sores, and rashes. In some embodiments, the inflammation is caused by another disease or disorder.

[0251] 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), gastrointestinal 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).

[0252] In some embodiments, the disease or condition is an inflammatory disease and / or an autoimmune disease. A wide variety of inflammatory and / or autoimmune diseases are treatable according to the methods described herein. In some embodiments, 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 combinations thereof.

[0253] In some embodiments, the target protein activates the immune response. In some embodiments, the target protein inhibits the immune response. In some embodiments, the immune response is an innate immune response (e.g., a humoral and / or cell-mediated immune response). In some embodiments, the immune response is an adaptive immune response (e.g., a humoral and / or cell-mediated immune response). Non-limiting examples of immune responses include T cell-mediated immune responses (e.g., cytokine production and cytotoxicity), B cell-mediated immune responses, humoral immune responses, and activation of cytokine-responsive cells (e.g., macrophages).

[0254] In some embodiments, the target protein enhances signals involved in T cell activation and / or survival. In some embodiments, the target protein activates stimulating checkpoint molecules. Non-limiting examples of stimulating checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, glucocorticoid-induced TNFR family-associated genes (GITR), and inducible T cell costimulators (ICOS). In a particular embodiment, the target protein is an agonist targeting CD28.

[0255] In some embodiments, the target protein reduces signals involved in T cell incompetence and / or exhaustion. In some 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), cytotoxic cell immunoglobulin-like receptor (KIR), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), T cell activation V domain Ig inhibitor (VISTA), sialic acid-binding immunoglobulin lectin 7 (SIGLEC 7), and sialic acid-binding immunoglobulin lectin 9 (SIGLEC 9). In a particular embodiment, the target protein is an inhibitor of PD-1.

[0256] In some embodiments, the effective amount is sufficient to adjust (e.g., increase or decrease): a) Development of high endothelial cell venules (HEV) and / or tertiary lymphoid organs (TLO); b) Immune cell activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) Immune cells efflux from lymph nodes and / or homing; d) Immune cell tumor efflux and / or homing; e) Cytokine production; f) Antigen presentation; g) Target protein expression; or h) Autoantibody levels or a combination thereof.

[0257] In some embodiments, the effective amount is sufficient to increase: a) Development of HEV and / or TLO; b) Immune cell activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) Immune cells efflux from lymph nodes and / or homing; d) Immune cell tumor efflux and / or homing; e) Cytokine production; f) Antigen presentation; g) Target protein expression; or h) Autoantibody levels or a combination thereof.

[0258] 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 approximately 10%-99%, for example, approximately: 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, the effective amount is sufficient to reduce: a) Development of HEV and / or TLO; b) Immune cell activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) Immune cells efflux from lymph nodes and / or homing; d) Immune cell tumor efflux and / or homing; e) Cytokine production; f) Antigen presentation; g) Target protein expression; or h) Autoantibody levels or a combination thereof. 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 approximately 10%-99%, for example, approximately: 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%.

[0260] In some embodiments, the effective amount is sufficient: a) Increase organ function; b) Regulate (e.g., increase or decrease) inflammation; c) Reduce the level of autoantibodies; d) Reduce the rate and / or frequency of relapses and / or outbreaks; e) Reduce viral load; or f) Reduce (e.g., control) infection. Or a combination thereof.

[0261] In some embodiments, the effective amount is sufficient to increase organ function, inflammation, or a combination thereof. 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 approximately 10%-99%, for example, approximately: 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%.

[0262] In some embodiments, the effective amount is sufficient to reduce inflammation, autoantibody levels, the rate and / or frequency of relapses and / or outbreaks, viral load, or infection, or a combination of the foregoing. 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 approximately 10%-99%, for example, approximately: 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75%, or 70%-75%.

[0263] In some embodiments, the target protein activates immune cells. In some embodiments, the target protein inhibits immune cells. Immune cells are cells that play a role in the immune response. Immune cells have a hematopoietic origin and include 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 may be expressed on cancer cells (e.g., metastatic cancer cells), in the tumor microenvironment (e.g., on stromal cells), or on non-malignant cells (e.g., immune cells).

[0264] In some embodiments, the effective amount is sufficient to increase the immune response. 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 approximately 10%-99%, for example, approximately: 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75%, or 70%-75%.

[0265] In some embodiments, the effective amount is sufficient to reduce the immune response. 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 approximately 10%-99%, for example, approximately: 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%.

[0266] In some embodiments, the effective amount is sufficient to reduce the inflammatory response. 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 approximately 10%-99%, for example, approximately: 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75%, or 70%-75%.

[0267] In some embodiments, the effective amount is sufficient to reduce autoimmunity. 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 approximately 10%-99%, for example, approximately: 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75%, or 70%-75%.

[0268] In some embodiments, the effective amount is sufficient to regulate the expression of the target protein in immune cells.

[0269] In some embodiments (e.g., immune and / or inflammatory treatments), an effective amount is sufficient to modulate (e.g., increase or decrease) the migration of immune cells (e.g., antigen-presenting cells such as dendritic cells and / or macrophages) and / or T cells, the proliferation of immune cells, the recruitment of immune cells (e.g., antigen-presenting cells such as dendritic cells and / or macrophages, monocytes, T cells and / or B cells), the lymph node homing of immune cells (e.g., dendritic cells and / or T cells), the lymph node efflux of immune cells (e.g., dendritic cells and / or T cells), the differentiation of immune cells, the activation of immune cells, the polarization of immune cells, the production of cytokines (e.g., increasing pro-inflammatory cytokines, decreasing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, decreasing anti-inflammatory cytokines), the degranulation of immune cells, the maturation of immune cells, antigen presentation, target protein expression, inflammation, autoantibody levels; increase organ function; reduce the rate and / or number of relapses or outbreaks, viral load; control infection, or a combination of the foregoing. aging

[0270] The methods described herein are applicable to the treatment of aging, age-related conditions, and / or age-related disorders or diseases. In some embodiments, the methods described herein include the treatment of general health status, body temperature, weight, height, waist circumference, fertility, body fat, heart rate, blood pressure, pulse rate, blood oxygen level, respiratory rate, respiratory pattern, blood glucose level, blood pH, cardiac output, heart rhythm, and the concentration of certain substances in the blood. In some embodiments, the treatment may be assessed by measuring complete blood cell count, red blood cells, white blood cells, platelets, hemoglobin, hematocrit, mean corpuscular volume, basal metabolic panel, blood glucose, calcium, electrolyte tests, renal function, blood enzyme tests, troponin, creatine kinase, lipoprotein panel, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, coagulation panel, and / or bone marrow tests.

[0271] In some embodiments, the effective amount is sufficient to slow down aging. In some embodiments, the slowdown 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 approximately 10%-99%, for example, approximately: 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75%, or 70%-75%. senescence

[0272] 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 related to photosensitivity or photoaging. In still still other embodiments, the diseases or disorders may be selected from arthritis, Alzheimer's disease, asthma, blindness, cancer, chronic bronchitis, chronic kidney disease, chronic obstructive pulmonary disease, coronary heart disease, deep vein thrombosis, dementia, depression, diabetes, epilepsy, heart failure, high cholesterol, hypertension, motor neuron disease, multiple sclerosis, osteoporosis, Paget's disease, Parkinson's disease, shingles, and stroke.

[0273] In some embodiments, the effective amount is sufficient to reduce aging. 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 approximately 10%-99%, for example, approximately: 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75%, or 70%-75%. fibrosis

[0274] In some embodiments, the methods disclosed herein relate to the treatment of fibrosis. In some embodiments, the methods relate to the treatment of conditions associated with or caused by fibrosis. In some embodiments, 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.

[0275] In some embodiments, the effective amount is sufficient to reduce fibrosis. 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 approximately 10%-99%, for example, approximately: 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75%, or 70%-75%. Infect

[0276] In some embodiments, this disclosure relates to the treatment of an infection. In some aspects, the infection is a bacterial infection, a protozoan infection, a viral infection, a fungal 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, salmonella, Lyme disease, meningococcal disease, influenza, measles, mumps, rubella (e.g., rubella), pneumonia, sexually transmitted diseases (e.g., syphilis, chlamydia, gonorrhea), chronic sinusitis, whooping cough, pertussis, or a combination thereof.

[0277] Unless otherwise defined, all terms, symbols, and other scientific or specialized terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some instances, terms with their commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the commonly understood meaning 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 field and / or as otherwise defined herein.

[0278] The specialized terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0279] As used in this article, the indefinite articles “a”, “an”, and “the” should be understood to include plural indicators unless the context otherwise indicates otherwise.

[0280] Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise" and its variations such as "comprises" and "comprising" shall be understood to mean including, for example, one stated whole or step or a group of wholes or steps, but not excluding any other whole or step or a group of wholes or steps. When used herein, the term "comprise" may be replaced by the terms "containing" or "including".

[0281] As used herein, “consisting of” excludes any element, step, or component not specified in the elements of the claims. When used herein, “consisting substantially of” does not exclude materials or steps that do not substantially affect the essential and novel features of the claims. Whenever used herein in the context of any aspect or embodiment of this disclosure, any of the terms “comprising,” “containing,” “including,” and “having” may be replaced in some embodiments by the terms “consisting of” or “substantially of” to change the scope of this disclosure.

[0282] As used herein, the connecting term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, in the case 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 and second elements together. Any of these options is understood to fall within the meaning described herein and therefore satisfies the requirement of the term "and / or" as used herein. The simultaneous applicability of more than one option is also understood to fall within the meaning described herein and therefore satisfies the requirement of the term "and / or".

[0283] When presenting a list, unless otherwise stated, it should be understood that each individual element of this list and each combination of this list is a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C". Example Example 1: Validation of target protein overexpression as a checkpoint

[0284] This example demonstrates the ability of the disclosed target proteins to act as checkpoint immunomodulators. In this example, human target proteins (including positive controls PD-1 and CTLA-4) cDNA were cloned into the pLVX-TetOne vector (Clontech) under the doxycycline-inducible TRE3G promoter. Human checkpoint ligands PD-L1 and PD-L2 cDNA were cloned into the pLVX-IRES-Puro vector (Clontech) under the constitutive CMV promoter. T cell lines were constructed by infecting Jurkat cells with the lentiviral vector pLVX-TetOne-target protein. B cell lines were constructed by infecting Raji cells with the lentiviral vectors pLVX-IRES-Puro-target protein or pLVX-IRES-Puro-PD-L2. Anti-CD3 beads were prepared internally by coating anti-human CD3 antibodies from eBioscience onto Dynabeads M-450 Epoxy beads from Invitrogen using a coating protocol from Invitrogen.

[0285] On day 1, target protein expression was induced in Jurkat and Raji cell lines by adding doxycycline to a concentration of 250 ng / mL. Two days after doxycycline induction, Jurkat and Raji cells were washed and diluted to 1 x 10⁻⁶ in RPMI + 10% FBS. 6 Cells. Anti-CD3 beads were added to Jurkat cells, and in parallel, control cells (doxycycline negative) were used to assess IL-2 production in unsuppressed T cells. Jurkat cells and beads [and antibody] were incubated together at room temperature for 30 min. 500 μl of Raji cells were added to the Jurkat and beads. The final ratio was 500kJJurkat:500kRaji:2000k beads or 1:1:4. Using a multichannel pipette, 200 μl of the cell:bead mixture was transferred to a 96-well plate (U-bottom). This was incubated at 37°C for 22 h.

[0286] The next day, the plate was centrifuged, and the supernatant was transferred to a new 96-well plate for IL-2 ELISA (R&D Systems, D2050). IL-2 detection was expected to decrease in exhausted T cells. This is how checkpoint target proteins are selected. Example 2. Validation of secretory target proteins that stimulate or inhibit cytokine release. CBA (Cytochemistry) experiments were used to study pro-inflammatory cytokines produced by PBMCs treated with secretory peptides.

[0287] This example demonstrates the validation of the target protein (SEQ ID NO:33885) as an anti-inflammatory immunomodulator suitable for the treatment of autoimmune diseases such as psoriasis, arthritis and multiple sclerosis.

[0288] Peripheral blood mononuclear cells were inoculated with CD3 / CD8 and + / - peptides for 24-48 hours. Cytokines were measured from the supernatant after incubation using a CBA cytometer.

[0289] Materials and Methods:

[0290] Thawing PBMCs: Thaw PBMCs in 20 ml aliquots (one 50 ml Erlenmeyer flask per donor) using pre-warmed TexMACS medium (Miltenyi Biotech). After warming the medium to 37°C, remove the cells from liquid nitrogen and place them in a tissue culture hood. For each donor, add 1 ml of medium from the warmed aliquot to the frozen vial and mix. Then add the thawed cells back into the 50 ml Erlenmeyer flask. Repeat this process until all cells are thawed and added to the 50 ml tube. Continue with other donors (if used). Rotate the cells at 400 x g for 5 min. Aspirate the supernatant and resuspend the cells at approximately 10 x 10^6 cells / ml (see vial for cell count).

[0291] PBMC Plating: Frozen PBMCs from healthy human volunteers (StemCell Technologies) were thawed and plated at 250,000 cells / well. After thawing and resuspending the PBMCs at 10 × 10⁶ cells / ml, the cells were counted (Countess III, Thermo Scientific) and adjusted to 10 × 10⁶ cells / well based on the count. For example, the number of PBMCs determined by the number of wells used in the experiment would require a total of 2.5 × 10⁶ cells or 0.250 ml for 10 wells. 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 / 190 µL of medium. For example, a 10-well plate will require a total of 1.9 ml (190 µL x 10 wells) of medium minus 0.250 ml of cells (1.9 ml - 0.250 ml), or 1.65 ml of medium added to 0.250 ml of cells. Add 190 µL of cells and medium to each 96-well U-plate. Incubate in an incubator until dilution is prepared and ready.

[0292] Peptide diluents: Ideally, all peptides should start at a concentration of at least 10 mM, especially those in DMSO (ATCC), to achieve a final concentration of 1 μM (H2O) or 0.5 μM (DMSO). All test item diluents should be prepared at 20 times the final concentration; for example, a final concentration of 1 μM peptide will need to be prepared at 20 μM.

[0293] Reagent dilution: Prepare three 10 μL / well (30 μL, 20x) dilutions in TexMACS medium. The activator—CD3 / CD28 (TransAct; Miltenyi Biotechnology)—or cytokine will be at 21X and added after 45 minutes of incubation with the peptide. Ensure relevant controls are included—positive: TransAct + cells, negative: mediator + cells. Dilutions should also be prepared in ultra-low binding plates (Corning).

[0294] Assay Procedure: Peptide Treatment: The assay was initiated as described above by thawing the cells and plate them in 96-well U-bottom tissue culture plates (Corning). The cell culture plates were placed in a tissue incubator (5% CO2, 37°C). While the cells were in the incubator, dilutions were prepared as described above (ideally 1 h or less). The cell culture plates were removed from the incubator, and 10 μL of 20X dilution was added to the relevant wells. After adding the inhibitor, the cell culture plates were placed in the tissue incubator for 45 minutes. The plates were removed from the incubator, and 10 μL of 21X TransAct was added to the relevant wells*. The plates were then returned to the incubator and incubated for 24 hours. After 24 hours, the plates were rotated at 400 x g for 5 minutes.

[0295] *Activation: This assay can be performed in two different forms (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 a single peptide.

[0296] Flow Cytometry Bead Array (CBA): After rotation, the supernatant (approximately 150 μL) is transferred to a new ultra-low binding U substrate. The supernatant is then frozen at -20°C until thawed for use in CBA (BD) to determine the levels of human IL-2, TNFα, IL-1β, IP-10, IL-10, and IFNg. CBA is performed according to the manufacturer's protocol.

[0297] SEQ ID NO:33885 significantly blocked the release of TNF-α from activated PBMCs, but did not block the release of another inflammatory cytokine, IL-1β. Specific inhibition of TNF-α release would reduce the inflammatory response. Figure 1 and Figure 2). Sprout Table: X = Sprouts exhibiting significant activation or inhibition of related cytokine release. Inhibition occurred when the peptide was applied in combination with an anti-CD3 / CD28 T cell activator. Activation occurred when the peptide was applied to PBMCs alone (see Materials and Methods). Significance = P = 0.05 Example 3. Validation of target proteins as regulators of innate immune responses.

[0298] This example demonstrates the validation of the target protein (SEQ ID NO:37413) as a regulator of the innate immune response. Specifically, the target protein inhibits the response of various Toll-like receptors to their specific agonists. Therefore, the target protein is suitable for treating autoimmune diseases such as lupus, psoriasis, arthritis, and multiple sclerosis. As a Toll-like receptor (TLR) inhibitor, SEQ ID NO:37413 could be particularly useful for the treatment of lupus.

[0299] To determine the effect of the target protein on TLR activity, the target protein, unrelated proteins, and controls (+ / -) of previously used TLR agonists were applied to commercial reporter cell lines to monitor two major signal transduction pathways in response to TLR activation. To determine which immune cells were most responsive to the target protein, the target protein was applied to peripheral blood mononuclear cells (PBMCs), and then the PBMCs were subjected to single-cell RNA-seq using cell hashing 24 hours later.

[0300] Methods and Materials:

[0301] Dual reporter assay: From the example: NF-κB-SEAP and IRF-Lucia luciferase reporter monocytes (THP1) (InvivoGen) 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, followed by treatment with the target protein (final concentration 10 μM), or left untreated.

[0302] After treatment, THP1 cells were cultured for 24 hours, and then reporter activity was measured. Both reporter proteins were measurable in the cell culture supernatant when using QUANTI-Blue (Avivegan) (SEAP assay) and QUANTI-Luc (Avivegan) (luciferase assay). QUANTI-Blue is a colorimetric enzyme assay developed to determine the activity of any alkaline phosphatase in biological samples, such as cell culture supernatant. QUANTI-Luc is a lyophilized assay containing all the components necessary for the quantitative measurement of the activity of Lucia luciferase and other luciferases utilizing coelenterates. An increase in reporter activity was observed after treatment with the target protein in cells where the target protein is immunostimulatory. Conversely, a decrease in reporter activity was observed after treatment with the target protein in cells where the target protein is immunosuppressive.

[0303] scRNA-seq: From the example: Assessment of changes in cell population after treatment with target protein by single-cell RNA-seq (scRNA-seq). The target protein (e.g., listed in the sequence listing) was synthesized using solid-phase peptide synthesis (SPPS) via fluorenylmethoxycarbonyl (Fmoc) protecting group chemistry. Human primary blood mononuclear cells were seeded at 1 x 10^6 PBMCs in each well of a 12-well plate containing 1 ml RPMI-1640 (10% FBS). The PBMCs were treated with the target protein at a final concentration of 1 μg / mL LPS (Sigma-Aldrich) plus or minus 10 μM for 24 h, or left untreated. 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 in 1 ml of cell staining buffer (BioLegend). 5 μl of human TruStain FcX (BioLegend) was added to the cells, and the cells were incubated at 4 °C for 10 min. Add 1 μg of single-cell smear antibody (TotalSeq, Baijin) along with approximately 50 μl of cell staining buffer to the cells to obtain a total volume of 100 μl. Incubate this at 4°C for 30 min, then wash three times with 400 x g. Combine 10 different cell smear samples into a tube with the desired cell count (approximately 1000 cells / μl).

[0304] Single cells were processed using the Chromium Next GEM Single Cell 3'_ Kit (Dual Indexed) (10X Genomics CG000317Rev C) according to the protocol provided by 10X Genomics. In short, the sample was processed through GEM generation and barcoding, post-GEM-RT cleanup and cDNA amplification, 3' gene expression library construction, cell surface protein library construction, and final sequencing.

[0305] After sequencing, reads were aligned to the human reference genome (GENCODE34 / GRCH38). Reads were demultiplexed using DNA barcoding antibodies and ran through quality controls (e.g., removal of double cells, selection of single cells with less than 15% mitochondrial contamination at the RNA level, and reads containing more than 500 genes). Raw data were normalized. Principal component analysis was performed, and cell clusters were annotated. Differential gene expression analysis was performed between controls and those treated with the target protein. Changes in cell population dynamics and / or gene expression observed via principal component analysis after treatment with the target protein in the supernatant validated that the target protein acts as a circulating factor.

[0306] SEQ ID NO:37413 significantly blocks IRF signaling pathway responses from several TLRs. Upregulation of the IRF pathway is associated with many autoimmune diseases, particularly lupus. Consistent with its association with innate immune-related diseases (like lupus), SEQ ID NO:37413 strongly affects active monocytes and dendritic cells, while having a relatively small effect on T cells, B cells, and NK cells. Figure 3 , Figure 4 , Figure 5A , Figure 5B ). ORFID Relative activity SEQ ID NO:33341 +++ Example 4.

[0307] Inhibiting NK cell function by blocking the function of co-stimulatory ligands and cytokines is a promising therapeutic strategy for treating alloimmune responses and graft rejection. This example demonstrates validation of target proteins (SEQ ID NO:33482, SEQ ID NO:37776, and SEQ ID NO:35050) as novel co-stimulatory ligands that enhance NK-mediated cytotoxicity in human B-cell lymphoma.

[0308] We used a CRISPR system to generate a pooled KO library containing 4,708 novel proteins and 998 known proteins within 2,127 lncRNAs in Raji cells (human B-cell lymphoma). To understand the roles of these proteins in regulating NK cell cytotoxicity, we performed a cell-killing assay using primary human NK cells. We examined changes in the initial library representation in Raji cells using the library representation after 24 hours of co-incubation with NK cells. We used known NK cell cytotoxicity modulators as references to establish the significance of the spurs. The novel co-stimulators (enhancers) of NK-mediated cytotoxicity contained targets that significantly inhibited Raji cell killing after CRISPR KO, similar to known controls—ICAM1 and FAS.

[0309] Materials and Methods:

[0310] CRISPR KO generation. CRISPR guide RNA libraries have been designed at ProFound Therapeutics to target 4,708 different predicted novel proteins located within 2,127 lncRNAs, 998 known proteins (including positive controls for NK cell function (inhibitors, such as B2M, TAP1, TAP2, HLA-DM) and negative controls (co-stimulators, such as ICAM1, FAS, IL7, DGKE) and 900 neutral controls (non-targeted and intergenic guide RNAs)). The CRISPR guide RNA libraries were packaged into lentiviruses, transduced into the Raji Cas9 line at an MOI of 0.3, and selected using puromycin selection, as described in [Sanjana NE, Shalem O, Zhang F. Nat. Methods. [Nature Methods] 2014 Aug; 11(8):783-4].

[0311] NK cell isolation and expansion. Primary human NK cells were isolated from frozen PBMCs using the EasySep Human NK Cell Isolation Kit (StemCell, catalog 17955). Primary human NK cells were expanded according to the manufacturer's protocol using the ImmunoCult NK Cell Expansion Kit (StemCell, catalog 100-0711).

[0312] Screening setup. Screening was performed using four biological replicates. NK cell cytotoxicity was assessed by co-culturing 20 million Raji CRISPR KO library cells with 60 million primary NK cells in T75 tissue culture flasks for 24 hours. NK cells were removed using a positive CD56 selection kit (Stem Cells, catalog 17855). Dead cells were removed using an EasySep dead cell removal (annexin V) kit (Stem Cells, catalog 17899). The remaining Raji CRISPR KO library cells were processed to extract genomic DNA. NGS library preparation and Illumina sequencing were performed on these cells and Raji cells prior to exposure to NK cells, as described in [Sanjana NE, Shalem O, Zhang F. Nat. Methods. [Nature Methods] 2014 Aug; 11(8):783-4].

[0313] Data analysis. Screening and interpretation were performed using the ProFound Tx custom data analysis tool and confirmed with MAGeCK-VISPR, as described in [Li W, Koster J, Xu H, Chen CH, Xiao T, Brown M, Liu S. Genome Biology 2015(281)].

[0314] We found that several lncRNA-encoded proteins inhibited Raji cell killing in the presence of primary NK cells after loss of function. The effect size was comparable to that of control proteins, which are known modulators of NK cell function, such as components of ICAM1 and FAS. SEQ ID NO:33482, SEQ ID NO:37776, and SEQ ID NO:35050 are examples of novel proteins with strong phenotypes, suggesting their role in enhancing NK cell cytotoxicity and predicted cell surface localization. Another novel protein with validated co-stimulatory activity against NK cell cytotoxicity and predicted cell surface localization:

[0315] SEQ ID NO:34013, SEQ ID NO:34021, SEQ ID NO:33482, SEQ ID NO:35972, SEQ ID NO:36456, SEQ ID NO:37776, SEQ ID NO:33345, SEQ ID NO:35531, SEQ ID NO:36975, SEQ ID NO:37561, SEQ ID NO:35050、SEQ ID NO:34728、SEQ ID NO:34076 Novel proteins with validated co-stimulatory effects on NK cell cytotoxicity and predicted secretion:

[0316] SEQ ID NO:37151, SEQ ID NO:37187, SEQ ID NO:37199, SEQ ID NO:38297, SEQ ID NO:34018, SEQ ID NO:37016, SEQ ID NO:33395, SEQ ID NO:33372, SEQ ID NO:38018, SEQ ID NO:36168, SEQ ID NO:34490, SEQ ID NO:35219, SEQ ID NO:38378, SEQ ID NO:37411, SEQ ID NO:37782, SEQ ID NO:36984, SEQ ID NO:37938, SEQ ID NO:34395, SEQ ID NO:34586, SEQ ID NO:34589, SEQ ID NO:35295, SEQ ID NO:36972, SEQ ID NO:37564, SEQ ID NO:37158, SEQ ID NO:38247, SEQ ID NO:38249, SEQ ID NO:37863, SEQ ID NO:33904, SEQ ID NO:38163, SEQ ID NO:38189, SEQ ID NO:38195, SEQ ID NO:38196, SEQ ID NO:38206 Example 5.

[0317] The impact of NK cell dysfunction on the tumor microenvironment is becoming increasingly recognized, for example, in cancer patients refractory to T-cell checkpoint therapy. The identification of novel inhibitory ligands upregulated on tumor cells to induce NK cell dysfunction offers promising therapeutic opportunities, through which antagonists of these inhibitory ligands (such as blocking antibodies) can reactivate NK cell cytotoxicity and promote cancer cell killing.

[0318] This example demonstrates the validation of target proteins (SEQ ID NO:2013, SEQ ID NO:35586 and SEQ ID NO:36545) as novel inhibitory ligands for inhibiting NK-mediated cytotoxicity in human B-cell lymphoma.

[0319] We used a CRISPR system to generate a pooled KO library containing 4,708 novel proteins and 998 known proteins within 2,127 lncRNAs in Raji cells (human B-cell lymphoma). To understand the roles of these proteins in regulating NK cell cytotoxicity, we performed a cell-killing assay using primary human NK cells. We examined changes in the initial library representation in Raji cells using the library representation after 24 hours of co-incubation with NK cells. We used known NK cell cytotoxicity modulators as references to establish the significance of the spurs. The novel inhibitors of NK-mediated cytotoxicity contained targets that significantly enhanced Raji cell killing after CRISPR KO, similar to known controls—B2M and TAP1.

[0320] Materials and Methods:

[0321] CRISPR KO generation. CRISPR guide RNA libraries have been designed by Bofang Therapeutics to target 4,708 different predicted novel proteins located within 2,127 lncRNAs, 998 known proteins (including positive controls for NK cell function (inhibitors, such as B2M, TAP1, TAP2, HLA-DM) and negative controls (co-stimulators, such as ICAM1, FAS, IL7, DGKE) and 900 neutral controls (non-targeted and intergenic guide RNAs)). The CRISPR guide RNA libraries were packaged into lentiviruses, transduced into the Raji Cas9 line at an MOI of 0.3, and selected using puromycin selection, as described in [Sanjana NE, Shalem O, Zhang F. Nat. Methods. [Nature Methods] 2014 Aug; 11(8):783-4].

[0322] NK cell isolation and expansion. Primary human NK cells were isolated from frozen PBMCs using the EasySep Human NK Cell Isolation Kit (Stem Cell Corporation, Catalog No. 17955). Primary human NK cells were expanded according to the manufacturer's protocol using the ImmunoCult NK Cell Expansion Kit (Stem Cell Corporation, Catalog No. 100-0711).

[0323] Screening setup. Screening was performed using four biological replicates. NK cell cytotoxicity was assessed by co-culturing 20 million Raji CRISPR KO library cells with 60 million primary NK cells in T75 tissue culture flasks for 24 hours. NK cells were removed using a positive CD56 selection kit (Stem Cells, catalog 17855). Dead cells were removed using an EasySep dead cell removal (annexin V) kit (Stem Cells, catalog 17899). The remaining Raji CRISPR KO library cells were processed to extract genomic DNA. NGS library preparation and Illumina sequencing were performed on these cells and Raji cells prior to exposure to NK cells, as described in [Sanjana NE, Shalem O, Zhang F. Nat. Methods. [Nature Methods] 2014 Aug; 11(8):783-4].

[0324] Data analysis. Screening and interpretation were performed using the ProFound Tx custom data analysis tool and confirmed with MAGeCK-VISPR, as described in [Li W, Koster J, Xu H, Chen CH, Xiao T, Brown M, Liu S. Genome Biology 2015(281)].

[0325] We found that several lncRNA-encoded proteins enhanced Raji cell killing in the presence of primary NK cells after loss of function. The effect sizes of these proteins were comparable to those of control proteins, which are known regulators of NK cell function, such as MHC components (B2M, TAP1, TAP2). SEQ ID NO:2013, SEQ ID NO:35586, and SEQ ID NO:36545 are examples of novel proteins with strong phenotypes and predicted cell surface localization. Figure 8 and Figure 9 ). Another novel protein with validated inhibitory activity against NK cell cytotoxicity and predicted cell surface localization:

[0326] SEQ ID NO:35361, SEQ ID NO:33359, SEQ ID NO:33807, SEQ ID NO:33817, SEQ ID NO:35236, SEQ ID NO:36386, SEQ ID NO:37561 A novel protein with proven inhibitory activity against NK cell cytotoxicity and predicted secretion: SEQ ID NO:38261, SEQ ID NO:33454, SEQ ID NO:33510, SEQ ID NO:33225, SEQ ID NO:33247, SEQ ID NO:36705, SEQ ID NO:38128, SEQ ID NO:35759, SEQ ID NO:36769, SEQ ID NO:36771, SEQ ID NO:34633, SEQ ID NO:36461, SEQ ID NO:37622, ​​SEQ ID NO:37640, SEQ ID NO:38369, SEQ ID NO:37377, SEQ ID NO:37958, SEQ ID NO:35900, SEQ ID NO:37565, SEQ ID NO:37099, SEQ ID NO:33521, SEQ ID NO:33548, SEQ ID NO:33683, SEQ ID NO:33704, SEQ ID NO:34374、SEQ ID NO:35661、SEQ ID NO:37816

[0327] All teachings of patents, published applications, and references cited herein are incorporated in their entirety by reference.

[0328] While exemplary embodiments have been specifically shown and described, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the embodiments covered 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, 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 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 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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