Compositions and Methods for Modulating the Immune Response

Abstract

The present disclosure provides compositions such as polypeptides, polynucleotides, gene editing systems, small molecules, vectors, or host cells that include and / or modulate the expression or activity of immunoregulatory-related proteins in various embodiments. The present disclosure also provides, in various embodiments, methods of treating aging, senescence, fibrosis, autoimmunity, cancer, infectious diseases, and / or immune disorders using agents that include and / or modulate the expression or activity of immunoregulatory-related proteins, as well as methods of identifying said agents.

Description

Technical Field

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

[0002] Incorporation by reference of materials into XML This application incorporates by reference the Sequence Listing contained in the following Extensible Markup Language (XML) file, which is filed concurrently with this specification. a) File name: 57081055002.xml; created on May 23, 2023, size 40,525,913 bytes.

Background Art

[0003] The main function of the immune system is to mediate defensive immunity against pathogens such as viruses, bacteria, fungi, parasites, and tumors. Various immune deficiency mechanisms are often enhanced between cancer and chronic infections. An insufficient immune response can lead to immunodeficiency, susceptibility to infection, and cancer (e.g., tumor) progression. However, without tight regulation, the immune system can also promote acute and chronic inflammation and lead to immunopathology or autoimmunity. Therefore, it is extremely important to identify additional novel therapeutic targets for the regulation of immune cells.

Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

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

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

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

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

[0009] In another aspect, the present disclosure provides a method for detecting a disease or condition in a subject or detecting the likelihood of developing a disease or condition in a subject, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject, wherein the level of expression or activity of the target protein in the sample indicates the likelihood of developing a disease or condition in the subject, and the disease or condition is selected from aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), infection, immune disease (e.g., inflammation and / or autoimmune disease), or a combination thereof.

[0010] In another aspect, the present disclosure provides a method for preparing a sample useful for determining the likelihood of developing a disease or condition in a subject, the method comprising: a) obtaining or having obtained a sample from the subject; b) adding a protease inhibitor, a control peptide, a standard peptide, or a combination thereof to the sample to prepare a sample useful for detecting the likelihood of developing cancer; and c) quantifying the expression or activity of a target protein in the sample prepared in step b). The method 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.

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

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

[0013] In another aspect, the present disclosure provides a method of selecting a subject suitable for treatment of a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject and selecting a subject suitable for treatment of the disease or condition according to the level of the expression or activity of the target protein in the sample, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), infectious disease, immune disease (e.g., inflammation and / or autoimmune disease), or a combination thereof.

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

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

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

[0017]

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Mode for Carrying Out the Invention

[0018] The description of exemplary embodiments continues below.

[0019] Target Protein In one aspect, the present disclosure provides a target protein identified herein (e.g., a target protein comprising an amino acid sequence 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 protein identified herein” and “target protein of the present disclosure” include the polypeptides disclosed in the sequence listing. The target protein can be produced recombinantly (e.g., via DNA or mRNA) or synthetically.

[0020] In some embodiments, the target protein is an extracellular protein (e.g., a secreted protein, such as a ligand). In certain 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 the membrane but not transmembrane.

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

[0022] Some of the target proteins in the Sequence Listing have been identified as being differentially expressed (e.g., upregulated or downregulated) in a disease and / or condition selected from aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), infection, immune disease (e.g., inflammation and / or autoimmune disease), or a combination thereof, such that modulation of the level and / or activity of the target protein acts to treat, ameliorate, and / or prevent the onset of the disease or condition, as compared to a reference state (e.g., a normal state).

[0023] As used herein, the term "differentially expressed" refers to at least one recognizable difference in protein expression. It can be a quantitatively measurable, semi-quantitatively estimable, or qualitatively detectable difference in protein expression. Thus, a protein that is differentially expressed, i.e., a "DEP", can have a higher expression level in a reference state (e.g., a normal state) than in a disease state in which the DEP has a lower expression level or is not expressed at all. Conversely, a DEP can have a higher expression level in a disease state than in a reference state (e.g., a normal state) in which the DEP has a lower expression level or is not expressed at all. Further, expression can be considered differential if the DEP changes recognizably (e.g., mutates) between the two states being compared. Recognizable changes can include amino acid substitutions, insertions, and / or deletions, including N-terminal and C-terminal truncations, as well as modifications (e.g., post-translational modifications).

[0024] As used herein, the term "reference" refers to a standard used for comparison purposes. One of ordinary skill in the art can select an appropriate reference for a particular comparison purpose. Thus, for example, a reference for a disease state may be a normal healthy state; a reference for a mutant protein may be a non-mutant protein; a reference for disease treatment may be no treatment or a standard treatment. In some embodiments, particularly those including methods for identifying an agent that modulates the expression and / or activity of a target protein, the reference is the activity and / or expression of the target protein in the absence of the agent. In some embodiments, the reference is based on a predetermined level, such as functional expression or an empirical assay. In some embodiments, the reference is obtained from one cell, sample or subject (e.g., a healthy subject, a cell or sample from a subject without a particular disease; a healthy subject, a subject without a particular disease). In some embodiments, the reference is obtained from two or more cells, samples or subjects (e.g., a population thereof) (e.g., a healthy subject, a cell or sample from a subject without a particular disease; a healthy subject, a subject without a particular disease), such as 2, 3, 4, 5, 10, 20, 30, 50, 100 or more, or a statistically significant number of cells, samples or healthy subjects. A reference obtained from two or more cells, samples or subjects can be expressed as a statistic (e.g., an average or median).

[0025] In some embodiments, the protein is used as a marker for an immune and / or disease state, such as exhausted T cells, CD4 + T cells (e.g., Th1, Th2, Th17), CD8 + T cells, or a combination thereof.

[0026] In certain embodiments, the target protein has a higher expression level in activated immune cells. In some embodiments, the target protein has an expression level in activated immune cells that is at least about 0.5-fold higher, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher (e.g., 50-fold higher, 100-fold higher) than the target protein expression level in a reference (e.g., the same type of non-activated immune cells).

[0027] In certain embodiments, the target protein has a lower expression level in activated immune cells. In some embodiments, the target protein has an expression level in activated immune cells that is at least about 0.5-fold lower, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) than the target protein expression level in a reference (e.g., the same type of non-activated immune cells).

[0028] In some embodiments, the target protein has an expression level that is at least about 0.5-fold higher, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher (e.g., 50-fold higher, 100-fold higher) in a disease (e.g., determined from a sample from a cell or tissue of a subject having the disease) than the target protein expression level in a reference (e.g., a sample from a cell or tissue of a subject without the disease).

[0029] In some embodiments, the target protein has an expression level in a disease (e.g., determined from a sample comprising or obtained from a cell or tissue of a subject having the disease) that is at least about 0.5-fold lower than the target protein expression level in a reference (e.g., a sample from a cell or tissue of a subject without the disease), such as at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold lower (e.g., 50-fold lower, 100-fold lower). In some embodiments, the target protein is not expressed or is expressed at undetectable levels in the disease (e.g., when determined from a sample comprising or obtained from a cell or tissue of a subject having the disease).

[0030] In some embodiments, the target protein has a transcript level in a disease (e.g., determined from a sample from a cell or tissue of a subject having the disease) that is at least about 0.5-fold higher than the target protein transcript level in a reference (e.g., a sample from a cell or tissue of a subject without the disease), such as at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher (e.g., 50-fold higher, 100-fold higher). In certain embodiments, the increase in the transcription level of the target protein contributes to (e.g., results in) the disease or condition described herein.

[0031] In some embodiments, the target protein has a transcript level that is at least about 0.5-fold lower, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) in a disease (e.g., as determined from a sample comprising or obtained from the cells or tissue of a subject having the disease) than the target protein transcript level in a reference (e.g., a sample from cells or tissue of a subject without the disease). In some embodiments, the transcription of the target protein is not expressed or is expressed at undetectable levels in a disease (e.g., as determined from a sample comprising or obtained from the cells or tissue of a subject having the disease). In certain embodiments, the decrease in the transcription level of the target protein contributes to (e.g., results in) the disease or condition described herein.

[0032] In certain embodiments, the gene encoding the target protein comprises at least one mutation (e.g., fusion of amino acid repeats, deletion, insertion, point mutation, and / or expansion) in the disease described herein.

[0033] Non-limiting examples of (biological) samples include blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., biopsy or micro-biopsy), pancreatic juice, chorionic villus samples, and cells isolated from a subject, among others.

[0034] In some embodiments, the target protein is translated from a non-coding RNA. In some embodiments, the non-coding RNA is a long intergenic non-coding RNA (lincRNA). In certain embodiments, the non-coding RNA is a long non-coding RNA (lncRNA). In some embodiments, the non-coding RNA is a microRNA (miRNA or miR).

[0035] In some embodiments, the target protein is translated from non-exon elements in unprocessed precursor mRNA (pre-mRNA). In some embodiments, the non-exon element is an intron in the pre-mRNA. In some embodiments, the non-exon element is the 5' untranslated region (5'-UTR) in the pre-mRNA. In some embodiments, the non-exon element is the 3' untranslated region (3'-UTR) in the pre-mRNA.

[0036] In some embodiments, the target protein has a length of 2,000 amino acids or less, such as 1000 amino acids or less, 750 amino acids or less, 500 amino acids or less, 250 amino acids or less, 150 amino acids or less, or 100 amino acids or less. In some embodiments, the target protein has a length of 7 amino acids or more, such as 8, 9, 10, 15, 18, 25, 50, 75, or 100 amino acids or more. In certain embodiments, the target protein has a length of about 50 to about 200 amino acids, such as about 100 to about 150 amino acids. In certain embodiments, the target protein has a length of 7 amino acids or more. In more certain embodiments, the target protein has a length of about 18 amino acids.

[0037] In some embodiments, the target protein of the present disclosure is a modulator of one or more GPCRs. In some embodiments, the target protein is an agonist of one or more GPCRs. In some embodiments, the target protein is an antagonist of one or more GPCRs. In some embodiments, the target protein is a direct modulator of one or more GPCRs, such as a ligand of one or more GPCRs. In some embodiments, the target protein is an indirect modulator of one or more GPCRs.

[0038] The expression and / or activity of various GPCRs are associated with various diseases / disorders, conditions, and indications, including those shown in Table A (e.g., see Kenakin, T., Biased Receptor Signaling in Drug Discovery, Pharmacol Rev 71:267-315, April 2019; Harmar, A.J., et al., IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels, Nucleic Acids Research, 2009, Vol. 37; and Davenport AP, Scully CCG, de Graaf C, Brown AJH, and Maguire JJ. Advances in therapeutic peptides targeting G protein-coupled receptors. Nat Rev Drug Discov. 2020 Jun. 19(6):389-413; the entire contents of each are hereby incorporated by reference in their entirety). Accordingly, in some embodiments, the target proteins disclosed herein that are modulators of GPCRs are useful for treating and / or diagnosing one or more diseases / disorders, conditions, and / or indications known to be associated with GPCR expression and / or activity, such as cancer or precancerous conditions, or any of the diseases / disorders, conditions, and indications listed in Table A.

[0039]

Table A-1

[0040]

Table A-2

[0041]

Table A-3

[0042]

Table A-4

[0043]

Table A-5

[0044] Agents that regulate the target protein In the present specification, agents are provided that regulate the expression of the target proteins disclosed herein, such as the target proteins in the Sequence Listing, their variants or fragments (e.g., biologically active fragments of the target proteins). The expression of the target protein or its variant or fragment can be regulated by a wide range of processes that directly or indirectly result in an increase or decrease in the target protein level. Non-limiting examples include changes in the copy number of the gene encoding the target protein, transcription initiation, elongation or termination, RNA processing, RNA stability (e.g., mRNA stability), RNA degradation, translation initiation, post-translational modification of the protein, protein stability, proteolysis (e.g., cleavage such as protease cleavage), or combinations thereof.

[0045] In some embodiments, the agent regulates (e.g., increases or decreases) the expression of the gene or gene transcript encoding the target protein. In some embodiments, the agent regulates the expression or activity of the target protein. In some embodiments, the agent decreases (e.g., inhibits, reduces or neutralizes) the activity of the target protein. In some embodiments, the agent increases (e.g., activates) the activity of the target protein. In some aspects, the agent decreases (e.g., inhibits or down-regulates) the expression of the target protein. In other embodiments, the agent increases (e.g., activates or up-regulates) the expression of the target protein.

[0046] As used herein, the term "increasing" or "increase" refers to a modulation that results in a higher level of expression, activity, function, or a combination or metric thereof (e.g., cancer cell death or DNA methylation at a target site) of a target protein as compared to a reference (e.g., the level prior to or in the absence of modulation by an agent). In some embodiments, the agent increases the expression or activity or metric of the target protein by at least about 5% relative to the reference, e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to the reference.

[0047] As used herein, the term "decreasing" or "decrease" refers to a modulation that results in a lower level of expression, activity, function, or a combination or metric thereof (e.g., cancer cell death or DNA methylation at a target site) of a target protein as compared to a reference (e.g., the level prior to or in the absence of modulation by an agent). In some embodiments, the agent decreases the expression or activity or metric of the target protein by at least about 5% relative to the reference, e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to the reference.

[0048] Non-limiting examples of metrics include energy production or conversion in the liver (e.g., regulation of ATP synthesis, β-oxidation, oxidation of metabolites derived from glycolysis, oxidation of metabolites derived from amino acids), mitochondrial transcription, mitochondrial ribosome assembly, mitochondrial translation, mitochondrial thermogenesis, hormonal signaling (e.g., mitochondrial estrogen receptor (mtER) signaling), redox maintenance (e.g., NADH and / or FADH 2) Cell cycle regulation, cell migration, cell morphology, apoptosis, necrosis, membrane potential, ion (e.g., calcium or zinc) storage, ion (e.g., calcium or zinc) homeostasis, metabolite synthesis (e.g., heme biosynthesis or steroid biosynthesis), nutrient sensing, endoplasmic reticulum stress response pathway, signal transduction processes (e.g., calcium signaling) are included.

[0049] In some embodiments, the level of expression, activity, function or combination thereof, or metric of the target protein is measured, for example, after initiating a treatment regimen, after contacting the agent (e.g., with cells) or administering (e.g., to a subject) the agent 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.

[0050] In some embodiments, the agent comprises, consists essentially of, or consists of a polypeptide, polynucleotide, gene editing system, small molecule, or cell (e.g., cell therapy).

[0051] In some embodiments, the target protein activates immune cells and the agent modulates (e.g., increases or decreases) the level of expression, activity, function or combination thereof of the target protein. In some embodiments, the target protein inhibits immune cells (e.g., inhibits activation of immune cells, induces immune cell death (e.g., apoptosis), or a combination thereof), and the agent modulates (e.g., increases or decreases) the level of expression, activity, function or combination thereof of the target protein.

[0052] In certain embodiments, the agent modulates (e.g., increases or decreases) the expression, activity, function, or combinations thereof of a target protein in cancer cells (e.g., metastatic cancer cells), cells in the tumor microenvironment (e.g., stromal cells), target cells of an inflammatory response (e.g., epithelial cells, endothelial cells, stem cells or non-immune cells), immune cells (e.g., effector T cells, helper T cells, Th1 cells, Th2 cells, Th17 cells, B cells, natural killer (NK) cells, innate lymphoid cells (e.g., ILC1 cells, ILC2 cells, ILC3 cells), macrophages (e.g., M1 macrophages, M2 macrophages), monocytes, and / or antigen-presenting cells (e.g., dendritic cells), or combinations thereof. In certain embodiments, the agent modulates the expression, activity, function, or combinations thereof of a target protein in a tumor, tumor microenvironment, metastatic site, lymph node, spleen, secondary lymphoid organ, tertiary lymphoid organ, barrier tissue, skin, intestine, airway, wound, other immune tissue, non-immune tissue, or combinations thereof.

[0053] In some embodiments, the agent modulates (e.g., increases or decreases) inflammation, decreases the level of autoantibodies, increases organ function, decreases the rate or number of relapses or recurrences, decreases the viral load, controls infection, or combinations thereof.

[0054] In some embodiments, the agent induces downregulation of the target protein (e.g., increases target protein degradation); prevents multimerization (e.g., dimerization) of the target protein; sequesters the target protein (e.g., secreted target protein); modulates (e.g., agonizes, antagonizes, or disrupts) a known function of the target protein; decreases the binding between the target protein and a binding partner (e.g., via steric hindrance); modulates (e.g., increases or decreases) downstream cell signaling; induces phagocytosis of cells expressing the target protein; or combinations thereof. In certain embodiments, the agent lacks agonist activity against the target protein. In certain embodiments, the agent has agonist activity against the target protein. In some embodiments, the agent lacks antagonist activity against the target protein. In some embodiments, the agent has antagonist activity against the target protein. In certain embodiments, the agent binds to at least one residue of the target protein involved in binding to a binding partner. In some embodiments, the agent binds to one or more binding sites and / or domains of the target protein involved in binding of the target protein to a binding partner.

[0055] Non-limiting examples of binding partners include cytokine receptors and immune signaling receptors.

[0056] In some embodiments, the agent induces downregulation of the binding partner of the target protein; sequesters the binding partner of the target protein (e.g., secreted binding partner); prevents multimerization (e.g., dimerization) of the binding partner of the target protein; sequesters the binding partner of the target protein (e.g., secreted binding partner); modulates (e.g., agonizes, 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 performs a combination of the foregoing. In certain embodiments, the agent lacks agonist activity against the binding partner of the target protein. In certain embodiments, the agent has agonist activity against the binding partner of the target protein. In some embodiments, the agent lacks antagonist activity against the binding partner of the target protein. In some embodiments, the agent has antagonist activity against the binding partner of the target protein. In certain embodiments, the agent further binds to at least one residue of the binding partner of the target protein involved in the binding between the target protein and the binding partner. In more specific embodiments, the agent further binds to one or more binding sites and / or domains of the binding partner of the target protein involved in the binding between the target protein and the binding partner.

[0057] In some embodiments, the agent modulates (e.g., activates or inhibits) immune signaling, cytokine signaling, inflammatory signaling, or a combination of the foregoing.

[0058] In some embodiments, the agent enhances signals involved in T cell activation and / or survival. In certain embodiments, the agent activates stimulatory checkpoint molecules. Non-limiting examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, GITR, inducible T cell co-stimulator (ICOS). In certain embodiments, the agent is an agonist for CD28.

[0059] In some embodiments, the agent reduces signals involved in T cell anergy and / or exhaustion. In certain embodiments, the agent inhibits inhibitory checkpoint molecules. Non-limiting examples of inhibitory checkpoint molecules include PD-1, PD-L1, PD-L2, TIM-3, LAG-3, CTLA-4, A2AR, CD276, B7-H4, BTLA, IDO, KIR, NOX2, VISTA, SIGLEC 7, and SIGLEC 9. In certain embodiments, the agent is an inhibitor (e.g., a blocking antibody) against PD-1.

[0060] In some embodiments, the agent further modulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of checkpoint proteins other than the target protein.

[0061] In certain embodiments, the agent modulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of a variant of the target protein disclosed herein. In some embodiments, the variant comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the target protein disclosed herein. For example, the sequence identity to the variant can be at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the sequence identity is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the sequence identity is about 70-99%, 75-99%, 75-95%, 80-99%, 80-98%, 80-95%, 80-90%, 85-98%, 85-97%, 85-90%, 90-97%, 90-96%, 90-85%, 90-80%, or 95-99%. In some embodiments, the variant comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to the amino acid sequence of the target protein disclosed herein.

[0062] As used herein, the term "sequence identity" refers to the degree to which two nucleotide sequences or two amino acid sequences have the same residue at the same position when the sequences are aligned to achieve the maximum level of identity expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as the reference sequence and is compared to the test sequence. The sequence identity between the reference sequence and the test sequence is expressed as the percentage of positions over the full length of the reference sequence at which the reference sequence and the test sequence share the same nucleotide or amino acid to achieve the maximum level of identity when the reference sequence and the test sequence are aligned. As an example, when, upon alignment to achieve the maximum level of identity, the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the full length of the reference sequence, the two sequences are considered to have 70% sequence identity.

[0063] The alignment of the comparison arrays to achieve the maximum level of identity can be readily performed by one of ordinary skill in the art using an appropriate alignment method or algorithm. In some cases, the alignment may include gaps introduced to provide the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (generally, see Ausubel et al., Current Protocols in Molecular Biology).

[0064] When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, coordinates are then specified as necessary, and the sequence algorithm program parameters are specified. Next, the sequence comparison algorithm calculates the percent sequence identity of the test sequence to the reference sequence based on the specified program parameters. A commonly used tool for determining percent sequence identity is the Protein Basic Local Alignment Search Tool (BLASTP) available from the National Center for Biotechnology Information of the National Library of Medicine of the National Institutes of Health. (Altschul et al., 1990).

[0065] In some embodiments, the amino acid sequence of a variant of the target polypeptide disclosed herein comprises at least one amino acid substitution relative to the amino acid sequence of the target protein. In some embodiments, the number of amino acid substitutions in the variant relative to the amino acid sequence of the target protein disclosed herein is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, the number of amino acid substitutions is at least about 5, 6, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60. In some embodiments, the number of amino acid substitutions is at most about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 6, or 5. In some embodiments, the number of amino acid substitutions is about 1-60, 1-55, 2-55, 2-50, 3-50, 3-45, 4-45, 4-40, 5-40, 5-35, 6-35, 6-30, 7-30, 7-25, 8-25, 8-20, 9-20, 9-15, 10-15, 5-60, 10-60, 10-55, 15-55, 15-50, 20-50, 20-45, 25-45, 25-40, or 30-40. In some embodiments, the number of amino acid substitutions is about 10-35, 10-33, 11-33, 11-31, 12-31, 12-29, 13-29, 13-27, 14-27, or 14-25.

[0066] The amino acid substitutions in the variant can be substitutions with standard amino acids or non-standard amino acids. Non-standard amino acids include, but are not limited to, D-amino acids such as the D-version of standard L-amino acids.

[0067] In some embodiments, the amino acid substitution is a conservative substitution. The term "conservative amino acid substitution" or "conservative substitution" refers to an amino acid substitution having a value of 0 or more in BLOSUM62.

[0068] In some embodiments, the amino acid substitutions are highly conservative substitutions. The term "highly conservative amino acid substitution" or "highly conservative substitution" refers to an amino acid substitution having a value of at least 1 (e.g., at least 2) in BLOSUM62.

[0069] In some embodiments, variants of the target protein of the present disclosure contain from about 5 to 60 amino acid substitutions relative to the amino acid sequence of the target protein disclosed herein. In some embodiments, the amino acid substitutions include at least one conservative substitution. In some embodiments, the amino acid substitutions include at least one highly conservative substitution.

[0070] A. Polypeptide agent The terms “polypeptide,” “peptide,” or “protein” mean a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A protein, peptide, or polypeptide can include any suitable L- and / or D-amino acids, such as common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine), 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). Amino, carboxyl, and / or other functional groups on a peptide may be free (e.g., unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed, for example, in Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. The functional groups of a protein, peptide, or polypeptide can also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label such as a fluorophore or hapten) using methods known in the art. A protein, peptide, or polypeptide can optionally include one or more modifications (e.g., amino acid linker, acylation, acetylation, amidation, methylation, terminal modification factor (e.g., cyclization modification), N-methyl-α-amino group substitution). Further, a protein, peptide, or polypeptide can be an analog of a known and / or natural peptide, such as a peptide analog having conservative amino acid residue substitutions.

[0071] In some embodiments, the agent comprises a polypeptide. In some embodiments, the polypeptide is an isolated polypeptide (e.g., isolated or extracted from a biological sample or source). In some embodiments, the polypeptide is a recombinant polypeptide. In some embodiments, the polypeptide is an inhibitor (e.g., a direct inhibitor or an indirect inhibitor) of the expression and / or activity of a target protein disclosed herein. In some embodiments, the polypeptide is an activator (e.g., a direct activator or an indirect activator) of the expression and / or activity of a target protein disclosed herein. In some embodiments, the polypeptide decreases the expression or activity of a target protein disclosed herein. In other embodiments, the polypeptide increases the expression or activity of a target protein disclosed herein. In some embodiments, the polypeptide is a target protein disclosed herein or a portion thereof (e.g., its biologically active portion, e.g., a biologically active fragment of the target protein).

[0072] In some embodiments, the polypeptide is an immunoglobulin molecule, such as an antibody (e.g., a whole antibody, an intact antibody) or an antigen-binding fragment of an antibody. In some embodiments, the antibody or its antigen-binding fragment binds to a target protein. In some embodiments, the antibody or its antigen-binding fragment binds to a protein that can regulate the expression or activity of a target protein.

[0073] In some embodiments, the polypeptide is an antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" refers to a full-length antibody comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or multimers thereof (e.g., IgM). Each heavy chain H) and a heavy chain constant region (including domains CH1, hinge, CH2, and CH3). Each light chain comprises a light chain variable region (V L ) and a light chain constant region (CL). V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that are interspersed within framework regions (FRs). V H and V L each contain three CDRs and four FR segments and are arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibody can be of any species, e.g., rodent (e.g., mouse, rat, guinea pig) antibody, human antibody, or the antibody can be a humanized or chimeric antibody.

[0074] 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.

[0075] In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is human or chimeric. In some embodiments, the antibody is primatized (e.g., humanized). In some embodiments, the antibody is multispecific, e.g., bispecific, trispecific, or tetravalent. In some embodiments, the antibody is a heteroconjugate antibody.

[0076] 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 part of an immunoglobulin molecule (e.g., an antibody) that retains the antigen-binding properties of the parent full-length antibody. Non-limiting examples of antigen-binding fragments include V H region, V L region, Fab fragment, F(ab’) 2 fragment, Fd fragment, Fv fragment, and domain antibodies (dAbs) consisting of one V H domain or one V L domain. VH and VL domains can be linked to each other via a synthetic linker to form various types of single-chain antibody designs where the V H / V L domains pair within the molecule or, when the V H and V L domains are expressed by separate chains, pair intermolecularly, forming a monovalent antigen-binding site such as a single-chain Fv (scFv) or a diabody. In some embodiments, the polypeptides disclosed herein are antigen-binding fragments selected from Fab, Fab’, F(ab’) 2 , Fd, Fv, disulfide-bonded Fv (sdFv, e.g., diabody, triabody or tetrabody), scFv, SMIP or rlgG. In some embodiments, the polypeptide is an scFv. Antigen-binding fragments can be generated by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.

[0077] Polypeptide agents (e.g., monoclonal antibodies) can be monovalent, divalent or multivalent. Monoclonal antibodies can be monospecific or multispecific (e.g., bispecific). Monospecific antibodies bind to one antigen epitope. Multispecific antibodies such as bispecific or trispecific antibodies are included within the term monoclonal antibody.

[0078] "Multispecificity" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes within an antigen, such as 3, 4, or 5 different antigens or epitopes. "Bispecificity" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen.

[0079] "Isolated antibody" refers to an antibody or antigen-binding fragment thereof that is substantially free of other antibodies having different antigen specificities (e.g., an isolated anti-target protein antibody is substantially free of antibodies that specifically bind to antigens other than the target protein). In the case of a bispecific antibody, the bispecific antibody specifically binds to the two antigens of interest and is substantially free of antibodies that specifically bind to antigens other than the two antigens of interest. In some embodiments, the polypeptide agent (e.g., a monoclonal antibody) is at least 80% pure, e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0080] In some embodiments, the polypeptide is an antagonist antibody that binds to a target protein (e.g., a target protein whose expression or activity is elevated in a cancer state compared to a reference state). In some embodiments, the antibodies described herein are antagonist antibodies that bind to a protein that can modulate the expression or activity of a target protein. As used herein, the term "antagonist antibody" refers to an antibody that, when bound to an antigen (e.g., a target protein or a protein that can modulate the expression or activity of a target protein), reduces (e.g., inhibits) the function of the antigen. In some embodiments, the antigen is a receptor and the antagonist antibody binds to the ligand-binding domain of the receptor. In some embodiments, the antigen is a transmembrane protein and the antagonist antibody binds to the extracellular region of the transmembrane protein. In some embodiments, the antigen is an enzyme or a signaling molecule and the antagonist antibody reduces the activity of the enzyme or attenuates the signaling pathway mediated by the signaling molecule. In some embodiments, the antagonist antibody reduces the antigen function by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 99%.

[0081] In some embodiments, the polypeptide is an agonist antibody that binds to a target protein (e.g., a target protein whose expression or activity is reduced in a cancerous state compared to a reference state). In some embodiments, the antibody is an agonist antibody that binds to a protein that can regulate the expression or activity of the target protein. As used herein, the term "agonist antibody" refers to an antibody that, when bound to an antigen (e.g., a target protein or a protein that can regulate the expression or activity of a target protein), increases the function of the antigen. In some embodiments, the antigen is a receptor and the agonist antibody binds to the ligand-binding domain of the receptor. In some embodiments, the antigen is a transmembrane protein and the agonist antibody binds to the extracellular region of the transmembrane protein. In some embodiments, the antigen is an enzyme or a signaling molecule and the agonist antibody increases the activity of the enzyme or activates a signaling pathway mediated by the signaling molecule. In some embodiments, the agonist antibody increases the antigen function by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1,000%.

[0082] In some embodiments, the agonist antibody does not exhibit at least one of the following functional properties: reducing (e.g., inhibiting) the activity of the antigen; inducing antibody-dependent cell death of cells expressing the antigen (e.g., by natural killer (NK) cells, monocytes, macrophages, neutrophils, dendritic cells, or eosinophils); inducing phagocytosis of cells expressing the antigen (e.g., by macrophages); inducing opsonization of cells expressing the antigen; and inducing downregulation of the antigen on the cell surface (e.g., by cross-linking or clustering the antigen to induce internalization and degradation).

[0083] Appropriate techniques, assays, and reagents for making and using therapeutic antibodies against antigens are known in the art. See, for example, methods for making recombinant antibodies, including antibody engineering, use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis; antibody testing and characterization; pharmacokinetics and pharmacodynamics of antibodies; antibody purification and storage; and screening and labeling techniques in Therapeutic Monoclonal Antibodies: From Bench to Clinic (Zhiqiang An eds., 1st ed. 2009); Antibodies: A Laboratory Manual (Edward A. Greenfield eds., 2d ed. 2013); Ferrara et al., Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker, PLoS ONE 7(11):e49535 (2012).

[0084] In some embodiments, the polypeptide is an antibody mimetic that binds to a target protein disclosed herein. The term "antibody mimetic" refers to a polypeptide that can mimic the ability of an antibody to bind an antigen, but has a structure that is structurally different from a native antibody structure. Non-limiting examples of antibody mimetics include adnectin, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, nanobody, nanoCLAMP, and Versabody.

[0085] In some embodiments (e.g., when the expression or activity of the target protein is reduced in the disease state as compared to the reference state), the agent is a polypeptide (e.g., an isolated polypeptide) comprising an amino acid sequence that is at least 70% identical to at least a portion of the target protein (e.g., a biologically active portion or fragment). For example, the percent identity can be at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the full-length target protein or a biologically active portion or fragment thereof. In some embodiments, the polypeptide comprises the amino acid sequence of the full-length target protein. In some embodiments, the polypeptide comprising the amino acid sequence of the full-length target protein is a recombinant polypeptide. In some embodiments, the polypeptide comprising the amino acid sequence of the full-length target protein is a synthetic polypeptide.

[0086] In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence having at least one amino acid substitution relative to the target protein. For example, the number of amino acid substitutions can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the amino acid substitution is a highly conservative substitution.

[0087] In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence that is at least 70% identical to at least a portion of a protein that can regulate the expression or activity of a target protein. For example, the percent identity can be at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the polypeptide comprises the amino acid sequence of a protein that can regulate the expression or activity of a target protein.

[0088] 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 that can regulate the expression or activity of a target protein. For example, the number of amino acid substitutions can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about 1 - 20, 1 - 19, 2 - 19, 2 - 18, 2 - 17, 3 - 17, 3 - 16, 4 - 16, 4 - 15, 5 - 15, 5 - 14, 6 - 14, 6 - 13, 7 - 13, 7 - 12, 8 - 12, 8 - 11 or 9 - 11. In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the amino acid substitutions are highly conservative substitutions.

[0089] In some embodiments, the polypeptide is a cell-penetrating peptide. In certain embodiments, the polypeptide is linked to a cell-penetrating peptide. Suitable cell-penetrating peptide sequences can be protein-derived, designed, or chimeric (modified). See, for example, Regberg, et al., Applications of cell-penetrating peptides for tumor targeting and future cancer therapies, Pharmaceuticals 5(9):991-1007 (2012). Non-limiting examples of cell-penetrating peptides include TAT(48-60), Penetratin, pVEC, MPG8, Transportan, Transportan10, PepFect3, PepFect 6, PepFect 14, polyarginine, stearyl-polyarginine, Pep-1, Pep-3, CADY, YTA2, YTA4, SynB1, SynB3, Maurocalcine, and PTD4.

[0090] In some embodiments, the polypeptide is a circulating factor (e.g., a cytokine).

[0091] In some embodiments, the polypeptide (e.g., an isolated polypeptide) and the biological properties (e.g., biological activity or half-life) of the target protein are similar. Non-limiting examples of biological activity include, inter alia, enzyme activity or properties (e.g., selectivity, steady state or kinetics), binding activity (e.g., nucleic acid (DNA, RNA) binding protein binding) or properties (e.g., specificity, affinity or kinetics), cell signaling activity, immunological activity, and structural activity (e.g., cell adhesion). Non-limiting examples of enzyme activity include transferase activity (e.g., transferring a functional group from one molecule to another), oxidoreductase activity (e.g., catalyzing an oxidation-reduction reaction), hydrolase activity (e.g., cleaving a chemical bond by hydrolysis), lyase activity (e.g., generating a double bond), ligase activity (e.g., joining two molecules via a covalent bond) and isomerase activity (e.g., catalyzing an intramolecular structural change from one isomer to another).

[0092] In some embodiments, the polypeptide (e.g., an isolated polypeptide) is a recombinant protein. In other embodiments, the polypeptide (e.g., an isolated polypeptide) is a synthetic protein. Methods for producing therapeutic polypeptides are known in the art. See, for example, Therapeutic Proteins: Methods and Protocols (Mark C. Smales & David C James eds., 2005); Pharmaceutical Biotechnology: Fundamentals and Applications (Daan J.A. Crommelin, Robert D. Sindelar & Bernd Meibohm eds., 2013). The polypeptide can be recombinantly expressed using, for example, mammalian cells, insect cells, yeast or bacteria under the control of an appropriate promoter.

[0093] In some embodiments, the polypeptides described herein (e.g., a target protein or a portion thereof, a polypeptide agent that modulates a target protein) are modified, for example, by cleavage (e.g., protease cleavage) or post-translational modification. In certain embodiments, the modification affects the activity of the polypeptide, for example, by activating an inactive polypeptide or by changing the activity level of the polypeptide (e.g., increasing, decreasing). In certain embodiments, the polypeptides described herein are provided as prodrugs that can be converted, for example, in vivo (e.g., by proteolytic cleavage, post-translational modification) into active polypeptides. In some embodiments, the polypeptide comprises a post-translational modification or other chemical modification. Non-limiting examples of post-translational modifications include acetylation, amidation, formylation, glycosylation, hydroxylation, methylation, myristoylation, phosphorylation, deamidation, prenylation (e.g., farnesylation, geranylation, etc.), ubiquitination, ribosylation, and sulfation. Phosphorylation can occur on amino acids such as tyrosine, serine, threonine, or histidine.

[0094] In some embodiments, the polypeptide is attached to a heterologous peptide or protein, such as a conjugate or fusion protein, via a covalent bond (e.g., a peptide bond) or a non-covalent bond. In some embodiments, the polypeptide comprises a tag (e.g., a detectable label such as a fluorophore or an enzyme, or a purification tag such as an epitope tag).

[0095] 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 arising from a target protein described herein. In some aspects, the neoantigen is a cancer-specific neoantigen. There are various ways to produce neoantigens. For example, a neoantigen can be produced in vitro as a polypeptide before being formulated into a neoplasm vaccine or immunogenic pharmaceutical composition. In some embodiments, the immunogenic pharmaceutical composition comprises an effective amount of one or more neoantigens or a pharmaceutically acceptable salt thereof. In some embodiments, the immunogenic pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, adjuvant, or additive.

[0096] Alternatively, a neoantigen can be produced in vivo by introducing a polynucleotide or expression vector (e.g., a viral expression vector) encoding the neoantigen into cells or tissues (e.g., of a subject in need thereof). In certain embodiments, the polypeptide comprises at least two neoantigens. In some embodiments, the polypeptide comprises a T cell enhancer amino acid sequence. In some embodiments, the T cell enhancer is selected from the group consisting of an invariant chain, the leader sequence of tissue-type plasminogen activator, a PEST sequence, a cyclin destruction box, a ubiquitination signal, and a SUMOylation signal.

[0097] B. Polynucleotide agents In some embodiments, the agent comprises a polynucleotide or an analog or derivative thereof. In some embodiments, the polynucleotide or an analog or derivative thereof is an inhibitor of a target protein. In some embodiments, the polynucleotide or an analog or derivative thereof is an activator of a target protein. In some embodiments, the polynucleotide or an analog or derivative thereof reduces (e.g., reduces or neutralizes) the expression or activity of a target protein. In other embodiments, the polynucleotide or an analog or derivative thereof increases the expression or activity of a target protein.

[0098] A polynucleotide can have a sequence containing naturally occurring ribonucleotide or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or combinations thereof. Thus, a polynucleotide can contain, for example, nucleotides containing naturally occurring bases (e.g., A, G, C, or T) and nucleotides containing modified bases (e.g., 7-deazaguanosine, inosine, or methylated nucleotides such as 5-methyl dCTP and 5-hydroxymethylcytosine). In some embodiments, the polynucleotide contains at least one modified nucleotide. Non-limiting examples of modified nucleotides include 2'-fluoro, 2'-O-methyl, 2'-deoxy, locked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'-thiouridine, and 2'-deoxyuridine. In some embodiments, the modification increases nuclease resistance, increases serum stability, decreases immunogenicity, or combinations thereof.

[0099] 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).

[0100] In some embodiments, the polynucleotide contains a polynucleotide analog or derivative. In some embodiments, the analog or derivative is a peptide nucleic acid (PNA). In some embodiments, the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative contains one or more phosphorothioate linkages. In some embodiments, the agent contains deoxyriboguanidine (DNG) nucleotides. In some embodiments, the agent contains riboguanidine (RNG) nucleotides.

[0101] In some embodiments, the polynucleotide regulates the expression and / or activity of a nucleic acid encoding a target protein (e.g., the target protein of the Sequence Listing) or a portion thereof (e.g., a biologically active portion or a fragment thereof) disclosed herein.

[0102] In some embodiments, the polynucleotide comprises a nucleotide sequence that is complementary (e.g., fully or partially complementary) to at least a portion of a gene or gene transcript encoding a target protein disclosed herein, such that the polynucleotide sequence can hybridize or anneal to the gene or gene transcript (e.g., under physiological conditions). In other embodiments, the polynucleotide comprises a nucleotide sequence that is complementary to at least a portion of a gene or gene transcript encoding a protein that can regulate the expression or activity of a target protein disclosed herein.

[0103] In some embodiments, the polynucleotide encodes a target protein disclosed herein, or a variant thereof (e.g., a biologically active variant thereof), or a portion thereof (e.g., a biologically active portion or a fragment thereof).

[0104] In some embodiments, the nucleic acid encoding the 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 coding 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 coding nucleic acid is an mRNA molecule or a portion thereof. In some embodiments, the coding nucleic acid is present in a non-coding RNA (e.g., long intergenic non-coding RNA (lincRNA), long non-coding RNA (lncRNA), or miRNA).

[0105] The coding nucleic acid can include a standard open reading frame (ORF) or a non-standard ORF. In certain embodiments, the coding nucleic acid includes a non-standard ORF.

[0106] The polynucleotide can be single-stranded (ss) or double-stranded (ds). In some embodiments, the polynucleotide is double-stranded (ds). In some embodiments, the length of the ds polynucleotide is about 15 to 50 base pairs, such as about 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 18 to 50, 18 to 45, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 50, 35 to 45, 35 to 40 or 40 to 50 base pairs. In some embodiments, the length of the polynucleotide is about 19 to 23 base pairs. In some embodiments, the length of the polynucleotide is about 21 base pairs.

[0107] In some embodiments, the polynucleotide is single-stranded (ss). In some embodiments, the length of the ss polynucleotide is about 15 to 50 nucleotides, such as about 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 18 to 50, 18 to 45, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 50, 35 to 45, 35 to 40 or 40 to 50 nucleotides.

[0108] In some embodiments, the polynucleotide inhibits the maturation of a newly generated nuclear RNA transcript into mRNA for transcription. In some embodiments, the polynucleotide comprises a nucleotide sequence complementary to a sequence at the intron-exon boundary.

[0109] In some embodiments, a polynucleotide (e.g., an antisense oligonucleotide) can hybridize to an mRNA encoding a target protein (e.g., under physiological conditions). In some embodiments, the length of the polynucleotide is at least about 10 nucleotides, such as at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or about 10-30, 15-30, 15-25, 20-25 nucleotides. In some embodiments, the polynucleotide is at least 75% identical to the antisense sequence of the same target transcript, such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical.

[0110] In some embodiments, the polynucleotide further comprises an overhang sequence (e.g., unpaired overhang nucleotides that are not directly involved in the formation of a duplex structure by the core sequence). In some embodiments, the polynucleotide comprises a 3' overhang, a 5' overhang, or both. In some embodiments, the overhang is about 1-5 nucleotides. In some embodiments, the overhang comprises modified ribonucleotides or deoxynucleotides, such as phosphorothioate, phosphorothioate or deoxynucleotide inversion (linked 3' to 3') nucleotides.

[0111] Non-limiting examples of polynucleotide agents suitable for use in the compositions, kits and methods described herein include small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antagomir, antisense DNA, antisense RNA, morpholino nucleic acid (MNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), aptamer and guide RNA (gRNA).

[0112] In some embodiments, the polynucleotide inhibits gene expression (e.g., via the biological process of RNA interference (RNAi)). Polynucleotides suitable for RNA interference can be readily designed and produced by those skilled in the art using techniques, assays, and reagents known in the art, including computational tools. See, for example, Pei et al. 2006, Reynolds et al. 2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005, Chalk et al. 2004, Amarzguioui et al. 2004.

[0113] In some embodiments, the polynucleotide is a miRNA. In some embodiments, the miRNA is about 22 nucleotides in length. The miRNA binds to a target site on the mRNA molecule and silences the mRNA, for example, by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA.

[0114] In some embodiments, the polynucleotide is a siRNA. In some embodiments, the siRNA contains a nucleotide sequence identical to about 15 - 25 contiguous mRNA sequences encoding the target protein. In some embodiments, the siRNA is a double-stranded RNA molecule having about 19 - 25 base pairs. In some embodiments, the siRNA begins with the dinucleotide AA. In some embodiments, the siRNA has a GC content of about 30 - 70%, such as about 30 - 65%, 30 - 60%, 30 - 55%, 30 - 50%, 40 - 70%, 40 - 65%, 40 - 60%, 40 - 55%, 45 - 70%, 45 - 65%, 45 - 60%, or 45% - 55%.

[0115] In some embodiments, the polynucleotide is shRNA. shRNA is an RNA molecule containing a hairpin turn that reduces the expression of a target gene via RNAi. shRNA can be delivered to cells in the form of a plasmid, such as a viral vector or a bacterial vector, for example, by transfection, electroporation, or transduction.

[0116] siRNAs and shRNAs are similar to intermediates in the processing pathway of endogenous microRNA (miRNA) genes (see, e.g., Bartel, Cell 116:281-97 (2004)). In some embodiments, siRNAs function as miRNAs; in other embodiments, miRNAs function as siRNAs (see, e.g., Zeng et al., Mol Cell 9:1327-33 (2002); Doench et al., Genes Dev 17:438-42 (2003)). MicroRNAs such as siRNAs use RISC to downregulate target genes, but unlike siRNAs, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein output through translational repression or polyA removal and mRNA decay (see, e.g., Wu et al., Proc Natl Acad Sci USA 103:4034-39 (2006)). Known miRNA binding sites are within the mRNA 3’UTR; miRNAs are thought to target sites with near-perfect complementarity to nucleotides 2-8 from the 5’ end of the miRNA (see, e.g., Rajewsky, Nat Genet 38 Suppl:S8-13 (2006) and Lim et al., Nature 433:769-73 (2005)). This region is known as the seed region. Since siRNAs and miRNAs are interchangeable, exogenous siRNAs downregulate mRNAs with seed complementarity to the siRNA (see, e.g., Birmingham et al., Nat Methods 3:199-204 (2006)). Multiple target sites within the 3’UTR result in stronger downregulation (see, e.g., Doench et al., Genes Dev 17:438-42 (2003)).

[0117] In some embodiments, the polynucleotide is a messenger RNA (mRNA) or circular RNA (circRNA) encoding a target protein or a variant thereof (e.g., a variant that is at least about 70% identical to the wild-type protein, e.g., at least about 75%, 80%, 85%, 90%, 95%, 98% or 99% identical) disclosed herein. In some embodiments, the mRNA is codon-optimized (e.g., to improve the efficiency of protein synthesis and limit mRNA destabilization by rare codons; see, e.g., Presnyak et al., Cell. 160(6):1111-24(2015) and Thess et al., Mol Ther. 23(9):1456-64(2015)).

[0118] In some embodiments, the polynucleotide comprising RNA is chemically synthesized. In some embodiments, the polynucleotide comprising RNA is recombinantly expressed. In some embodiments, the RNA is transcribed in vitro. The production and use of RNA therapeutics are known in the art. See, for example, RNA Therapeutics: Function, Design, and Delivery (Mouldy Sioud eds., 2010) and Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Medicine 9:60(2017).

[0119] In some embodiments, the mRNA is produced by in vitro transcription. In some embodiments, the mRNA is modified to optimize its activity. In some embodiments, the mRNA comprises modified bases, a 5' cap, a 5' cap analog, an anti-reverse cap analog (ARCA), or a combination thereof.

[0120] In some embodiments, the mRNA comprises a poly(A) tail. In some embodiments, the poly(A) tail is about 100 to 200 nucleotides. In some embodiments, the poly(A) tail improves the expression and / or stability of the mRNA (see, e.g., Kaczmarek et al., Genome Medicine 9:60 (2017)).

[0121] In some embodiments, the mRNA comprises a 5' cap. In some embodiments, the mRNA comprises a 5' cap analog. In some embodiments, the 5' cap analog is a 1,2-dithiophosphate-modified cap (see, e.g., Strenkowska et al., Nucleic Acids Res. 44:9578-90 (2016)).

[0122] In some embodiments, the mRNA comprises a modified 3' untranslated region (UTR), 5' UTR, or both. In some embodiments, the modified UTRs comprise sequences involved in the recruitment of RNA-binding proteins (RBPs) and miRNAs to enhance the level of protein production (see, e.g., Kaczmarek et al., Genome Medicine 9:60 (2017)). In some embodiments, the 3' UTR, 5' UTR, or both are modified to encode regulatory elements. In some embodiments, the regulatory elements include a K-turn motif, miRNA binding sites, or a combination thereof for controlling RNA expression in a cell-specific manner (see, e.g., Wroblewska et al., Nat Biotechnol. 33:839-41 (2015)).

[0123] In some embodiments, the mRNA comprises an RNA base modification. In some embodiments, the mRNA comprises pseudouridine. In some embodiments, the mRNA comprises N1-methyl-pseudouridine (e.g., to mask immune-stimulatory activity and enhance translation initiation, see, for example, Andries et al., J Control Release 217:337-44 (2015) and Svitkin et al., Nucleic Acids Res. 45:6023-36 (2017)).

[0124] In some embodiments, the RNA (e.g., mRNA) is circular RNA.

[0125] Compositions and methods for producing mRNA are described, for example, in WO 2016 / 011306, WO 2016 / 014846, WO 2016 / 022914, WO 2016 / 077123, WO 2016 / 164762, WO 2016 / 201377, WO 2017 / 049275, US Patent No. 9,937,233, US Patent No. 8,710,200, US Patent No. 10,022,425, US Patent No. 9,878,056, US Patent No. 9,572,897, WO 2010 / 084371, US Patent No. 9,353,153, WO 2015 / 034925, and WO 2019 / 236673. See, for example, Jemielity et al., RNA 9(9):1108-22(2003); Mockey et al., Biochem Biophys Res Commun. 340:1062-88(2006); Strenkowska et al., Nucleic Acids Res. 44:9578-90(2016); Presnyak et al., Cell 160:1111-24(2015) and Kaczmarek et al., Genome Medicine 9:60(2017)). In some embodiments, the mRNA is prepared in a lipid nanoparticle (LNP) formulation (see, for example, for in vivo delivery, US Patent No. 9,764,036, US Patent No. 9,682,139, Kauffman et al., Nano Lett. 15:7300-6(2015) and Fenton et al., Adv Mater. 28:2939-43(2016)).

[0126] In some embodiments, the polynucleotide is an aptamer. In certain embodiments, the aptamer binds to a target protein disclosed herein. In certain embodiments, the aptamer binds to a binding partner of a target protein disclosed herein.

[0127] In some embodiments, the polynucleotide is linked (e.g., by a covalent bond) to a delivery polymer. In some embodiments, the linkage between the polynucleotide and the delivery polymer is reversible. In some embodiments, the polynucleotide is linked to the delivery polymer via a physiologically labile linker. In some embodiments, the physiologically labile linker is a disulfide bond.

[0128] In some embodiments, the polynucleotide is conjugated to the polymer in the presence of an excess of the polymer. In some embodiments, the excess polymer is removed prior to administration (e.g., to a cell or subject).

[0129] One of ordinary skill in the art can readily make appropriate polynucleotide agents for use in the compositions, kits, and methods described herein using the locus information of the protein sequences included in the sequence listing incorporated herein, such as chromosomal location, start nucleotide position, and end nucleotide position, as well as polymorphism identification.

[0130] C. Agent Comprising a Gene Editing System In some embodiments, the agent comprises a gene editing system. In some embodiments, the gene editing system results in a nucleotide deletion, nucleotide substitution, nucleotide addition, or a combination thereof in the gene encoding the target protein.

[0131] 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.

[0132] In some embodiments, a gene editing system (e.g., a CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates the expression of a target protein (e.g., via gene knockout). In some embodiments, a gene editing system (e.g., a CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates the expression of a protein that can regulate the expression or activity of a target protein (e.g., via gene knockout). In some embodiments, a gene editing system (e.g., a CRISPR / Cas system) increases the expression of a target protein (e.g., via gene knock-in or gene replacement). In some embodiments, a gene editing system (e.g., a CRISPR / Cas system) increases the expression of a protein that can regulate the expression or activity of a target protein (e.g., via gene knock-in or gene replacement).

[0133] In some embodiments, the CRISPR system specifically catalyzes the cleavage of the gene encoding the target protein, thereby inactivating the gene. Repair of nucleic acid strand breaks via non-homologous end joining (NHEJ) often results in changes in the DNA sequence at the cleavage site, resulting in small insertions or deletions (Indels). In some embodiments, NHEJ is used to knockout the gene encoding the target protein. In some embodiments, homologous recombination repair (HDR) is used to simultaneously inactivate the gene encoding the target protein and insert a heterologous sequence at the inactivated locus. Cells in which knockout and / or knock-in events have occurred can be identified and / or selected by methods well known in the art.

[0134] 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., from Streptococcus Pyogenes). In some embodiments, the single Cas endonuclease is Cpf1. In some embodiments, Cpf1 is AsCpf1 (from the Acidaminococcus species) or LbCpf1 (from the Lachnospiraceae species). The selection of the nuclease and gRNA is typically determined according to whether nucleotide deletions, substitutions, or additions to the target sequence are desired.

[0135] In some embodiments, the type II Cas endonuclease is Cas9 from (e.g., Streptococcus pyogenes). In some embodiments, the modified Cas9 is nickase Cas9, dead Cas9 (dCas9), or eSpCas9. In some embodiments, the nickase Cas9 is Cas9 D10A. In some embodiments, the dCas9 is D10A or H840A. In some embodiments, the gene editing system includes dual nickase Cas9 (see, e.g., Ran et al., Cell 154:1380-89 (2013) for achieving more accurate genome editing). Wild-type Cas9 generates a double-strand break (DSB) at a specific DNA sequence targeted by the gRNA. Nickase Cas9 generates only single-strand breaks. dCas9 is catalytically inactive. In some embodiments, the dCas9 is fused to a nuclease (e.g., FokI for generating DSBs at target sequences homologous to two gRNAs). Various CRISPR / Cas9 plasmids are publicly available from the Addgene repository (Addgene, Cambridge, MA: addgene.org / crispr / ).

[0136] In some embodiments, the gene editing system comprises: a) a wild-type or modified type II Cas endonuclease, or a polynucleotide encoding a wild-type or modified type II Cas endonuclease; b) a CRISPR RNA (“crRNA”); and c) a trans-activating crRNA (“tracrRNA”) comprising.

[0137] In some embodiments, the crRNA comprises at least one "guide RNA" (sgRNA), such as at least 2, 3, or 4 gRNAs. In some embodiments, the gRNA comprises a sequence identical to a portion of the gene sequence of the target protein. In some embodiments, the gRNA comprises a sequence identical to a portion of the gene sequence of a protein capable of regulating the expression or activity of the target protein. In some embodiments, the gRNA is at least about 16 nucleotides, such as at least about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; or about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; or about 16 - 24, 17 - 24, 17 - 23, 18 - 23, 18 - 22, 19 - 22, or 19 - 21, or 19, 20, or 21 nucleotides. In some embodiments, the sgRNA is chemically modified.

[0138] The design of gRNA sequences for gene editing is known in the art. See, for example, Cong et al., Science, 339:819 - 23 (2013) and Ran et al., Nature Protocols 8:2281 - 308 (2013). Cas9 requires a gRNA sequence of at least about 16 or 17 nucleotides to cleave DNA, and Cpf1 requires a gRNA sequence of at least about 16 nucleotides to cleave DNA. In practice, the gRNA sequence has a length of about 17 - 24 nucleotides (e.g., about 19, 20, or 21 nucleotides) and is complementary to the target gene. Custom gRNA generators and algorithms are commercially available. Chemically modified sgRNAs have also been demonstrated to be effective for genome editing (see, for example, Hendel et al., Nature Biotechnol., 985 - 91 (2015)).

[0139] In some embodiments, the crRNA further comprises a sequence capable of binding to the tracrRNA. When bound, the partially double-stranded structure is cleaved by RNase III, and the resulting crRNA / tracrRNA hybrid instructs the Cas9 endonuclease to recognize and cleave the target DNA sequence.

[0140] In some embodiments, the target DNA sequence is proximal to a "protospacer adjacent motif" ("PAM") specific to the Cas endonuclease. The PAM sequence appears throughout a given genome. CRISPR endonucleases of various prokaryotic species have unique PAM sequence requirements. Non-limiting examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endonucleases, such as the Cas9 endonuclease, associate with a G-rich PAM site, such as 5'-NGG, and perform blunt-end cleavage of the target DNA at a position 3 nucleotides upstream (5') of the PAM site.

[0141] In some embodiments, the gene editing system is: a) a wild-type or modified type II Cas endonuclease, or a polynucleotide encoding a wild-type or modified type II Cas endonuclease; and b) a crRNA comprising.

[0142] Cpf1-related CRISPR arrays are processed into mature crRNAs without the need for tracrRNA. The Cpf1 endonuclease associates with a T-rich PAM site, such as 5'-TTN. Cpf1 can also recognize a 5'-CTA PAM motif. Cpf1 introduces offset or staggered double-strand breaks with 5'-nucleotide overhangs of 4 or 5 nucleotides, for example, by cleaving target DNA having an offset or staggered cut of 5 nucleotides located 18 nucleotides downstream (3') of the PAM site on the coding strand and 23 nucleotides downstream of the PAM site on the complementary strand. The 5-nucleotide overhangs resulting from such offset cuts enable more accurate genome editing by DNA insertion via homologous recombination rather than by insertion into blunt-ended cut DNA. See, for example, Zetsche et al., Cell 163:759-71 (2015).

[0143] In some embodiments, the gene editing system activates or suppresses transcription of a target gene. In some embodiments, the gene editing system comprises: a) a chimeric protein comprising dCas9 and one or more effector domains; and b) one or more sgRNAs comprising.

[0144] In some embodiments, the chimeric protein suppresses the expression of a target protein (CRISPRi). In some embodiments, the chimeric protein activates the expression of a 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.

[0145] The effector domain contains the biologically active portion of an effector protein (e.g., a transcriptional activator or a transcriptional repressor). In some embodiments, the gene editing system comprises one effector domain. In some embodiments, the gene editing system comprises at least two effector domains, such as 2, 3, or 4 effector domains. In some embodiments, the effector domain comprises KRAB. In some embodiments, the effector domain comprises VP64. In some embodiments, the effector domain comprises VP64, p65, and Rta. In some embodiments, dCas9 is D10A. In some aspects, dCas9 is H840A.

[0146] Since dCas9 is catalytically inactive, it does not cleave target DNA and interferes with transcription by steric hindrance. dCas9 chimeric proteins (e.g., dCas9-VPR) are guided by one or more gRNAs to sequences upstream of the transcription start site (TSS) of a target gene and regulate the transcription of the target gene. For example, Gilbert et al., CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes, Cell 154, 442-51 (2013); Cheng et al., Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system, Cell Res. 23:1163-71 (2013); Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation, Cell 159:647-61 (2014); Tanenbaum et al., A protein-tagging system for signal amplification in gene expression and fluorescence imaging, Cell 159:635-46 (2014); Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Nature 517:583-88 (2015); Chavez et al., Highly efficient Cas9-mediated transcriptional programming, Nat.Methods.12:326-28 (2015); Zalatan et al., Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Cell 160:339-50 (2015); Horlbeck et al.See Chavez et al., Comparison of Cas9 activators in multiple species, Nat Methods. 7:563-67 (2016); eLife. 5:e19760 (2016); Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.

[0147] CRISPR technology for editing eukaryotic genes is disclosed in US Patent Application Publication No. 2016 / 0138008A1, US Patent Application Publication No. 2015 / 0344912A1, US Patent No. 8,697,359, US Patent No. 8,771,945, US Patent No. 8,945,839, US Patent No. 8,999,641, US Patent No. 8,993,233, US Patent No. 8,895,308, US Patent No. 8,865,406, US Patent No. 8,889,418, US Patent No. 8,871,445, US Patent No. 8,889,356, US Patent No. 8,932,814, US Patent No. 8,795,965, and US Patent No. 8,906,616. The Cpf1 endonuclease and corresponding guide RNA and PAM site are disclosed in US Patent Application Publication No. 2016 / 0208243A1. CRISPR technology for causing mtDNA dysfunction in the mitochondrial genome is disclosed in Jo et al., BioMed Res. Int. 2015:305716 (2015). Co-delivery of Cas9 and sgRNA with nanoparticles is disclosed in Mout et al., ACS Nano 11(3):2452-58 (2017).

[0148] In some embodiments, the agent comprises a transposon-based gene editing system. An example of a transposon-based gene editing system suitable for use in the disclosure provided herein is the Gene Writer system described in International Publication No. WO 2020 / 047124, published March 5, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0149] In some embodiments, the agent comprises a transcription activator-like effector nuclease (TALEN) system. TALEN-based systems include proteins that contain a TAL effector DNA binding domain and an enzyme domain. They are created by fusing the TAL effector DNA binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). The above-mentioned FokI restriction enzyme is an exemplary enzyme domain suitable for use in TALEN-based gene regulatory systems.

[0150] TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when infecting plants. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence with different 12th and 13th amino acids. These two positions, called repeat variable diresidues (RVDs), are highly variable and strongly correlate with specific nucleotide recognition. Thus, the TAL effector domain can be engineered to bind to a specific target DNA sequence by selecting a combination of repeat segments containing appropriate RVDs. The nucleic acid specificities for combinations of RVDs are as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (however, in some embodiments, NN can also bind to adenine with lower specificity).

[0151] In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to the target DNA sequence of the target protein. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to the target DNA sequence defined by a set of genomic coordinates.

[0152] In some embodiments, the gene regulatory system comprises two or more TAL effector fusion proteins, each comprising a TAL effector domain, and at least one of the TAL effector domains binds to a target DNA sequence of the target protein. In some embodiments, at least one of the two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to the target DNA sequence defined by a set of genomic coordinates.

[0153] Methods and compositions for assembling TAL-effector repeats are known in the art. See, for example, Cermak et al, Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting, Nucleic Acids Res 39(12):e82(2011). Plasmids for construction of TAL-effector repeats are commercially available, for example, from Addgene.

[0154] In some embodiments, the agent comprises a zinc finger nuclease (ZFN) system. The ZFN domain can be generated using commercially available plasmids, for example, plasmid pairs from Sigma Aldrich (St. Louis, MO), such as CSTZFN-1KT COMPOZR® Custom Zinc Finger Nuclease (ZFN) R-3257609. The plasmids can be prepared using commercially available systems according to the manufacturer's protocol (e.g., NEB Monarch Miniprep (catalog number T1010), New England Biolabs, Ipswich, MA).

[0155] In some embodiments, the agent comprises a vector designed to deliver conventional gene therapy (e.g., gene knockout or knock-in by homologous recombination). Non-limiting examples of such vectors include retroviruses (e.g., lentivirus 5), adenoviruses, adeno-associated viruses, herpes simplex viruses, nanoparticles, and DNA transposons.

[0156] D. Small molecule agents In some embodiments, the agent comprises a small molecule. In some embodiments, the small molecule binds to a target protein. In some embodiments, the small molecule binds to a protein that can regulate the expression or activity of the target protein. In some embodiments, the small molecule is an inhibitor of the target protein (e.g., a direct inhibitor, an indirect inhibitor). In some embodiments, the small molecule is an activator of the target protein (e.g., a direct activator, and an indirect activator).

[0157] Examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic or inorganic compounds. The atoms in a small molecule are typically linked to each other via covalent and / or ionic bonds. In certain embodiments, the small molecule is an organic small molecule. The arrangement of atoms in an organic small molecule may represent a chain (e.g., a carbon-carbon chain or a carbon-heteroatom chain), or a ring containing carbon atoms, such as benzene or a polycyclic system, or a combination of carbon and heteroatoms, i.e., a heterocycle such as pyrimidine or quinazoline. Small molecules can have a wide range of molecular weights, but generally include molecules having a molecular weight of less than about 5,000 Daltons. For example, such small molecules can have a molecular weight of less than about 1,000 Daltons, preferably less than about 750 Daltons, or more preferably less than about 500 Daltons. Small molecules can be found naturally (e.g., identified, isolated, purified) and / or produced synthetically (e.g., by traditional organic synthesis, biocatalyzed synthesis, or a combination thereof). See, for example, Ganesan, Drug Discov. Today 7(1):47-55 (January 2002); Lou, Drug Discov. Today, 6(24):1288-1294 (December 2001). Examples of naturally occurring small molecules include, but are not limited to, hormones, neurotransmitters, nucleotides, amino acids, sugars, lipids, and derivatives thereof.

[0158] In certain embodiments, the agent includes a proteolysis targeting chimera (PROTAC).

[0159] Small molecules suitable for use in the compositions, kits and methods of the present disclosure can be identified by one of ordinary skill in the art using any of the screening methods disclosed herein.

[0160] E. Therapeutic Cells and Cell-Based Therapies In some embodiments, the agent comprises therapeutic cells. In certain embodiments, the therapeutic cells express and / or are engineered to express a target protein (e.g., the target protein of the Sequence Listing or a variant thereof), a polypeptide (e.g., an antibody, antigen-binding fragment or polypeptide comprising an amino acid sequence that is at least 70% identical to at least a portion of the target protein), a polynucleotide (e.g., recombinant DNA, RNA, such as mRNA or siRNA) and / or a gene editing system (e.g., the CRISPR / Cas system) as described herein.

[0161] In some embodiments, the polypeptides (e.g., antibodies or antigen-binding fragments) disclosed herein are incorporated into cell-based therapies. In some embodiments, the polypeptide is an engineered T cell receptor. In some embodiments, the polypeptide is a chimeric antigen receptor (CAR) (e.g., expressed on T (CAR-T) cells, natural killer (CAR-NK) cells or macrophage (CAR-M) cells). In some embodiments, the CAR comprises a transmembrane domain and an antigen recognition portion that binds to a target protein. In certain embodiments, the polypeptide is expressed by therapeutic cells (e.g., CAR-T, CAR-NK or CAR-M cells). In certain embodiments, the polypeptide is a cytokine receptor expressed on the membrane of CAR-T, CAR-NK or CAR-M cells. In more specific embodiments, the polypeptide is a cytokine secreted from CAR-T, CAR-NK or CAR-M cells.

[0162] Therapeutic cells suitable for use in the compositions, kits and methods of the present disclosure can be generated, identified and / or enriched by methods known to those of skill in the art. Non-limiting examples of such methods include purifying, expanding and / or differentiating cells from a subject (e.g., human) into specific cell products; engineering somatic cells for gene therapy; immortalizing cells; ex vivo gene modification of cells (e.g., using viral vector and / or lipid nanoparticle delivery techniques); in vivo gene modification of cells (e.g., using viral vector and / or lipid nanoparticle delivery techniques); 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.

[0163] Expression Vectors and Hosts In another aspect, the present disclosure provides an expression vector comprising a polynucleotide described herein.

[0164] The term "expression vector" refers to a replicable nucleic acid capable of expressing one or more proteins when the expression vector is transformed into a suitable expression host cell.

[0165] In some embodiments, the expression vector comprises an expression control polynucleotide sequence operably linked to a polynucleotide, a polynucleotide sequence encoding a selectable marker, or both. In some embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In some embodiments, the expression control polynucleotide sequence comprises an inducible promoter sequence. The term "promoter" refers to the region of DNA to which RNA polymerase binds to initiate transcription of a gene. The term "operably linked" means that a nucleic acid is positioned in a recombinant polynucleotide, such as a vector, in such a way that it enables expression of the nucleic acid under the control of the element (e.g., promoter) to which it is linked. The term "selectable marker element" is an element that confers a trait suitable for artificial selection. A selectable marker element can be a negative or positive selectable marker. Non-limiting examples of expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Molecular Cloning: A Laboratory Manual (Michael R. Green & Joseph Sambrook eds., 4th ed. 2012).

[0166] In another aspect, the disclosure provides an expression host cell comprising any one or more of the polynucleotides or expression vectors described herein.

[0167] The term "expression host cell" refers to a cell useful for receiving, maintaining, replicating, and / or amplifying a vector.

[0168] Non-limiting examples of expression host cells include mammals such as hybridoma cells, baby hamster kidney fibroblasts (BHK cells), Chinese hamster ovary (CHO) cells, COS cells, HeLa cells, and human embryonic kidney (HEK), yeast cells such as Pichia pastoris cells, or bacterial cells such as DH5α. For example, for the process of host cell culture for producing protein therapeutics, see Mammalian Cell Cultures for Biologics Manufacturing (Weichang Zhou & Anne Kantardjieff eds., 2014), and for the purification of protein therapeutics, see Protein Biotechnology: Isolation, Characterization, and Stabilization (Felix Franks eds., 2013) and Protein Purification Protocols (Paul Cutler eds., 2010); and for the formulation of therapeutic proteins, see Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic (Brian K Meyer eds., 2012).

[0169] The polynucleotides or expression vectors described herein can be introduced into appropriate or desired host cells using techniques known in the art, including transformation, electroporation, and transduction. The introduced nucleic acid can be extrachromosomal within the host cell or integrated into the genome of the host cell.

[0170] Pharmaceutical composition In another aspect, the present disclosure provides a pharmaceutical composition comprising an agent disclosed herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a composition having a pharmacological activity or other direct effect in the alleviation, treatment, or prevention of cancer, or a finished dosage form or formulation thereof.

[0171] In some embodiments, the composition (e.g., pharmaceutical composition) includes a pharmaceutically acceptable carrier, excipient, stabilizer, diluent, or tonic (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations used. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents, or tonics 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®), PLURONICS® and polyethylene glycol (PEG).

[0172] In some embodiments, the agent of the pharmaceutical composition (e.g., polypeptide, polynucleotide, or small molecule) is modified, e.g., conjugated to a heterologous moiety. The term "conjugated" refers to being bound via a covalent or non-covalent interaction. Conjugation can be performed using any suitable linker; non-limiting examples include peptide linkers, compound linkers, and chemical crosslinking agents.

[0173] In some embodiments, the heterologous moiety is a marker (e.g., a fluorescent marker or a radioactive marker), a molecule that stabilizes an agent, a molecule that targets an agent (e.g., to a specific cell or tissue, such as to facilitate or prevent passage across the blood-brain barrier), or a combination thereof.

[0174] In some embodiments, the heterologous moiety is polyethylene glycol (PEG), hexadecanoic acid, a hydrogel, a nanoparticle, a multimerization domain, and a carrier peptide. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is a polymeric nanoparticle. In some embodiments, the polymer is an amphiphilic polymer. In other embodiments, the polymer is a hydrophobic or hydrophilic polymer. Non-limiting examples of polymers include poly(lactic acid)-poly(ethylene glycol), poly(lactic-co-glycolic acid)-poly(ethylene glycol), poly(lactic-co-glycolic acid) (PLGA), poly(lactic-co-glycolic acid)-d-α-tocopheryl polyethylene glycol succinate, poly(lactic-co-glycolic acid)-ethylene oxide fumarate, poly(glycolic acid)-poly(ethylene glycol), polycaprolactone-poly(ethylene glycol), or any salt thereof. In some embodiments, the polymeric nanoparticle comprises poly(lactic-co-glycolic acid) (PLGA).

[0175] In some embodiments, the composition (e.g., a pharmaceutical composition) is formulated for an appropriate dosing schedule and route. Non-limiting examples of administration routes include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical. In some embodiments, the composition (e.g., a pharmaceutical composition) is stored in the form of an aqueous solution or a dry formulation (e.g., lyophilized). In some embodiments, the composition is formulated to be administered by infusion (e.g., intravenous infusion).

[0176] In some embodiments, the composition is formulated to be administered 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 "administered in combination" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. Non-limiting examples of additional agents or treatments include biologics (e.g., antibodies, peptides), cell therapies, gene therapies, immunotherapies, and small molecules that affect immune-mediated diseases or conditions.

[0177] The treatment regimen defines the dosage and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, two or more agents are administered sequentially as part of a prescription regimen. In other embodiments, the delivery of two or more agents is simultaneous or co-temporaneous. In some embodiments, two or more agents are co-formulated. In some embodiments, by administering two or more agents or treatments in combination, a reduction in other parameters associated with the symptom or disorder is greater than that observed when one agent or treatment is delivered alone or in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Each of the two or more therapeutic agents can be administered by any suitable route including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The two or more therapeutic agents can be administered by the same route or different routes.

[0178] In some embodiments, the agent or pharmaceutical composition of the disclosure is delivered by a viral vector, e.g., by contacting cells with the viral vector, administered locally to a tumor (e.g., by injection), or systemically to a subject (e.g., a human patient) (e.g., by intravenous or oral administration).

[0179] The viral genome provides a rich source of vectors that can be used for the efficient delivery of exogenous genes into mammalian cells. The viral genome is a particularly useful vector for gene delivery because the polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Non-limiting examples of viral vectors include retroviruses (e.g., retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses, such as picornaviruses and alphaviruses, and double-stranded DNA viruses, such as adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-deficient herpesviruses), and poxviruses (e.g., vaccinia, modified vaccinia virus Ankara (MVA), fowlpox and canarypox). Further non-limiting examples include, for example, norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus.Non-limiting examples of retroviruses include avian leukosis sarcoma, avian C-type virus, mammalian C-type, B-type virus, D-type virus, oncovirus, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (see, e.g., Coffin JM. Retroviridae: The viruses and their replication. In: Fields BN, Knipe DM, Howley PM et al, eds. Fundamental Virology. 3rd ed. Philadelphia: Lippincott-Raven Publishers, 1996:763-843). Further non-limiting examples include murine leukemia virus, murine sarcoma virus, murine mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, rhesus 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.

[0180] In some embodiments, the agents or pharmaceutical compositions of the present disclosure are formulated to be delivered by a membrane-based carrier in vivo, in vitro, ex vivo, or in situ. In some embodiments, the membrane-based carrier is a cell-based carrier (e.g., a mammal such as a human cell). In some embodiments, the membrane-based carrier is a vesicle-based carrier. In some embodiments, the membrane-based carrier comprises one or more vectors (e.g., a plasmid, virus, virus-like particle, or virion) described herein.

[0181] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more liposomes. A liposome is a spherical vesicular structure composed of a single or multiple lipid bilayers surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible and non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB) (see, for example, Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)).

[0182] Vesicles can be made from several different types of lipids; phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, for example, U.S. Patent No. 6,693,086, the teachings of which regarding the preparation of multilamellar vesicle lipids are incorporated herein by reference). The formation of vesicles can be spontaneous when lipid membranes are mixed with an aqueous solution, but can also be facilitated by applying force in the form of agitation using a homogenizer, sonicator or extrusion device (see, for example, Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)). The extruded lipids can be prepared by extrusion through a small-sized filter as described in Templeton et al., Nature Biotech, 15:647-52 (1997), the teachings of which regarding the preparation of extruded lipids are incorporated herein by reference).

[0183] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by lipid nanoparticles (LNPs). In one embodiment, the LNP preparation containing the agent or pharmaceutical composition of the present disclosure has one or more of the following characteristics: (a) the LNP preparation contains a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid, and (b) the LNP preparation has an average particle size of 80 nm to 160 nm.

[0184] Nanostructured lipid carriers (NLCs) are SLNs that retain the characteristics of modified solid lipid nanoparticles (SLNs), improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are important components for drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier combining liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs are composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, the two components enhance drug encapsulation efficiency, facilitate surface modification, and prevent leakage of water-soluble drugs. See, for example, Li et al., Nanomaterials 7(6):122 (2017).

[0185] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by a carbohydrate carrier (e.g., anhydride-modified phytoglycogen or glycogen-like material). Non-limiting examples of carbohydrate carriers include octenyl succinic acid phytoglycogen, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin.

[0186] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by a protein carrier (e.g., a protein covalently linked to a cyclic polynucleotide). Non-limiting examples of protein carriers include human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), and globulin.

[0187] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by a cationic carrier (e.g., a cationic lipopolymer or transfection reagent). Non-limiting examples of cationic carriers include Lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimer, chitosan, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HC1), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC).

[0188] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by exosomes, adipocytes, and / or red blood cells. See, for example, Ha et al., Acta Pharm Sin B. 6(4):287-96(2016).

[0189] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more fusosomes. The fusosomes are engineered to confer target cell specificity for fusion and payload delivery, thereby enabling the generation of a delivery vehicle with programmable cell specificity. See, for example, WO 2020014209 pamphlet, the teachings of which regarding the design, preparation, and use of fusosomes are incorporated herein by reference.

[0190] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by ex vivo differentiated red blood cells. See, for example, WO 2015073587 pamphlet; WO 2017123646 pamphlet; WO 2017123644 pamphlet; WO 2018102740 pamphlet; WO 2016183482 pamphlet; WO 2015153102 pamphlet; WO 2018151829 pamphlet; WO 2018009838 pamphlet; Shi et al., PNAS, 111(28):10131-36(2014); US Patent No. 9,644,180; Huang et al., Nature Communications 8:423(2017).

[0191] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more microsomes, virus-like particles (VLPs), or plant nanovesicles and plant messenger packs (PMPs). See, for example, WO 2011 / 097480, WO 2013 / 070324, WO 2017 / 004526, and WO 2020 / 041784.

[0192] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more anellosomes. The production and use of anellosomes for the delivery of therapeutic products are described in U.S. Patent No. 11,166,996, and its teachings regarding the design, preparation, and use of anellosomes are incorporated herein by reference.

[0193] Method for regulating a target protein In another aspect, the present disclosure provides a method for regulating the expression or activity of a target protein or a variant thereof identified in the Sequence Listing in a cell (target cell, cell of a target tissue), the method comprising contacting the cell (e.g., in vitro, ex vivo, or in vivo) with an agent that includes and / or regulates the expression or activity of the target protein identified herein, or a pharmaceutical composition comprising the agent.

[0194] In some embodiments, the target cell is a cancer cell (e.g., a metastatic cancer cell), a cell in the tumor microenvironment (e.g., a stromal cell), or a combination thereof. In certain embodiments, the target cell is a metastatic cancer cell.

[0195] In some embodiments, the target cell is associated with and / or involved in inflammation. In certain embodiments, the target cell is an epithelial cell, an endothelial cell, a stem cell, a non-immune cell, or a combination thereof.

[0196] In some embodiments, the target cells are associated with and / or involved in fibrosis, aging, and / or senescence. In certain embodiments, the target cells are epithelial cells, endothelial cells, stem cells, non-immune cells, or combinations thereof.

[0197] In some embodiments, the target cells are immune cells. In certain embodiments, the target cells are effector T cells, helper T cells, Th1 cells, Th2 cells, Th17 cells, B cells, natural killer (NK) cells, innate lymphoid cells (e.g., ILC1 cells, ILC2 cells, ILC3 cells), macrophages (e.g., M1 macrophages, M2 macrophages), monocytes, and / or antigen-presenting cells (e.g., dendritic cells), or combinations thereof.

[0198] In some embodiments, the target protein of the present disclosure is used to mediate depletion of a cell population (e.g., a population of cancer cells such as tumor cells; a population of immune cells). In some embodiments, the target protein of the present disclosure promotes cell targeting (e.g., delivering a therapeutic agent in a cell-type specific manner), for example as a binder of a surface marker.

[0199] The target tissue can be any tissue of the body.

[0200] In certain embodiments, the target tissue includes a tumor, a tumor microenvironment, a metastatic site, or combinations thereof.

[0201] In some embodiments, the target tissue is immune tissue. In some embodiments, the target cells are non-immune tissue. In some embodiments, the target tissue includes lymph nodes, spleen, secondary lymphoid organs, tertiary lymphoid organs, barrier tissues, skin, intestine, airway, wound, immune tissue, non-immune tissue, or combinations of the foregoing.

[0202] In certain embodiments, the effective amount is sufficient to reduce the expression of the target protein in the target cells and / or target tissue. In some embodiments, the reduction is at least about 10%, for example, at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10-99%, for example, about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0203] In certain embodiments, the effective amount is sufficient to increase the expression of the target protein in the target cells and / or target tissue. In some embodiments, the increase is at least about 10%, for example, at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is about 10-99%, for example, about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%. In some embodiments, the increase is about 1-100 fold, 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 fold.

[0204] In some embodiments, the effective amount is sufficient to modulate 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 of the foregoing. In some embodiments, the effective amount is sufficient to modulate a signaling pathway involving the Janus kinase (JAK) signaling family (e.g., JAK1, JAK2, JAK3, and TYK2), members of the signal transducer and activator of transcription (STAT) protein family (e.g., STAT1, STAT3), members of the protein kinase B family (e.g., RAC-α, RAC-β, or RAC-γ serine / threonine-protein kinase), members of the interferon regulatory factor (IRF) family, mitogen-activated protein kinase (MAPK), or a combination of the foregoing.

[0205] Methods of diagnosis and treatment In another aspect, the disclosure provides a method of detecting a disease or condition in a subject or predicting the likelihood (or risk level) of developing a disease or condition in a subject, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject, wherein the level of expression or activity of the target protein in the sample indicates the likelihood of developing a disease or condition in the subject, and 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.

[0206] In another aspect, the disclosure provides a method of classifying a subject based on the predicted likelihood of developing a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject; predicting the likelihood of developing a disease or condition based on the expression or activity of the target protein in the sample; and classifying the subject based on the predicted likelihood, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), infection, immune disease (e.g., inflammation and / or autoimmune disease), or a combination thereof.

[0207] In another aspect, the present disclosure provides a method of stratifying a set of subjects having a disease or condition, comprising: quantifying the expression and / or activity of a target protein in a sample from an individual subject within the set; and stratifying the set of subjects with respect to treatment according to the level of expression and / or activity of the target protein in the sample for each individual subject, 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.

[0208] In some embodiments, a higher expression or activity level of the target protein in a sample from a subject, as compared to an appropriate control (e.g., a reference standard), indicates the presence of a disease or condition or the likelihood of developing a disease or condition. In some embodiments, a lower expression or activity level of the target protein in a sample from a subject, as compared to an appropriate control (e.g., a reference standard), indicates the presence of a disease or condition or the likelihood of developing a disease or condition.

[0209] In some embodiments, the method further comprises administering to a subject determined or predicted to be at risk of developing a disease or condition an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein.

[0210] In some embodiments, the method further comprises administering to a subject determined or predicted to be at risk of developing a disease or condition an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein.

[0211] In another aspect, the present disclosure provides a method of preparing a sample useful for detecting the likelihood of developing a disease or condition in a subject, comprising: a) obtaining or having obtained a sample from the subject; b) adding a protease inhibitor, a control peptide, a standard peptide, or a combination thereof to the sample to prepare a sample useful for detecting the likelihood of developing cancer; c) quantifying the expression or activity of the target protein in the sample prepared in step b) comprising d) providing a method, wherein the disease or condition is selected from aging, senescence, fibrosis, autoimmunity, cancer (e.g., tumor), infectious disease, immune disease (e.g., inflammation and / or autoimmune disease), or a combination thereof

[0212] In another aspect, the disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein

[0213] In another aspect, the disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein, wherein the subject has an altered level of expression and / or activity of a target protein disclosed herein

[0214] As used herein, the term "treatment" or "treating" refers to the medical management of a subject with the purpose of improving, alleviating, stabilizing (i.e., without worsening), preventing or curing a disease, medical condition, or disorder. "Treatment" includes active treatment (treatment directed at improving a disease, medical condition, or disorder), causal treatment (treatment directed at the cause of a related disease, medical condition, or disorder), palliative treatment (treatment designed to relieve symptoms), prophylactic treatment (treatment directed at minimizing or partially or completely inhibiting the onset of a related disease, medical condition, or disorder); and adjuvant treatment (treatment utilized to supplement another therapy). Treatment includes a detectable or undetectable decrease in the degree of a disease or condition; prevention of the spread of a disease or condition; delay or deceleration of the progression of a disease or condition; improvement or alleviation of a disease or condition; and remission (partial or complete). "Improving" or "alleviating" a disease or condition means that the degree and / or undesirable clinical symptoms of the disease, disorder, or condition are reduced and / or the time course of progression is decelerated or lengthened as compared to the degree or time course in the absence of treatment. "Treatment" also includes prolonging survival as compared to expected survival in the absence of treatment. Persons in need of treatment include those who already have the condition or disorder, those who are susceptible to having the condition or disorder, or those in whom the condition or disorder should be prevented.

[0215] 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 neonate. In some embodiments, the human is a pediatric patient. In some embodiments, the human is a young person. In some embodiments, the human is an adult. In some embodiments, the human is under 18 years old. In some embodiments, the human is at least 18 years old. In some embodiments, the human is between 18 and 25 years old. In some embodiments, the human is at least 25 years old, such as at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 years old.

[0216] As used herein, the terms "effective amount", "therapeutically effective amount" or "sufficient amount" refer to an amount sufficient to effect a treatment (e.g., produce a beneficial or desired result), including an effect at the cellular, tissue or clinical level, when administered to a subject (e.g., a mammal such as a human cancer patient). Thus, the term depends on the context in which it is applied. For example, in the context of treating cancer, it is an amount of an agent sufficient to achieve a response as compared to the response obtained without administration of the agent. The amount of a given composition described herein corresponding to such an amount will vary depending on various factors such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, subject (e.g., age, sex, weight, etc.) or identity of the host being treated, but can nonetheless be routinely determined by one of ordinary skill in the art. In some embodiments, a "therapeutically effective amount" of the compositions of the present disclosure is an amount that produces a beneficial or desired result in a subject (e.g., as compared to a control). The therapeutically effective amount of the compositions of the present disclosure can be readily determined by one of ordinary skill in the art by routine methods known in the art. The dosing regimen can be adjusted to provide an optimal therapeutic response.

[0217] The therapeutic agents described in this specification can be administered via various routes of administration, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intraarterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion, and inhalation (e.g., intratracheal, intranasal, or oral inhalation, intranasal instillation), depending on the compound and the particular disease or condition being treated. Administration can be local or systemic, as indicated. The preferred mode of administration can vary depending on the particular compound selected.

[0218] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more additional therapeutic agents (e.g., a second therapeutic agent).

[0219] Administration of two or more therapeutic agents includes co - administering the therapeutic agents substantially simultaneously, such as in a combination pharmaceutical. Alternatively, such administration includes co - administering each therapeutic agent in multiple or separate containers (e.g., capsules, powders, and liquids). Such administration also includes using the therapeutic agents in a sequential manner, either almost simultaneously or at different times. When two or more therapeutic agents are administered, the therapeutic agents can be administered via the same route of administration or via different routes of administration.

[0220] In another aspect, the present disclosure provides a method of modulating the expression or activity of a target protein or a variant thereof identified in the Sequence Listing in a cell, the method comprising contacting the cell with an agent disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cell is within a subject.

[0221] In another aspect, the present disclosure provides a method of identifying an agent that modulates the expression and / or activity of a target protein (e.g., the target protein of the Sequence Listing), the method comprising: a) contacting a sample containing the target protein (e.g., a biological sample such as a cell or tissue) with an agent (e.g., a candidate agent being tested for its ability to modulate the expression and / or activity of the target); and b) determining whether the agent modulates the expression or activity of the target protein comprising There is provided a method indicating that a difference in the expression or activity of a target protein contacted with an agent, as compared to a reference, indicates that the agent modulates the expression or activity of the target protein.

[0222] In some embodiments, a difference of at least about 10% in the expression or activity of a protein contacted with an agent, as compared to a reference, indicates that the agent modulates the expression or activity of the protein. In some embodiments, the difference is at least about 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% or more.

[0223] In some embodiments, a decrease in the expression or activity of a target protein contacted with an agent, as compared to a reference, indicates that the agent inhibits the expression or activity of the target protein. In some embodiments, an increase in the expression or activity of a protein, as compared to a reference, indicates that the agent activates the expression or activity of the protein.

[0224] Indications Cancer A variety of cancers are treatable according to the methods described herein. In some embodiments, the cancer includes solid tumors (e.g., tumors of the breast, lung, prostate, colon, bladder, ovary, kidney, stomach, colon, rectum, testis, head and / or neck, pancreas, brain, skin). Thus, in some embodiments, the cancer is a solid tumor cancer. Solid tumor cancers treatable according to the methods described herein include breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, gastric cancer, colorectal cancer, rectal cancer, colon-rectal cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, and skin cancer. In some embodiments, the cancer is a blood cancer (e.g., leukemia, lymphoma, myeloma). Blood cancers 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.

[0225] Examples of cancers treatable according to the methods described herein include acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; pediatric adrenocortical carcinoma; AIDS-related cancers (e.g., Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma); anal cancer; appendiceal cancer; pediatric astrocytoma; pediatric atypical teratoid / rhabdoid tumor, central nervous system; basal cell carcinoma of the skin; bile duct cancer; bladder cancer; pediatric bladder cancer; bone cancer (including Ewing sarcoma, osteosarcoma, and malignant fibrous histiocytoma); brain tumor / cancer; breast cancer; Burkitt lymphoma; carcinoid tumor (gastrointestinal); pediatric carcinoid tumor; pediatric heart (cardiac) tumor; pediatric embryonal tumor; pediatric germ cell tumor; primary CNS lymphoma; cervical cancer; pediatric cervical cancer; cholangiocarcinoma; pediatric chordoma; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); chronic myeloproliferative tumor; colorectal cancer; pediatric colorectal cancer; pediatric craniopharyngioma; cutaneous T-cell lymphoma (e.g., mycosis fungoides and Sézary syndrome); ductal carcinoma in situ (DCIS); embryonal tumor, pediatric central nervous system,; endometrial cancer (uterine cancer); pediatric ependymoma; esophageal cancer; pediatric esophageal cancer; adherent neuroblastoma; Ewing sarcoma; pediatric extracranial embryonal tumor; extragonadal embryonal tumor; eye (ocular) cancer; pediatric intraocular melanoma; intraocular melanoma; retinoblastoma; fallopian tube cancer; fibrous histiocytoma, malignant, and osteosarcoma of bone; gallbladder cancer; stomach (gastric cancer); pediatric stomach (gastric cancer); gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); pediatric gastrointestinal stromal tumor; germ cell tumor; pediatric central nervous system embryonal tumor (e.g., pediatric extracranial embryonal tumor, extragonadal embryonal tumor, ovarian embryonal tumor, testicular cancer); gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; pediatric heart tumor; hepatocellular (liver) cancer; histiocytosis, Langerhans cell; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; pediatric intraocular melanoma; islet cell tumor, pancreatic neuroendocrine tumor; Kaposi sarcoma; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia; lip and oral cavity cancer; liver cancer; lung cancer (non-small cell and small cell); pediatric lung cancer; lymphoma; male breast cancer; malignant fibrous histiocytoma of bone and osteosarcoma; melanoma; pediatric melanoma; melanoma, intraocular (ocular); pediatric intraocular melanoma; Merkel cell carcinoma; malignant mesothelioma; pediatric mesothelioma; metastatic cancer; metastatic squamous neck cancer of unknown primary; midline carcinoma with NUT gene alteration; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides;Myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms; chronic myeloid leukemia (CML); acute myeloid leukemia (AML); chronic myeloproliferative neoplasms; nasal and paranasal cavity cancers; nasopharyngeal cancer; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung cancer; oral cancer, lip cancer and oral cavity cancer as well as oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; pediatric ovarian cancer; pancreatic cancer; pediatric pancreatic cancer; pancreatic neuroendocrine tumors; papillomatosis (pediatric larynx); paraganglioma; pediatric paraganglioma; paranasal cavity cancer and nasal cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pediatric pheochromocytoma; pituitary tumor; plasma cell tumor / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; primary central nervous system (CNS) lymphoma; primary peritoneal cancer; prostate cancer; rectal cancer; recurrent cancer; renal cell (kidney) cancer; retinoblastoma; pediatric rhabdomyosarcoma; salivary gland cancer; sarcoma (e.g., pediatric rhabdomyosarcoma, pediatric hemangioma, Ewing sarcoma, Kaposi sarcoma, osteosarcoma (bone cancer), soft tissue sarcoma, uterine sarcoma); Sézary syndrome; skin cancer; pediatric skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; metastatic squamous neck cancer of unknown primary; stomach (gastric) cancer; pediatric gastric cancer; cutaneous T-cell lymphoma (e.g., mycosis fungoides and Sézary syndrome); testicular cancer; pediatric testicular cancer; throat cancer (e.g., nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer); thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; endometrial uterine cancer; uterine sarcoma; vaginal cancer; pediatric vaginal cancer; hemangioma; vulvar cancer; and Wilms tumor and other pediatric kidney tumors are included.;

[0226] Metastases of the aforementioned cancers can also be treated according to the methods described herein. In some embodiments, the cancer is metastatic cancer.

[0227] In some embodiments, the cancer is selected from lung cancer, breast cancer, Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary central nervous system lymphoma, chronic lymphocytic leukemia, epithelial ovarian cancer, prostate cancer, squamous cell carcinoma, non-melanoma skin cancer, nasal polyp, basal cell carcinoma, keratinocyte carcinoma, multiple myeloma, serous invasive ovarian cancer, hepatocellular carcinoma, small cell lung carcinoma, adenocarcinoma, lung adenocarcinoma, non-small cell lung cancer, ovarian cancer or colorectal cancer.

[0228] In some embodiments, the treatment is: a) inhibiting cancer cell growth, proliferation, metastasis, invasion or migration, or a combination of the foregoing; b) promoting cancer cell death; c) inducing autophagy in cancer cells, or a combination of the foregoing is.

[0229] In some embodiments, the effective amount is: a) inhibiting cancer cell growth, proliferation, metastasis, invasion or migration, or a combination of the foregoing; b) promoting cancer cell death; c) inducing autophagy in cancer cells, d) or a combination of the foregoing is sufficient to effect.

[0230] In some embodiments, the effective amount is sufficient to reduce cancer (e.g., tumor) growth, proliferation, metastasis, invasion, migration, autophagy or a combination of the foregoing. In certain embodiments, the reduction is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10 - 99%, e.g., about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0231] In some embodiments, the effective amount is sufficient to reduce cancer cell proliferation or tumor growth in a subject. In some embodiments, the reduction in cancer cell proliferation or tumor growth is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the reduction in cancer cell proliferation or tumor proliferation is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75% or 70 to 75%.

[0232] In some embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease) tumor autophagy, for example, by increasing at least one tumor inhibitory function of autophagy and / or decreasing at least one tumor promoting function of autophagy.

[0233] In some embodiments, the effective amount is sufficient to increase autophagy of cancer. In certain embodiments, the increase is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0234] In some embodiments, the effective amount is sufficient to reduce autophagy of cancer. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10-99%, such as about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0235] In some embodiments, the effective amount is sufficient to prevent death of the subject and thereby reduce cancer (e.g., tumor) mortality. In certain embodiments, the reduction in cancer (e.g., tumor) mortality is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction in cancer (e.g., tumor) mortality is about 10-99%, such as about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0236] In some embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease) the expression of a target protein in cancer (e.g., tumor) cells.

[0237] In some embodiments, an effective amount is sufficient to, for example, inhibit cancer growth, reduce cancer malignancy, inhibit cancer metastasis, promote remission, modulate (increase and / or decrease) an immune-mediated response associated with cancer, or regulate the body's response to cancer by the foregoing combinations.

[0238] Immuno - Oncology In some embodiments, the present disclosure provides a treatment that harnesses the immune system. In some embodiments, the method relates to immuno - oncology (e.g., cancer immunotherapy). In still further embodiments, the immune system is the innate immune system. In some embodiments, the immune system is the adaptive immune system. In still further embodiments, the treatment relates to humoral immunity or antibody - mediated immunity. In some embodiments, the treatment relates to cell - mediated immunity, such as cancer. In some embodiments, the method relates to treatment at the time of initial disease progression or prior to initial disease progression, while in other embodiments, the method relates to treatment or prevention of late - stage disease progression at the time of late - stage disease progression. In still further embodiments, the immuno - oncology effect results from stimulation of the immune system.

[0239] In some embodiments, an effective amount is sufficient to modulate (e.g., increase) the subject's immune system against cancer. In certain embodiments, the effective amount is the following: a) Immune cell - related readouts, immune cell activation, degranulation, maturation, migration, polarization, proliferation, and recruitment of immune cells (e.g., macrophages, monocytes, or dendritic cells); b) Lymph node activation, innervation, differentiation, egress, homing; c) Cytokine production; d) Antibody - dependent cell - mediated cytotoxicity (ADCC) and / or antibody - dependent cell - mediated phagocytosis (ADCP); e) Antigen presentation; f) Target protein expression, g) Or combinations of the foregoing is sufficient to modulate (e.g., increase or decrease) it.

[0240] In some embodiments, the adjustment is an increase. In certain embodiments, the increase is at least about 10%, for example, at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is from about 10% to 99%, for example, from about 10% to 98%, 15% to 98%, 15% to 97%, 20% to 97%, 20% to 96%, 25% to 96%, 25% to 95%, 30% to 95%, 30% to 94%, 35% to 94%, 35% to 93%, 40% to 93%, 40% to 92%, 45% to 92%, 45% to 91%, 50% to 91%, 50% to 90%, 55% to 90%, 55% to 85%, 60% to 85%, 60% to 80%, 65% to 80%, 65% to 75%, or 70% to 75%.

[0241] In some embodiments, the modulation is a decrease. In certain embodiments, the decrease 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 decrease is from about 10% to 99%, for example, from about 10% to 98%, 15% to 98%, 15% to 97%, 20% to 97%, 20% to 96%, 25% to 96%, 25% to 95%, 30% to 95%, 30% to 94%, 35% to 94%, 35% to 93%, 40% to 93%, 40% to 92%, 45% to 92%, 45% to 91%, 50% to 91%, 50% to 90%, 55% to 90%, 55% to 85%, 60% to 85%, 60% to 80%, 65% to 80%, 65% to 75%, or 70% to 75%.

[0242] In some embodiments (e.g., immuno-oncology specific therapies), an effective amount is sufficient to modulate (e.g., increase or decrease) immune cell-related readouts, migration of immune cells (e.g., antigen-presenting cells (e.g., dendritic cells and / or macrophages) and / or T cells), proliferation of immune cells, recruitment of immune cells (e.g., antigen-presenting cells (e.g., dendritic cells and / or macrophages), monocytes, T cells, and / or B cells), lymph node homing of immune cells (e.g., dendritic cells and / or T cells), lymph node egress of immune cells (e.g., dendritic cells and / or T cells), differentiation of immune cells, activation of immune cells, polarization of immune cells, cytokine production (e.g., increasing pro-inflammatory cytokines, decreasing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, decreasing anti-inflammatory cytokines), degranulation of immune cells, maturation of immune cells, ADCC of immune cells, ADCP of immune cells, antigen presentation, tumor homing of immune cells (e.g., T cells); increased tumor egress of immune cells (e.g., regulatory T cells), decreased tumor egress of immune cells (e.g., CD8 + T cells), target protein expression, or a combination of the foregoing.

[0243] Immunity / Inflammation In some embodiments, the methods of the disclosure relate to immunity. In some embodiments, the immunity relates to bacteria, parasites, viruses, fungi, and / or cancer cells. In some embodiments, the immunity is autoimmune and is the result of the immune system attacking self-molecules. In some embodiments, the methods relate to the treatment of immune tolerance.

[0244] In some embodiments of the present disclosure, the method relates to the treatment of inflammation. In some embodiments, the inflammation is acute or relatively short-term and lasts from a few minutes to a few hours. In still other embodiments, the inflammation is chronic or longer-term, lasting for weeks to months, and in some cases, years. In some embodiments, the methods disclosed herein relate to the treatment or prevention of conditions associated with inflammation. In certain embodiments, such conditions include, but are not limited to, skin flushing, pain or tenderness, swelling, heat, fatigue, fever, joint pain or stiffness, stomatitis, and rash. In some embodiments, the inflammation is caused by other diseases or disorders.

[0245] In some embodiments, the disclosed method relates to the treatment of autoimmune diseases. In some embodiments, autoimmune diseases can include diseases of the joints and muscles (e.g., psoriatic arthritis, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus), diseases of the gastrointestinal tract (e.g., Crohn's disease, celiac disease, ulcerative colitis, inflammatory bowel disease), diseases of the endocrine system (e.g., Graves' disease, Hashimoto's thyroiditis, Addison's disease), diseases of the skin (e.g., dermatomyositis, psoriasis, scleroderma), diseases of the nervous system (e.g., chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, multiple sclerosis), and other diseases (e.g., myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, autoimmune lymphoproliferative syndrome, type 1 diabetes), but are not limited thereto.

[0246] In some embodiments, the disease or condition is an inflammatory disease and / or an autoimmune disease. A wide variety of inflammatory and / or autoimmune diseases are treatable according to the methods described herein. In some embodiments, the inflammatory and / or autoimmune diseases include Alzheimer's disease, asthma, endometriosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease and ulcerative colitis), multiple sclerosis (MS), non-alcoholic fatty liver disease (e.g., non-alcoholic fatty liver disease (NAFLD)), obesity, Parkinson's disease, cancer, psoriasis, rheumatoid arthritis (RA), scleroderma, systemic lupus erythematosus (SLE), type 1 diabetes, type 2 diabetes, or a combination thereof.

[0247] In some embodiments, the target protein activates an immune response. In certain embodiments, the target protein inhibits an immune response. In some embodiments, the immune response is an innate immune response (e.g., a humoral and / or cell-mediated immune response). In certain embodiments, the immune response is an adaptive immune response (e.g., a humoral and / or cell-mediated immune response). Non-limiting examples of immune responses include T cell-mediated immune responses (e.g., cytokine production and cytotoxicity), B cell-mediated immune responses, humoral immune responses, and activation of cytokine-responsive cells (e.g., macrophages).

[0248] In some embodiments, the target protein enhances signals involved in T cell activation and / or survival. In certain embodiments, the target protein activates a stimulatory checkpoint molecule. Non-limiting examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, glucocorticoid-induced TNFR family-related gene (GITR), inducible T cell co-stimulator (ICOS). In certain embodiments, the target protein is an agonist for CD28.

[0249] In some embodiments, the target protein reduces signals involved in T cell anergy and / or exhaustion. In certain embodiments, the target protein inhibits an inhibitory checkpoint molecule. Non-limiting examples of inhibitory checkpoint molecules include programmed cell death protein 1 (PD-1), PD-L1, PD-L2, T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), B and T lymphocyte attenuator (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), V domain Ig suppressor of T cell activation (VISTA), sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC 7), and sialic acid-binding immunoglobulin-type lectin 9 (SIGLEC 9). In certain embodiments, the target protein is an inhibitor of PD-1.

[0250] In some embodiments, the effective amount is as follows: a) Development of high endothelial venules (HEV) and / or tertiary lymphoid organs (TLO); b) Activation, degranulation, differentiation, maturation, migration, polarization, proliferation, and / or recruitment of immune cells (e.g., macrophages, monocytes, or dendritic cells); c) Egress and / or homing of immune cells to lymph nodes; d) Egress and / or homing of immune cells to tumors; e) Cytokine production; f) Antigen presentation; g) Target protein expression; or h) Autoantibody levels, or a combination thereof is sufficient to modulate (e.g., increase or decrease) them.

[0251] In some embodiments, the effective amount is as follows: a) Development of HEV and / or TLO; b) Activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells (e.g., macrophages, monocytes or dendritic cells); c) Exit and / or homing of immune cells from lymph nodes; d) Exit and / or homing of immune cells from tumors; e) Cytokine production; f) Antigen presentation; g) Target protein expression; or h) Autoantibody levels, or a combination thereof is sufficient to increase.

[0252] In certain embodiments, the increase is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is about 10 - 99%, e.g., about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0253] In some embodiments, the effective amount is as follows: a) Development of HEV and / or TLO; b) Activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells; c) Exit and / or homing of immune cells from lymph nodes; d) Exit and / or homing of immune cells from tumors; e) Cytokine production; f) Antigen presentation; g) target protein expression; or h) autoantibody levels, or a combination thereof is sufficient to reduce.

[0254] In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10 - 99%, such as about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0255] In some embodiments, the effective amount is a) increasing organ function; b) modulating inflammation (e.g., increasing or decreasing); c) reducing the level of autoantibodies; d) reducing the rate and / or number of relapses and / or recurrences; e) reducing the viral load; or f) reducing (e.g., controlling) infection, or a combination of the foregoing is sufficient to effect.

[0256] In some embodiments, the effective amount is sufficient to increase organ function, inflammation, or a combination thereof. In certain embodiments, the increase is at least about 10%, for example, at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is about 10 - 99%, for example, about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0257] In some embodiments, the effective amount is sufficient to decrease inflammation, the level of autoantibodies, the rate and / or number of relapses and / or recurrences, the viral load or infection, or a combination of the foregoing. In certain embodiments, the decrease is at least about 10%, 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 decrease is about 10 - 99%, for example, about 10 - 98%, 15 - 98%, 15 - 97%, 20 - 97%, 20 - 96%, 25 - 96%, 25 - 95%, 30 - 95%, 30 - 94%, 35 - 94%, 35 - 93%, 40 - 93%, 40 - 92%, 45 - 92%, 45 - 91%, 50 - 91%, 50 - 90%, 55 - 90%, 55 - 85%, 60 - 85%, 60 - 80%, 65 - 80%, 65 - 75%, or 70 - 75%.

[0258] In some embodiments, the 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 are hematopoietic-derived cells including lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., basophils, eosinophils, granulocytes, macrophages, mast cells, and monocytes). The target protein can be expressed on cancer cells (e.g., metastatic cancer cells), in the tumor microenvironment (e.g., on stromal cells), or on non-malignant cells (e.g., immune cells).

[0259] In some embodiments, the effective amount is sufficient to increase the immune response. In certain embodiments, the increase is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is about 10-99%, e.g., about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0260] In some embodiments, the effective amount is sufficient to reduce the immune response. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0261] In some embodiments, the effective amount is sufficient to reduce the inflammatory response. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

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

[0263] In certain embodiments, the effective amount is sufficient to modulate the expression of the target protein in immune cells.

[0264] In some embodiments (e.g., for immune and / or inflammatory treatment), the effective amount is sufficient to regulate the migration, proliferation, recruitment, lymph node homing, lymph node egress, differentiation, activation, polarization, cytokine production (e.g., increasing pro-inflammatory cytokines, decreasing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, decreasing anti-inflammatory cytokines), degranulation, maturation, antigen presentation, target protein expression, inflammation, autoantibody levels (e.g., increasing or decreasing); increasing organ function; reducing the rate and / or number of relapses or recurrences, viral load; controlling infection or performing the aforementioned combinations in immune cells (e.g., antigen-presenting cells (e.g., dendritic cells and / or macrophages) and / or T cells).

[0265] Aging The methods described herein are applicable to the treatment of aging, aging-related conditions, and / or aging-related disorders or diseases. In some embodiments, the methods described herein include the treatment of overall health, body temperature, weight, height, abdominal circumference, reproductive ability, body fat, heart rate, blood pressure, pulse rate, blood oxygen level, respiratory rate, respiratory pattern, blood glucose level, blood pH, cardiac output, heart rhythm, and the concentration of certain substances in the blood. In some embodiments, the treatment can be evaluated by measurement of complete blood count, red blood cells, white blood cells, platelets, hemoglobin, hematocrit, mean corpuscular volume, basic metabolic panel, blood glucose, calcium, electrolyte tests, kidney function, blood enzyme tests, troponin, creatine kinase, lipoprotein panel, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, coagulation panel, and / or bone marrow examination.

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

[0267] Aging In some embodiments, the methods disclosed herein are for the treatment of aging. In still further embodiments, the methods disclosed herein relate to the treatment of aging-related diseases or disorders. In some embodiments, diseases or disorders include, but are not limited to, diabetes, metabolic syndrome, and obesity. In other embodiments, the disease or disorder is related to photosensitivity or photoaging. In still further embodiments, the disease or disorder can be selected from arthritis, Alzheimer's disease, asthma, blindness, cancer, chronic bronchitis, chronic kidney disease, chronic obstructive pulmonary disease, coronary heart disease, deep vein thrombosis, dementia, depression, diabetes, epilepsy, heart failure, hypercholesterolemia, hypertension, motor neuron disease, multiple sclerosis, osteoporosis, Paget's disease of bone, Parkinson's disease, shingles, and stroke.

[0268] In some embodiments, the effective amount is sufficient to reduce aging. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10-99%, such as about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0269] Fibrosis In some embodiments, the methods of the present disclosure relate to the treatment of fibrosis. In some embodiments, the methods relate to the treatment of conditions associated with or resulting from fibrosis. In some embodiments, fibrosis is pulmonary fibrosis, hepatic fibrosis, dermal fibrosis, renal fibrosis, pancreatic fibrosis, systemic sclerosis, cardiac fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, and / or macular degeneration.

[0270] In some embodiments, the effective amount is sufficient to reduce fibrosis. In certain embodiments, the reduction is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is from about 10 to 99%, such as from about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0271] Infectious disease In some embodiments, the present disclosure relates to the treatment of infectious diseases. In some aspects, the infectious disease is a bacterial infection, a protozoal infection, a viral infection, a fungal infection, or other pathogenic infection. In some embodiments, the infectious disease is AIDS or HIV, viral hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C), tuberculosis, Salmonella, Lyme disease, meningococcal disease, influenza, measles, mumps, rubella (e.g., German measles), pneumonia, sexually transmitted infections (e.g., syphilis, chlamydia, gonorrhea), chronic rhinitis, whooping cough, pertussis, or a combination thereof.

[0272] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used in this specification are intended to have the meanings commonly understood by those skilled in the art related to this disclosure. In some cases, terms with commonly understood meanings are defined in this specification for the sake of clarity and / or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as indicating a substantial difference from what is commonly understood in the art. It should be further understood that terms such as those defined in commonly used dictionaries should be construed to have meanings consistent with their meanings in the context of the relevant art and / or as otherwise defined in this specification.

[0273] The terms used in this specification are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0274] As used in this specification, the indefinite articles "a", "an", and "the" should be understood to include plural references unless the context clearly indicates otherwise.

[0275] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted to mean, for example, including the stated integer or step or group of integers or steps, but not excluding other integers or steps or group of integers or steps. As used in this specification, the term "comprising" can be replaced by the term "containing" or "including".

[0276] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claims. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claims. Whenever used in the context of an aspect or embodiment of the present disclosure, the terms "comprising", "containing", "including", and "having" can all, in some embodiments, be replaced with "consisting of" or "consisting essentially of" to change the scope of the disclosure.

[0277] As used herein, the connective term "and / or" between a plurality of recited elements is understood to encompass both the individual choices and the combined choices. For example, when two elements are connected by "and / or", the first choice refers to the applicability of the first element without the second element. The second choice refers to the applicability of the second element without the first element. The third choice refers to the applicability of the first element and the second element together. Any one of these choices falls within the scope of meaning and is thus understood to meet the requirements of the term "and / or" as used herein. The simultaneous applicability of multiple choices also falls within the scope of meaning and is thus understood to meet the requirements of the term "and / or".

[0278] When a list is presented, it should be understood that each individual element of that list, and all combinations of that list, are separate embodiments, unless otherwise stated. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

Examples

[0279] Illustration Example 1: Verification of Target Protein as a Checkpoint by Overexpression This example demonstrates the ability of the target protein of the present disclosure to act as a checkpoint immunomodulatory factor. In this example, human target protein (including positive control PD-1 and CTLA-4) cDNA is cloned into the pLVX-TetOne vector (Clontech) under the doxycycline (dox)-inducible TRE3G promoter. Human checkpoint ligand PD-L1 and PD-L2 cDNA are cloned into the pLVX-IRES-Puro vector (Clontech) under the expression of the constitutive CMV promoter. T cell lines are constructed by infecting Jurkat cells with the lentiviral vector pLVX-TetOne-target protein. B cell lines are constructed by infecting Raji cells with the lentiviral vector pLVX-IRES-Puro-target protein or pLVX-IRES-Puro-PD-L2. Anti-CD3 beads are prepared in-house by coating the anti-human CD3 antibody from eBioscience onto Invitrogen's Dynabeads M-450 epoxy beads using Invitrogen's coating protocol.

[0280] On day 1, target protein expression is induced in Jurkat and Raji cell lines by adding dox up to a concentration of 250 ng / mL. Two days after dox induction, Jurkat and Raji cells are washed and resuspended in RPMI + 10% FBS at 1 × 10 6 cells. Anti-CD3 beads are added to Jurkat cells, and in parallel, control cells (dox negative) are used to evaluate IL-2 production by non-suppressed T cells. Jurkat cells and beads [and antibody] are incubated at room temperature for 30 minutes. 500 μl of Raji cells are added to Jurkat and beads. The final ratio is 500k Jurkat:500k Raji:2000k beads or 1:1:4. Using a multi-channel pipette, 200 μl of the cell:bead mixture is transferred to a 96-well plate (U-bottom). This is maintained in an incubator at 37°C for 22 hours.

[0281] The next day, the plates are centrifuged and the supernatant is transferred to a new 96-well plate (R&D Systems #D2050) for IL-2 ELISA. A decrease in the detection of IL-2 is expected in exhausted T cells. This is the method for selecting checkpoint target proteins.

[0282] Example 2. Verification of secreted target proteins that stimulate or inhibit cytokine release. A CBA (Cytometry) experiment to examine pro-inflammatory cytokines produced by PBMCs treated with secreted peptides. This example demonstrates the verification 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.

[0283] Peripheral blood mononuclear cells were seeded for 24 - 48 hours with or without the T cell activator CD3 / CD8 and with or without the peptide. Cytokines were measured from the supernatant after incubation with a CBA Cytometer.

[0284] Materials and methods: PBMC thawing: PBMCs were thawed by preheating TexMACS medium (Miltenyi Biotech) in a conical 20 mL aliquot of 1 - 50 mL per donor. After warming the medium to 37 °C, the cells were removed from liquid nitrogen and transferred to a tissue culture hood. For each donor, 1 mL of medium from the warmed aliquot was added to the cryovial and mixed up and down. Then, the medium + thawed cells were returned to a 50 mL conical. This was repeated until all cells were thawed and added to the 50 mL tube. Continue with other donors if used. The cells were rotated at 400 x g for 5 minutes. The supernatant was aspirated and the cells were resuspended at approximately 10 × 10^6 cells / mL (refer to the vial for cell count).

[0285] PBMC Seeding: Cryopreserved PBMCs from normal, healthy human volunteers (StemCell Technologies) were thawed and seeded at 250,000 cells / well. After thawing the PBMCs and resuspending them at 10×10^6 cells / mL, the cells were counted (Countess III, Thermo Scientific) and adjusted to 10×10^6 cells / well based on the count. The number of PBMCs determined by the number of wells for the experiment was taken. For example, 10 wells would require a total of 2.5×10^6 cells or 0.250 mL. The cells required for the experiment were then transferred to another conical tube. Warmed TexMACS medium was added such that the final concentration was 250,000 cells / 190 μL of medium. For example, for 10 wells, the total medium volume is 1.9 mL (190 μL×10 wells), subtracting 0.250 mL of cells (1.9 mL - 0.250 mL), or 1.65 mL of medium needs to be added to 0.250 mL of cells. 190 μL of cells + medium was added to a 96-well U-bottom plate. It was placed in the incubator until dilution was carried out and it was ready.

[0286] Peptide Dilution: Ideally, all peptides should have a starting concentration of at least 10 mM in order for the peptides in DMSO (ATCC) to reach a final concentration of 1 μM (in H2O) or 0.5 μM (in DMSO). The dilution of all test substances should be carried out at 20-fold the final concentration. For example, the final concentration of 1 μM peptide needs to be diluted at 20 μM.

[0287] Reagent Dilution: Dilution with TexMACS medium is carried out to a sufficient extent to make 10 μL / well in triplicate (30 μL, 20-fold). The activating factors - CD3 / CD28 (TransAct; Miltenyi Biotech) or cytokines are at 21-fold and are added after a 45-minute incubation with the peptide. The relevant controls - positive: TransAct + cells, negative: vehicle + cells must be included. Dilution should also be carried out in an ultra-low binding plate (Corning).

[0288] Assay Procedure: Peptide Treatment: As described above, start the assay by thawing the cells and seeding them in a 96-well U-bottom tissue culture plate (Corning). Place the cell plate in a tissue culture incubator (5% CO2, 37°C). While the cells are in the incubator, start the preparation of the dilution (optimally within 1 hour or less) as described above. Remove the cell plate from the incubator and add 10 μL of a 20-fold dilution to the relevant wells. After the addition of the inhibitor, place the cell plate in the tissue culture incubator for 45 minutes. Remove the plate from the incubator and add 10 μL of 21-fold TransAct to the relevant wells*. Then, return the plate to the incubator and incubate for 24 hours. After 24 hours, spin the plate at 400 x g for 5 minutes.

[0289] *Activation: This assay can be performed in two different formats - activation and inhibition. The procedure is the same except that TransAct is not required in the peptide wells for activation. TransAct is only required in the control wells. The purpose of this procedure is to determine the possible response from the peptide alone.

[0290] Cytometric Bead Array (CBA): After spinning, transfer the supernatant (approx. 150 μL) to a new ultra-low binding U-bottom plate. Then, freeze the supernatant at -20°C until thawed for CBA (BD) to determine the levels of human IL-2, TNFα, IL-1b, IP-10, IL-10, and IFNg. CBA was performed according to the manufacturer's protocol.

[0291] SEQ ID NO: 33885 significantly blocks the release of TNF-α from activated PBMCs but does not block the release of another inflammatory cytokine, IL-1b. The specific inhibition of TNF-α release reduces the inflammatory response (Figures 1 and 2).

[0292] Table of Hits: X = Hits associated with significant activation or inhibition of related cytokine release. Inhibition occurs when the peptide is applied together with anti-CD3 / CD28 T cell activator. When the peptide is applied alone to PBMC, activation occurs (see Materials and Methods). Significance = P = 0.05

[0293]

Table 1

[0294] Example 3. Verification of target protein as a modulator of innate immune response. This example demonstrates the verification of a target protein, SEQ ID NO: 37413, as a modulator of innate immune response. Specifically, the target protein inhibits the response of multiple toll-like receptors to their specific agonists. Thus, the target protein is suitable for the treatment of autoimmune diseases such as lupus, psoriasis, arthritis, and multiple sclerosis. As a toll-like receptor (TLR) inhibitor, SEQ ID NO: 37413 would be particularly useful for the treatment of lupus.

[0295] To determine the effect of the target protein on TLR activity, the target protein, an irrelevant protein, and a control were applied to a commercially available reporter cell line that monitors two major signaling pathways in response to TLR activation, in the presence or absence of an established TLR agonist. To determine which immune cells are most responsive to the target protein, the target protein was applied to peripheral blood mononuclear cells (PBMC), and then, 24 hours later, the PBMC were subjected to single-cell RNA-seq using cell hashing.

[0296] Methods and Materials: Dual reporter assay: For example: NF-kB-SEAP and IRF-Lucia luciferase reporter monocytes (THP1) cells (InvivoGen) are cultured in RPMI 1640 medium supplemented with HEPES buffer (10 mM), sodium pyruvate (1 mM), glucose (4.5 g / L), fetal bovine serum (10%), penicillin (100 U / mL), streptomycin (100 μg / mL) and 2-mercaptoethanol (0.05 mM). THP1 cells are treated with 100 ng / mL LPS (Sigma-Aldrich) for 24 hours, with the target protein (final concentration 10 μM), or left untreated.

[0297] After treatment, THP1 cells are cultured for 24 hours and then assayed for reporter activity. When using QUANTI-Blue (InvivoGen), a SEAP detection reagent, and QUANTI-Luc (InvivoGen), a luciferase detection reagent, both reporter proteins can be measured in the cell culture supernatant. QUANTI-Blue is a colorimetric enzyme assay developed to determine any alkaline phosphatase activity in biological samples such as cell culture supernatants. QUANTI-Luc is a lyophilized assay reagent containing all the components necessary to quantitatively measure the activity of other coelenterazine-utilizing luciferases of Lucia Luciferase. An increase in reporter activity after treatment with the target protein is observed in cells where the target protein is immunostimulatory. Furthermore, a decrease in reporter activity after treatment with the target protein is observed in cells where the target protein is immunosuppressive.

[0298] scRNA-seq: For example, the shift of a cell population after treatment with a target protein is evaluated by single-cell RNA-seq (scRNA-seq). The target protein (e.g., as listed in the sequence listing) is synthesized by solid-phase peptide synthesis (SPPS) using fluorenylmethyloxycarbonyl (Fmoc) protecting group chemistry. Human primary blood mononuclear cells are seeded at 1×10 PBMC per well of a 12-well plate containing 1 ml of RPMI-1640 (10% FBS). PBMCs are treated with 1 μg / mL of LPS (Sigma-Aldrich) for 24 hours with or without the target protein at a final concentration of 10 μM, or left untreated. Single-cell suspensions are collected and centrifuged at 400×g for 5 minutes at 4°C. The medium is discarded and the cells are resuspended in 1 ml of cell staining buffer (BioLegend). 5 μl of Human TruStain FcX (BioLegend) is added to the cells and incubated at 4°C for 10 minutes. 1 μg of single-cell hashing antibody (BioLegend TotalSeq) is added to the cells together with approximately 50 μl of cell staining buffer to make a total volume of 100 μl. This is incubated at 4°C for 30 minutes and then washed 3 times at 400×g. 10 different cell hashing samples are pooled into one tube containing the desired number of cells (about 1000 cells / μl).

[0299] Single cells are processed through the Chromium Next GEM Single Cell 3’_Reagent Kit (Dual Index) (10X Genomics CG000317 Rev C) according to the protocol provided by 10X Genomics. Briefly, the sample is processed by GEM generation and barcoding, GEM-RT cleanup and cDNA amplification, followed by 3’ gene expression library construction, cell surface protein library construction, and finally sequencing.

[0300] After sequencing, reads are aligned to the human reference genome (GENCODE34 / GRCH38). Reads are demultiplexed using DNA barcoded antibodies and are run through quality control (e.g., doublets are removed, singlet cells are selected with less than 15% mitochondrial contamination at the RNA level, and reads contain more than 500 genes). Raw data is normalized. Principal component analysis is performed and cell clusters are annotated. Differential gene expression analysis is performed between treatment with control and target proteins. Shifts in cell population dynamics observed by principal component analysis and / or shifts in gene expression after treatment with the target protein in the supernatant demonstrate that the target protein acts as a circulating factor.

[0301] SEQ ID NO: 37413 significantly blocks the 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 immunity-related diseases such as lupus, SEQ ID NO: 37413 strongly affected activated monocytes and dendritic cells while having a relatively small effect on T cells, B cells, and NK cells (FIGS. 3, 4, 5A, and 5B).

[0302]

Table 2

[0303] Example 4. Inhibition of NK cell function by blocking the functions of costimulatory ligands and cytokines is a promising therapeutic strategy for treating allogeneic immune responses and graft rejection. This example demonstrates the validation of target proteins, SEQ ID NO: 33482, SEQ ID NO: 37776, and SEQ ID NO: 35050, as novel costimulatory ligands that enhance NK-mediated cytotoxicity in human B cell lymphoma.

[0304] Using the CRISPR system, a pooled KO library containing 4,708 individual novel proteins and 998 known proteins within 2,127 lncRNAs in Raji cells (human B cell lymphoma) was generated. To understand the role of these proteins in the regulation of NK cell cytotoxicity, the inventors performed cell killing assays using primary human NK cells. The inventors examined changes in the initial library representation in Raji cells in terms of library representation 24 hours after co-incubation with NK cells. The inventors used known regulators of NK cell cytotoxicity as a reference to establish the significance of hits. Novel co-stimulatory factors (enhancers) of NK-mediated cytotoxicity include targets that significantly inhibit Raji cell killing against CRISPR KO, similar to the known controls - ICAM1 and FAS.

[0305] Materials and Methods: CRISPR KO Generation. The CRISPR guide RNA library was designed by ProFound Therapeutics to target 4,708 different predicted novel proteins localized within 2,127 lncRNAs, 998 known proteins including positive (inhibitors, e.g., B2M, TAP1, TAP2, HLA-DM) and negative (co-stimulatory agents, e.g., ICAM1, FAS, IL7, DGKE) regulators of NK cell function, and 900 neutral controls (non-target and intergenic guide RNAs). The CRISPR guide RNA library was packaged into lentivirus and transduced into the Raji Cas9 strain at an MOI of 0.3 and selected using puromycin as described in Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783 - 4.

[0306] Isolation and Expansion of NK. Primary human NK cells were isolated from cryopreserved PBMCs using the EasySep Human NK Cell Isolation Kit (StemCell, catalog number 17955). Primary human NK cells were expanded using the immunoCult NK Cell Expansion Kit (StemCell, catalog number 100 - 0711) according to the manufacturer's protocol.

[0307] Screening setup. Screening was performed using four biological replicate experiments. The NK cell cytotoxicity assay was performed by co-culturing 20 million Raji CRISPR KO library cells with 60 million primary NK cells for 24 hours in a T75 tissue culture flask. NK cells were depleted using a positive CD56 selection kit (StemCell, catalog number 17855). Dead cells were removed using an EasySep dead cell removal (Annexin V) kit (StemCell, catalog number 17899). The remaining Raji CRISPR KO library cells were processed to extract genomic DNA. Prior to exposure to NK, NGS library prep and Illumina sequencing were performed on these cells and Raji cells as described in [Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783-4].

[0308] Data analysis. Screening analysis 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)].

[0309] We have found several lncRNA-encoded proteins that inhibit the killing of Raji cells in the presence of primary NK cells upon loss of function. The magnitude of the effect is comparable to control proteins that 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 a role in enhancing NK cell cytotoxicity and predicted cell surface localization.

[0310] Additional novel proteins for which a co-stimulatory effect on NK cell cytotoxicity with predicted cell surface localization was verified: Array No. 34013, Array No. 34021, Array No. 33482, Array No. 35972, Array No. 36456, Array No. 37776, Array No. 33345, Array No. 35531, Array No. 36975, Array No. 37561, Array No. 35050, Array No. 34728, Array No. 34076

[0311] Additional novel proteins for which the co-stimulatory effect on NK cell cytotoxicity with predicted secretion was verified: Array No. 37151, Array No. 37187, Array No. 37199, Array No. 38297, Array No. 34018, Array No. 37016, Array No. 33395, Array No. 33372, Array No. 38018, Array No. 36168, Array No. 34490, Array No. 35219, Array No. 38378, Array No. 37411, Array No. 37782, Array No. 36984, Array No. 37938, Array No. 34395, Array No. 34586, Array No. 34589, Array No. 35295, Array No. 36972, Array No. 37564, Array No. 37158, Array No. 38247, Array No. 38249, Array No. 37863, Array No. 33904, Array No. 38163, Array No. 38189, Array No. 38195, Array No. 38196, Array No. 38206

[0312] Example 5. The impact of NK cell dysfunction in the tumor microenvironment is being widely recognized, for example, in cancer patients resistant to T cell checkpoint therapy. Identification of novel inhibitory ligands upregulated on tumor cells to induce NK cell dysfunction presents a promising therapeutic opportunity as antagonists of these inhibitory ligands of NK cells, such as blocking antibodies, can reactivate NK cell cytotoxicity and promote cancer cell death.

[0313] This example demonstrates the verification of target proteins, Array No. 2013, Array No. 35586, and Array No. 36545, as novel inhibitory ligands that suppress NK-mediated cytotoxicity in human B cell lymphoma.

[0314] Using the CRISPR system, a pooled KO library containing 4,708 individual novel proteins and 998 known proteins within 2,127 lncRNAs in Raji cells (human B cell lymphoma) was generated. To understand the role of these proteins in the regulation of NK cell cytotoxicity, the inventors performed a cell killing assay using primary human NK cells. The inventors examined the change in the initial library representation in Raji cells in terms of library representation 24 hours after co-incubation with NK cells. The inventors used known regulators of NK cell cytotoxicity as a reference to establish the significance of hits. Novel inhibitors of NK-mediated cytotoxicity include targets that significantly enhance Raji cell death against CRISPR KO, similar to the known controls - B2M and TAP1.

[0315] Materials and Methods: CRISPR KO Generation. The CRISPR guide RNA library was designed by ProFound Therapeutics to target 4,708 different predicted novel proteins localized within 2,127 lncRNAs, 998 known proteins including positive (inhibitors, e.g., B2M, TAP1, TAP2, HLA-DM) and negative (costimulatory agents, e.g., ICAM1, FAS, IL7, DGKE) regulators of NK cell function, and 900 neutral controls (non-target and intergenic guide RNAs). The CRISPR guide RNA library was packaged into lentivirus and transduced into Raji Cas9 cells at an MOI of 0.3 and selected using puromycin as described in Sanjana NE, Shalem O, Zhang F. Nat.Methods. 2014 Aug;11(8):783 - 4.

[0316] Isolation and Expansion of NK. Primary human NK cells were isolated from cryopreserved PBMCs using the EasySep human NK cell isolation kit (StemCell, catalog number 17955). Primary human NK cells were expanded using the immunoCult NK cell expansion kit (StemCell, catalog number 100 - 0711) according to the manufacturer's protocol.

[0317] Screening setup. Screening was performed using four biological replicates. The NK cell cytotoxicity assay was performed by co-culturing 20 million Raji CRISPR KO library cells with 60 million primary NK cells for 24 hours in a T75 tissue culture flask. NK cells were depleted using a positive CD56 selection kit (StemCell, catalog number 17855). Dead cells were removed using an EasySep dead cell removal (Annexin V) kit (StemCell, catalog number 17899). The remaining Raji CRISPR KO library cells were processed to extract genomic DNA. Prior to exposure to NK, NGS libraries and Illumina sequencing were performed on these cells and Raji cells as described in [Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783-4].

[0318] Data analysis. Screening analysis 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)].

[0319] The inventors have found several lncRNA-encoded proteins that enhance the killing of Raji cells in the presence of primary NK cells upon loss of function. The magnitude of these effects is comparable to control proteins that are known modulators of NK cell function such as components of the MHC (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 (Figures 8 and 9).

[0320] Additional novel proteins for which an inhibitory effect on NK cell cytotoxicity with predicted cell surface localization was verified: Accession No. 35361, Accession No. 33359, Accession No. 33807, Accession No. 33817, Accession No. 35236, Accession No. 36386, Accession No. 37561

[0321] Additional novel proteins for which an inhibitory effect on NK cell cytotoxicity with predicted secretion was verified: Accession No. 38261, Accession No. 33454, Accession No. 33510, Accession No. 33225, Accession No. 33247, Accession No. 36705, Accession No. 38128, Accession No. 35759, Accession No. 36769, Accession No. 36771, Accession No. 34633, Accession No. 36461, Accession No. 37622, Accession No. 37640, Accession No. 38369, Accession No. 37377, Accession No. 37958, Accession No. 35900, Accession No. 37565, Accession No. 37099, Accession No. 33521, Accession No. 33548, Accession No. 33683, Accession No. 33704, Accession No. 34374, Accession No. 35661, Accession No. 37816

[0322] The teachings of all patents, published applications, and references cited herein are hereby incorporated by reference in their entirety.

[0323] Although exemplary embodiments have been specifically shown and described, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

[Claim 1] The invention described in the specification.

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