Compositions and Methods for the Regulation of Tumor Suppressor Genes and Oncogenes

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 cancer-related proteins in various embodiments. The present disclosure also provides methods for treating cancer using agents that include and / or modulate the expression or activity of cancer-related proteins in various embodiments and methods for identifying such agents. In some embodiments, the agent includes a target protein identified herein (e.g., a target protein listed in the Sequence Listing, Table A, or a variant of the foregoing).

Description

Technical Field

[0001] Incorporation by reference of materials This application claims the benefit of U.S. Provisional Application No. 63 / 345,756, filed May 25, 2022. The entire teachings of the above application are incorporated herein by reference.

[0002] Incorporation of materials into XML by reference This application incorporates by reference the Sequence Listing contained in the following Extensible Markup Language (XML) file, which was filed simultaneously with this specification. a) File name: 57081026002.xml; created on May 23, 2023, size 54,371,388 bytes.

Background Art

[0003] Cancer is the second leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018 or one in six deaths (www.who.int / health-topics / cancer). Cancer is caused by harmful genomic changes such as mutations that alter gene function and contribute to the malignant behavior of cancer cells. Considering the global cancer prevalence and the limited effectiveness of currently available therapeutic agents, it is extremely important to identify additional novel therapeutic targets for the treatment of various cancers.

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 treatment of cancer.

Means for Solving the Problems

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

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

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

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

[0009] In another aspect, the present disclosure provides a method for detecting cancer in a subject or determining the likelihood of developing cancer in a subject, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, wherein the level of expression or activity of the target protein in the sample indicates the likelihood of developing cancer in the subject.

[0010] In another aspect, the present disclosure provides a method for preparing a sample useful for determining the likelihood of developing cancer 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 present disclosure provides a method comprising the above steps.

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

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

[0013] In another aspect, the present disclosure provides a method for modulating the expression or activity of a target protein identified in the Sequence Listing, Table A or a variant thereof 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 a pharmaceutical composition comprising an agent or agents that includes and / or modulates the expression or activity of the target protein identified herein.

[0014] In another aspect, the present disclosure provides a method for identifying an agent that modulates the expression or activity of a target protein identified herein, comprising: a) contacting the target protein with the agent; and b) determining whether the agent modulates the expression or activity of the target protein wherein a difference in the expression or activity of the target protein 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.

[0015] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

Brief Description of the Drawings

[0016]

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Mode for Carrying Out the Invention

[0017] The description of the exemplary embodiments continues below.

[0018] Target Protein In one aspect, the present disclosure provides the target proteins identified herein. As used herein, the expressions "target proteins identified herein" and "target proteins of the present disclosure" include both the polypeptides disclosed in the Sequence Listing and the polypeptides disclosed in Table A herein. Target proteins can be produced recombinantly (e.g., via DNA or mRNA) or synthetically.

[0019] In some embodiments, the target protein is a tumor suppressor (e.g., SEQ ID NO: 37997). In some embodiments, the target protein is an oncogene (e.g., SEQ ID NO: 33586, 36829, and 38556). 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).

[0020] In some embodiments, the target protein is an intracellular protein. In some embodiments, the target protein is an extracellular protein (e.g., a secreted protein). In certain embodiments, the target protein is a transmembrane protein. 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 of the Sequence Listing or Table A. In various embodiments, the target protein is a protein comprising the amino acid sequence described in the Sequence Listing or Table A. In some embodiments, the target protein consists of the amino acid sequence described in the Sequence Listing or Table A. In some embodiments, the target protein comprises an amino acid sequence having one amino acid substitution relative to the amino acid sequence described in the Sequence Listing or Table A, and the substitution is a substitution of the methionine (Met) residue at the N-terminal residue in the amino acid sequence of the Sequence Listing or Table A. In some embodiments, the target protein consists of an amino acid sequence having one amino acid substitution relative to the amino acid sequence described in the Sequence Listing or Table A, and the substitution is a substitution of the methionine (Met) residue at the N-terminal residue in the amino acid sequence of the Sequence Listing or Table A. In some embodiments, the target protein comprises the amino acid sequence described in the Sequence Listing or Table A and further comprises a methionine (Met) residue at its N-terminus. In some embodiments, the target protein consists of the amino acid sequence described in the Sequence Listing or Table A and the methionine (Met) residue at its N-terminus.

[0022] [Table A-1]

[0023]

Table A-2

[0024] Some of the target proteins in the sequence listing or Table A have been identified as being differentially expressed (e.g., upregulated or downregulated) in a disease state (e.g., cancer, pre-cancerous state) as compared to a reference state (e.g., normal state) such that modulation of the level and / or activity of the target protein acts to treat, ameliorate and / or prevent the onset of the disease.

[0025] 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., 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., 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, as well as modifications (e.g., post-translational modifications) including N-terminal and C-terminal cleavage.

[0026] 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 can be a normal healthy state; a reference for a mutant protein can be a non-mutant protein; a reference for a disease treatment can 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 cell or sample from a healthy subject, 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) such as 2, 3, 4, 5, 10, 20, 30, 50, 100 or more (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) or a statistically significant number of cells, samples or healthy subjects. A reference obtained from two or more cells, samples or subjects can be represented as a statistic (e.g., an average or median).

[0027] In some embodiments, the target protein has an expression level that is at least about 0.5-fold higher, 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 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).

[0028] In some embodiments, the target protein has an expression level that is at least about 0.5-fold lower, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) in a disease (e.g., determined from a sample comprising or obtained from the cells or tissue of a subject having the disease) than the target protein expression level in a reference (e.g., a sample from cells or tissue of a subject without the disease). In some embodiments, the target protein is not expressed or is expressed at undetectable levels in a disease (e.g., when determined from a sample comprising or obtained from the cells or tissue of a subject having the disease).

[0029] 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, villus samples, and cells isolated from a subject, among others.

[0030] In some embodiments, the target protein is translated from non-coding RNA. In some embodiments, the non-coding RNA is a long intergenic non-coding RNA (lincRNA). In certain embodiments, the non-coding RNA is a long non-coding RNA (lncRNA). In some embodiments, the non-coding RNA is a microRNA (miRNA or miR).

[0031] In some embodiments, the target protein is translated from a non-exon element in an unprocessed precursor mRNA (pre-mRNA). In some embodiments, the non-exon element is an intron in the pre-mRNA. In some embodiments, the non-exon element is 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.

[0032] In some embodiments, the target protein has a length of 2,000 amino acids or less, such as 1,000 amino acids or less, 750 amino acids or less, 500 amino acids or less, 250 amino acids or less, 150 amino acids or less, or 100 amino acids or less. In some embodiments, the target protein has a length of 7 amino acids or more, such as 8, 9, 10, 15, 18, 25, 50, 75, or 100 amino acids or more. In certain embodiments, the target protein has a length of about 50 to about 200 amino acids, such as about 100 to about 150 amino acids. In certain embodiments, the target protein has a length of 7 amino acids or more. In more specific embodiments, the target protein has a length of about 18 amino acids.

[0033] Certain target proteins disclosed herein (e.g., SEQ ID NO: 30949; SEQ ID NO: 34229) have been identified as modulators of G protein-coupled receptors (GPCRs) (see, e.g., Example 9). Thus, in some embodiments, the target protein is a modulator of one or more GPCRs, such as GPCR CXCR4 or C3AR1. 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.

[0034] The expression and / or activity of various GPCRs are associated with various diseases / disorders, conditions, and indications, including those shown in Table B (e.g., see Kenakin, T., Biased Receptor Signaling in Drug Discovery, Pharmacol Rev 71:267-315, April 2019; Harmar, A.J., et al., IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels, Nucleic Acids Research, 2009, Vol. 37; and Davenport AP, Scully CCG, de Graaf C, Brown AJH, and Maguire JJ. Advances in therapeutic peptides targeting G protein-coupled receptors. Nat Rev Drug Discov. 2020 Jun. 19(6):389-413; the entire contents of each are hereby incorporated by reference in their entirety). Thus, in some embodiments, the target proteins disclosed herein that are modulators of GPCRs are useful for treating and / or diagnosing one or more diseases / disorders, conditions, and / or indications known to be associated with GPCR expression and / or activity, such as cancer or pre-cancerous conditions or any of the diseases / disorders, conditions, and indications listed in Table B.

[0035]

Table B-1

[0036]

Table B-2

[0037]

Table B-3

[0038]

Table B-4

[0039] 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, Table A, variants of the foregoing, or fragments of the foregoing (e.g., biologically active fragments of the target protein). 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.

[0040] 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.

[0041] As used herein, the term "increasing" or "increase" refers to an adjustment that results in a higher level of expression, activity, function, or a combination thereof or a metric (e.g., cancer cell death or DNA methylation at a target site) of a target protein as compared to a reference (e.g., the level before or in the absence of modulation by an agent). In some embodiments, the agent increases the expression or activity or metric of the target protein by at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% as compared to the reference.

[0042] As used herein, the term "decreasing" or "decrease" refers to an adjustment that results in a lower level of expression, activity, function, or a combination thereof or a metric (e.g., cancer cell death or DNA methylation at a target site) of a target protein as compared to a reference (e.g., the level before or in the absence of modulation by an agent). In some embodiments, the agent decreases the expression or activity or metric of the target protein by at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% as compared to the reference.

[0043] Non-limiting examples of metrics include energy production or energy 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.

[0044] In some embodiments, the level or metric of the expression, activity, function, or combination thereof of the target protein is measured, for example, after initiating a treatment regimen, after contacting the agent (e.g., with cells) or administering the agent (e.g., to a subject) for at least about 1 day, 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.

[0045] 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).

[0046] In some embodiments, the target protein is a tumor suppressor, and the agent increases the level of the expression, activity, function (e.g., tumor suppressor function), or combination thereof of the target protein. In other embodiments, the target protein is an oncogene, and the agent decreases the level of the expression, activity (e.g., carcinogenic activity), function, or combination thereof of the target protein.

[0047] In certain embodiments, the agent regulates the level of the expression, activity, function, or combination thereof of the target protein in cancer cells (e.g., metastatic cancer cells), cells in the tumor microenvironment (e.g., stromal cells), non-malignant cells, or a combination of the foregoing. In certain embodiments, the agent regulates the level of the expression, activity, function, or combination thereof of the target protein in a tumor, tumor microenvironment, metastatic site, stromal cells, or a combination of the foregoing.

[0048] 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); induces antibody-dependent cell cytotoxicity, phagocytosis and / or opsonization of cells expressing the target protein; or combinations thereof. In certain embodiments, the agent lacks agonist activity against the target protein. In certain embodiments, the agent has agonist activity against the target protein. In some embodiments, the agent lacks antagonist activity against the target protein. In some embodiments, the agent has antagonist activity against the target protein. In certain embodiments, the agent binds to at least one residue of the target protein involved in binding to 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 to a binding partner of the target protein.

[0049] In some embodiments, the agent induces downregulation of a binding partner of the target protein; sequesters a binding partner of the target protein (e.g., a secreted binding partner); prevents multimerization (e.g., dimerization) of a binding partner of the target protein; sequesters a binding partner of the target protein (e.g., a secreted binding partner); modulates (e.g., agonizes, antagonizes, or disrupts) a known function of a binding partner of the target protein; reduces the binding between the target protein and the binding partner (e.g., via steric hindrance); induces antibody-dependent cellular cytotoxicity, phagocytosis, and / or opsonization of cells expressing a binding partner of the target protein, or combinations of the foregoing. In certain embodiments, the agent lacks agonist activity against a binding partner of the target protein. In certain embodiments, the agent has agonist activity against a binding partner of the target protein. In some embodiments, the agent lacks antagonist activity against a binding partner of the target protein. In some embodiments, the agent has antagonist activity against a binding partner of the target protein. In certain embodiments, the agent further binds to at least one residue of a binding partner of the target protein involved in the binding between the target protein and the binding partner. In more certain embodiments, the agent further binds to one or more binding sites and / or domains of a binding partner of the target protein involved in the binding between the target protein and the binding partner.

[0050] In certain embodiments, the agent modulates (e.g., increases or decreases) the level of expression, activity, function, or combinations thereof of a variant of a target protein disclosed herein. In some embodiments, the variant comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the target protein disclosed herein. For example, the sequence identity to the variant 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.

[0051] 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 across the full length of the reference sequence at which the reference sequence and the test sequence share the same nucleotide or amino acid when aligned to achieve the maximum level of identity. As an example, when aligned to achieve the maximum level of identity, if the test sequence has the same nucleotide or amino acid residue at 70% of the same positions across the full length of the reference sequence, the two sequences are considered to have 70% sequence identity.

[0052] 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. Optionally, 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 (see generally Ausubel et al., Current Protocols in Molecular Biology).

[0053] 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 tool commonly used to determine 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).

[0054] 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.

[0055] 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.

[0056] In some embodiments, the amino acid substitutions are conservative substitutions. The term "conservative amino acid substitution" or "conservative substitution" refers to an amino acid substitution having a value of 0 or more in BLOSUM62.

[0057] In some embodiments, the amino acid substitutions are highly conserved substitutions. The term "highly conserved amino acid substitution" or "highly conserved substitution" refers to an amino acid substitution having a value of at least 1 (e.g., at least 2) in BLOSUM62.

[0058] In some embodiments, variants of the target proteins of the present disclosure contain about 5 to 60 amino acid substitutions relative to the amino acid sequence of the target proteins 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 conserved substitution.

[0059] 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). Proteins, peptides, or polypeptides 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 unnatural amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). Amino, carboxyl, and / or other functional groups on the peptide may be free (e.g., unmodified) or may be protected with suitable protecting groups. Suitable protecting groups for amino and carboxyl groups and methods for adding or removing protecting groups are known in the art and are disclosed, for example, in Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. 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. Proteins, peptides, or polypeptides 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, proteins, peptides, or polypeptides can be analogs of known and / or native peptides, such as peptide analogs having conservative amino acid residue substitutions.

[0060] 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).

[0061] 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.

[0062] 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, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The antibody can be of any species, such as a rodent (e.g., mouse, rat, guinea pig) antibody, a human antibody, or the antibody can be a humanized or chimeric antibody.

[0063] 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.

[0064] In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is human or chimeric. In some embodiments, the antibody is primatized (e.g., humanized). In some embodiments, the antibody is multispecific, such as bispecific, trispecific or tetravalent. In some embodiments, the antibody is a heteroconjugate antibody.

[0065] In some embodiments, the polypeptide agent is an antigen-binding fragment of an immunoglobulin molecule (e.g., an antibody). The term "antigen-binding fragment" refers to a portion of an immunoglobulin molecule (e.g., an antibody) that retains the antigen-binding properties of the parent full-length antibody. Non-limiting examples of antigen-binding fragments include the V H region, the V L region, Fab fragment, F(ab’) 2 fragment, Fd fragment, Fv fragment, and domain antibodies (dAbs) consisting of one V H domain or one V L domain. The 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 intramolecularly or intermolecularly when the V H and V L domains are expressed by separate chains, forming monovalent antigen-binding sites such as single-chain Fv (scFv) or diabodies. In some embodiments, the polypeptides disclosed herein are antigen-binding fragments selected from 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 scFv. Antigen-binding fragments can be generated by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in certain cases, chemical peptide synthesis procedures known in the art.

[0066] 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.

[0067] "Multispecificity" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes within an antigen, such as three, four, or five different antigens or epitopes. "Bispecificity" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen.

[0068] "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, such as about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0069] 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), decreases (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 decreases the activity of the enzyme or attenuates the signaling pathway mediated by the signaling molecule. In some embodiments, the antagonist antibody decreases 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%.

[0070] 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 the target protein), increases the function of the antigen. In some embodiments, the antigen is a receptor and the agonist antibody binds to the ligand-binding domain of the receptor. In some embodiments, the antigen is a transmembrane protein and the agonist antibody binds to the extracellular region of the transmembrane protein. In some embodiments, the antigen is an enzyme or a signaling molecule and the agonist antibody increases the activity of the enzyme or activates a signaling pathway mediated by the signaling molecule. In some embodiments, the agonist antibody increases the antigen function by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1,000%.

[0071] 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).

[0072] Appropriate techniques, assays, and reagents for making and using therapeutic antibodies against antigens are known in the art. 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 are described 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).

[0073] 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 that of a natural antibody. 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.

[0074] In some embodiments (e.g., when the expression or activity of the target protein is reduced in the cancer state 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.

[0075] 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.

[0076] In some embodiments, a polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence that is at least 70% identical to at least a portion of a protein that can modulate 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 modulate the expression or activity of a target protein.

[0077] In some embodiments, a polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence having at least one amino acid substitution relative to a protein that can modulate 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.

[0078] 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.

[0079] In some embodiments, the polypeptide is a circulating factor (e.g., a cytokine).

[0080] In some embodiments, the polypeptide (e.g., an isolated polypeptide) and the biological properties of the target protein (e.g., biological activity or half-life) are similar. Non-limiting examples of biological activity include, inter alia, enzyme activity or properties (e.g., selectivity, steady state or kinetics), binding activity (e.g., nucleic acid (DNA, RNA) binding protein binding) or properties (e.g., specificity, affinity or kinetics), cell signaling activity, immunological activity and structural activity (e.g., cell adhesion). Non-limiting examples of enzyme activity include transferase activity (e.g., transferring a functional group from one molecule to another), oxidoreductase activity (e.g., catalyzing an oxidation-reduction reaction), hydrolase activity (e.g., cleaving a chemical bond by hydrolysis), lyase activity (e.g., generating a double bond), ligase activity (e.g., joining two molecules via a covalent bond) and isomerase activity (e.g., catalyzing an intramolecular structural change from one isomer to another).

[0081] 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.

[0082] 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 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 post-translational modifications or other chemical modifications. Non-limiting examples of post-translational modifications include acetylation, amidation, formylation, glycosylation, hydroxylation, methylation, myristoylation, phosphorylation, deamidation, prenylation (e.g., farnesylation, geranylation, etc.), ubiquitination, ribosylation, and sulfation. Phosphorylation can occur on amino acids such as tyrosine, serine, threonine, or histidine.

[0083] In some embodiments, the polypeptide is coupled (e.g., via a covalent or non-covalent bond such as a peptide bond) to a heterologous peptide or protein such as a conjugate or fusion protein. In some embodiments, the polypeptide comprises a tag (e.g., a detectable label such as a fluorophore or an enzyme or a purification tag such as an epitope tag).

[0084] In some embodiments, the polypeptide comprises one or more neoantigens selected from the sequence listing, Table A, or variants thereof. As used herein, the term "neoantigen" refers to a tumor antigen arising from a target protein described herein. In some aspects, the neoantigen is a cancer-specific neoantigen. There are various methods for producing 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.

[0085] Alternatively, a neoantigen can be produced in vivo by introducing a polynucleotide or expression vector (e.g., a viral expression vector) encoding the neoantigen into cells or tissues (e.g., of a subject in need thereof). In certain embodiments, the polypeptide comprises at least two neoantigens. In some embodiments, the polypeptide comprises a T cell enhancer amino acid sequence. In some embodiments, the T cell enhancer is selected from the group consisting of an invariant chain, a leader sequence of tissue-type plasminogen activator, a PEST sequence, a cyclin destruction box, a ubiquitination signal, and a SUMOylation signal.

[0086] 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 decreases (e.g., reduces or neutralizes) the expression or activity of a target protein. In other embodiments, the polynucleotide or an analog or derivative thereof increases the expression or activity of a target protein.

[0087] A polynucleotide can have a sequence containing naturally occurring ribonucleotide or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or combinations thereof. Thus, a polynucleotide can include, for example, nucleotides containing naturally occurring bases (e.g., A, G, C, or T) and nucleotides containing modified bases (e.g., 7-deazaguanosine, inosine, or methylated nucleotides such as 5-methyl dCTP and 5-hydroxymethylcytosine). In some embodiments, the polynucleotide includes at least one modified nucleotide. Non-limiting examples of modified nucleotides include 2'-fluoro, 2'-O-methyl, 2'-deoxy, locked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'-thiouridine, and 2'-deoxyuridine. In some embodiments, the modification increases nuclease resistance, increases serum stability, decreases immunogenicity, or a combination thereof.

[0088] 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).

[0089] In some embodiments, the polynucleotide includes an analog or derivative of a polynucleotide. In some embodiments, the analog or derivative is a peptide nucleic acid (PNA). In some embodiments, the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative includes one or more phosphorothioate linkages. In some embodiments, the agent includes deoxyriboguanidine (DNG) nucleotides. In some embodiments, the agent includes riboguanidine (RNG) nucleotides.

[0090] In some embodiments, the polynucleotide regulates the expression and / or activity of a nucleic acid encoding a target protein (e.g., a target protein in the Sequence Listing or Table A), a variant thereof, or a portion thereof (e.g., a biologically active portion or a fragment thereof) disclosed herein.

[0091] 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.

[0092] In some embodiments, the polynucleotide encodes a target protein (e.g., a biologically active variant thereof) or a portion thereof (e.g., a biologically active portion or a fragment thereof) disclosed herein.

[0093] In some embodiments, the nucleic acid encoding the target protein or a variant 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).

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] In some embodiments, the polynucleotide (e.g., an antisense oligonucleotide) can hybridize to the mRNA encoding the target protein (e.g., under physiological conditions). In some embodiments, the length of the polynucleotide is at least about 10 nucleotides, such as at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or about 10 - 30, 15 - 30, 15 - 25, 20 - 25 nucleotides. In some embodiments, the polynucleotide is at least 75% identical to the antisense sequence of the same target transcript, such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0099] In some embodiments, the polynucleotide further comprises an overhang sequence (e.g., unpaired overhang nucleotides that are not directly involved in the formation of the double helix structure by the core sequence). In some embodiments, the polynucleotide comprises a 3' overhang, a 5' overhang, or both. In some embodiments, the overhang is about 1 - 5 nucleotides. In some embodiments, the overhang comprises modified ribonucleotides or deoxynucleotides, such as phosphorothioate, phosphorothioate, or deoxynucleotide inversion (linked 3' to 3') nucleotides.

[0100] 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).

[0101] 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.

[0102] 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 by, for example, causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA.

[0103] In some embodiments, the polynucleotide is a siRNA. In some embodiments, the siRNA comprises a nucleotide sequence identical to about 15 - 25 contiguous mRNA sequences encoding the target protein. In some embodiments, the siRNA is a double-stranded RNA molecule having about 19 - 25 base pairs. In some embodiments, the siRNA begins with the dinucleotide AA. In some embodiments, the siRNA has a GC content of about 30 - 70%, such as about 30 - 65%, 30 - 60%, 30 - 55%, 30 - 50%, 40 - 70%, 40 - 65%, 40 - 60%, 40 - 55%, 45 - 70%, 45 - 65%, 45 - 60%, or 45% - 55%.

[0104] In some embodiments, the polynucleotide is shRNA. shRNA is an RNA molecule that contains 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, by, for example, transfection, electroporation, or transduction.

[0105] 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 via translational repression or poly(A) tail 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 having 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)).

[0106] 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)).

[0107] In some embodiments, the polynucleotide comprising RNA is chemically synthesized. In some embodiments, the polynucleotide comprising RNA is recombinantly expressed. In some embodiments, the RNA is transcribed in vitro. The production and use of RNA therapeutics are known in the art. See, e.g., RNA Therapeutics: Function, Design, and Delivery (Mouldy Sioud eds., 2010) and Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Medicine 9:60 (2017).

[0108] 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.

[0109] In some embodiments, the mRNA comprises a poly(A) tail. In some embodiments, the poly(A) tail is about 100-200 nucleotides. In some embodiments, the poly(A) tail improves the expression and / or stability of the mRNA (see, e.g., Kaczmarek et al., Genome Medicine 9:60 (2017)).

[0110] 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)).

[0111] 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)).

[0112] In some embodiments, the mRNA comprises an RNA base modification. In some embodiments, the mRNA comprises pseudouridine. In some embodiments, the mRNA comprises N1-methyl-pseudouridine (e.g., to mask immunostimulatory activity and enhance translation initiation) (see, e.g., Andries et al., J Control Release 217:337-44 (2015) and Svitkin et al., Nucleic Acids Res. 45:6023-36 (2017)).

[0113] In some embodiments, the RNA (e.g., mRNA) is circular RNA.

[0114] 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)).

[0115] 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.

[0116] In some embodiments, the polynucleotide is linked to the delivery polymer (e.g., by a covalent bond). 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.

[0117] In some embodiments, the polynucleotide is conjugated to the polymer in the presence of excess polymer. In some embodiments, the excess polymer is removed prior to administration (e.g., to a cell or subject).

[0118] One of ordinary skill in the art can readily make appropriate polynucleotide agents for use in the compositions, kits, and methods described herein using the locus information of the protein sequences, such as chromosomal location, start nucleotide position, and end nucleotide position, and polymorphism identification, contained in the sequence listing and Table A incorporated herein.

[0119] C. Agents 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.

[0120] 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.

[0121] In some embodiments, the gene editing system (e.g., the CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates (e.g., via gene knockout) the expression of the target protein. In some embodiments, the gene editing system (e.g., the CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates (e.g., via gene knockout) the expression of a protein capable of regulating the expression or activity of the target protein. In some embodiments, the gene editing system (e.g., the CRISPR / Cas system) increases (e.g., via gene knock-in or gene replacement) the expression of the target protein. In some embodiments, the gene editing system (e.g., the CRISPR / Cas system) increases (e.g., via gene knock-in or gene replacement) the expression of a protein capable of regulating the expression or activity of the target protein.

[0122] 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.

[0123] In some embodiments, the gene editing system comprises a single Cas endonuclease or a polynucleotide encoding a single Cas endonuclease. In some embodiments, the single Cas endonuclease is Cas9, Cpf1, C2C1 or C2C3. In some embodiments, the single Cas endonuclease is Cas9 (e.g., of Streptococcus Pyogenes). In some embodiments, the single Cas endonuclease is Cpf1. In some embodiments, Cpf1 is AsCpf1 (derived from the Acidaminococcus species) or LbCpf1 (derived from the Lachnospiraceae species). The selection of the nuclease and gRNA is typically determined according to whether nucleotide deletions, substitutions or additions to the target sequence are desired.

[0124] In some embodiments, the type II Cas endonuclease is Cas9 (e.g., of Streptococcus pyogenes). In some embodiments, the modified Cas9 is nickase Cas9, dead Cas9 (dCas9) or eSpCas9. In some embodiments, the nickase Cas9 is Cas9 D10A. In some embodiments, dCas9 is D10A or H840A. In some embodiments, the gene editing system comprises dual nickase Cas9 (see, e.g., Ran et al., Cell 154:1380-89 (2013) for achieving more accurate genome editing). Wild-type Cas9 generates a double-strand break (DSB) at a specific DNA sequence targeted by the gRNA. Nickase Cas9 generates only single-strand breaks. dCas9 is catalytically inactive. In some embodiments, dCas9 is fused to a nuclease (e.g., FokI for generating DSBs at target sequences homologous to two gRNAs). Various CRISPR / Cas9 plasmids are publicly available from the Addgene repository (Addgene, Cambridge, MA: addgene.org / crispr / ).

[0125] 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; b) a CRISPR RNA (“crRNA”); and c) a trans-activating crRNA (“tracrRNA”) comprising.

[0126] 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.

[0127] 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)).

[0128] 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.

[0129] In some embodiments, the target DNA sequence is proximal to a "protospacer adjacent motif" ("PAM") specific to the Cas endonuclease. PAM sequences occur throughout a given genome. CRISPR endonucleases from 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 Cas9 endonuclease, associate with G-rich PAM sites, such as 5'-NGG, and perform blunt-end cleavage of the target DNA at a position three nucleotides upstream (5') of the PAM site.

[0130] 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.

[0131] 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 4- or 5-nucleotide 5' overhangs, for example, by cleaving target DNA such that the target DNA has an offset or staggered cleavage 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 cleavages enable more precise genome editing by DNA insertion via homologous recombination rather than by insertion of blunt-ended DNA. See, for example, Zetsche et al., Cell 163:759-71 (2015).

[0132] 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.

[0133] In some embodiments, the chimeric protein suppresses expression of the target protein (CRISPRi). In some embodiments, the chimeric protein activates expression of the target protein (CRISPRa). In some embodiments, the chimeric protein methylates the DNA sequence recognized by the sgRNA. In some embodiments, the chimeric protein demethylates the DNA sequence recognized by the sgRNA.

[0134] The effector domain comprises the biologically active portion of an effector protein (e.g., a transcriptional activator or a transcriptional repressor). In some embodiments, the gene editing system comprises one effector domain. In some embodiments, the gene editing system comprises at least two effector domains, 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.

[0135] Since dCas9 is catalytically inactive, it does not cleave the target DNA and interferes with transcription by steric hindrance. dCas9 chimeric proteins (e.g., dCas9-VPR) are guided by one or more gRNAs to sequences upstream of the transcription start site (TSS) of the target gene and regulate the transcription of the target gene. For example, Gilbert et al., CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes, Cell 154, 442-51 (2013); Cheng et al., Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system, Cell Res. 23:1163-71 (2013); Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation, Cell 159:647-61 (2014); Tanenbaum et al., A protein-tagging system for signal amplification in gene expression and fluorescence imaging, Cell 159:635-46 (2014); Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Nature 517:583-88 (2015); Chavez et al., Highly efficient Cas9-mediated transcriptional programming, Nat.Methods.12:326-28 (2015); Zalatan et al., Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Cell 160:339-50 (2015); Horlbeck et al.See Chavez et al., Comparison of Cas9 activators in multiple species, Nat Methods. 7:563-67 (2016); eLife. 5:e19760 (2016); Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.

[0136] CRISPR technology for editing eukaryotic genes is disclosed in US Patent Application Publication No. 2016 / 0138008A1, US Patent Application Publication No. 2015 / 0344912A1, and US Patents No. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. The Cpf1 endonuclease 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).

[0137] In some embodiments, the agent comprises a transposon-based gene editing system. An example of a transposon-based gene editing system suitable for use in the disclosure provided herein is the Gene Writer system described in International Publication No. WO 2020 / 047124, published on March 5, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0138] 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.

[0139] 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).

[0140] 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 of the target protein defined by a set of genomic coordinates.

[0141] 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.

[0142] 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.

[0143] In some embodiments, the agent comprises a zinc finger nuclease (ZFN) system. The ZFN domain can be generated using commercially available plasmids, such as plasmid pairs from Sigma Aldrich (St. Louis, MO) (CSTZFN-1KT COMPOZR® Custom Zinc Finger Nuclease (ZFN) R-3257609). The plasmids can be prepared using commercially available systems according to the manufacturer's protocol (e.g., NEB Monarch Miniprep (Catalog No. T1010), New England Biolabs, Ipswich, MA).

[0144] 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.

[0145] 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).

[0146] Examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic, or inorganic compounds. 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 can represent a chain (e.g., a carbon-carbon chain or a carbon-heteroatom chain) or a ring containing carbon atoms, such as benzene or a polycyclic system, or a combination of carbon and heteroatoms, i.e., a heterocycle such as pyrimidine or quinazoline. Small molecules can have a wide range of molecular weights, but generally include molecules having a molecular weight of less than about 5,000 Daltons. For example, such small molecules can be less than about 1,000 Daltons, preferably less than about 750 Daltons, or more preferably less than about 500 Daltons. Small molecules can be found in nature (e.g., identified, isolated, purified) and / or produced synthetically (e.g., by traditional organic synthesis, biocatalyzed synthesis, or a combination thereof). See, for example, Ganesan, Drug Discov. Today 7(1):47-55 (January 2002); Lou, Drug Discov. Today, 6(24):1288-1294 (December 2001). Examples of naturally occurring small molecules include, but are not limited to, hormones, neurotransmitters, nucleotides, amino acids, sugars, lipids, and derivatives thereof.

[0147] In certain embodiments, the agent includes a proteolysis-targeting chimera (PROTAC).

[0148] 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.

[0149] 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 the target proteins described herein (e.g., the target proteins of the Sequence Listing, Table A or variants thereof), polypeptides (e.g., antibodies, antigen-binding fragments or polypeptides comprising an amino acid sequence that is at least 70% identical to at least a part of the target protein), polynucleotides (e.g., recombinant DNA, RNA, such as mRNA or siRNA) and / or gene editing systems (e.g., CRISPR / Cas system).

[0150] 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.

[0151] 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., a human) into specific cell products; engineering somatic cells for gene therapy; cell immortalization; ex vivo gene modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technologies); in vivo gene modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technologies); genome editing; cell plasticity technologies; gene modification; and flow cytometry. In some embodiments, the therapeutic cells are autologous or syngeneic. In other embodiments, the therapeutic cells are allogeneic.

[0152] Expression Vectors and Hosts In another aspect, the present disclosure provides an expression vector comprising the polynucleotide described herein.

[0153] 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.

[0154] In some embodiments, the expression vector comprises an expression control polynucleotide sequence operably linked to the polynucleotide, a polynucleotide sequence encoding a selectable marker, or both. In some embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In some embodiments, the expression control polynucleotide sequence comprises an inducible promoter sequence. The term "promoter" refers to a 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 an element to which it is linked, such as a promoter. 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).

[0155] In another aspect, the present disclosure provides an expression host cell comprising any one or more of the polynucleotides or expression vectors described herein.

[0156] The term "expression host cell" refers to a cell useful for receiving, maintaining, replicating, and / or amplifying a vector.

[0157] 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).

[0158] The polynucleotides or expression vectors described herein can be introduced into a suitable or desired host cell 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.

[0159] Pharmaceutical composition In another aspect, the 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 pharmacological activity or other direct effect in the alleviation, treatment or prevention of cancer or a finished dosage form or formulation thereof.

[0160] In some embodiments, a composition (e.g., a pharmaceutical composition) includes a pharmaceutically acceptable carrier, excipient, stabilizer, diluent, or tonicity agent (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents, or tonicity agents include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, or dextrin); chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-protein complexes); nonionic surfactants (e.g., Tween®), PLURONICS® and polyethylene glycol (PEG).

[0161] In some embodiments, an agent (e.g., a polypeptide, polynucleotide, or small molecule) of a pharmaceutical composition is modified, e.g., conjugated to a heterologous moiety. The term “conjugated” refers to being joined via a covalent or non-covalent interaction. Conjugation can be carried out using any suitable linker; non-limiting examples include peptide linkers, compound linkers, and chemical cross-linking agents.

[0162] In some embodiments, the heterologous moiety is a marker (e.g., a fluorescent marker or a radioactive marker), a molecule that stabilizes the agent, a molecule that targets the agent to a particular cell or tissue (e.g., to facilitate or prevent passage across the blood-brain barrier), or a combination thereof.

[0163] In some embodiments, the heterologous moieties are polyethylene glycol (PEG), hexadecanoic acid, hydrogel, nanoparticles, multimerization domains, and carrier peptides. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are polymer nanoparticles. In some embodiments, the polymer is an amphiphilic polymer. In other embodiments, the polymer is a hydrophobic or hydrophilic polymer. Non-limiting examples of polymers include poly(lactic acid)-poly(ethylene glycol), poly(lactic-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 salts thereof. In some embodiments, the polymer nanoparticles comprise poly(lactic-co-glycolic acid) (PLGA).

[0164] In some embodiments, the composition (e.g., 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., 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).

[0165] In some embodiments, the composition is formulated to be administered as a combination therapy with one or more additional therapeutic agents (e.g., with a second therapeutic agent). As used herein, "combination therapy" or "administered in combination" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. Non-limiting examples of additional agents or treatments include biologics (e.g., antibodies, peptides), cell therapies, gene therapies, immunotherapies, and small molecules used in oncology (e.g., chemotherapeutic agents).

[0166] 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, the administration of two or more agents or treatments in combination results in a greater reduction in other parameters associated with the symptom or disorder than is 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.

[0167] In some embodiments, the agent or pharmaceutical composition of the disclosure is delivered by a viral vector, e.g., by contacting cells with the viral vector, and is administered locally to the tumor (e.g., by injection) or systemically to the subject (e.g., a human patient) (e.g., intravenously or orally).

[0168] 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, gibbon ape 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.

[0169] In some embodiments, the agents or pharmaceutical compositions of the present disclosure are formulated to be delivered by a membrane-based carrier in vivo, in vitro, ex vivo, or in situ. In some embodiments, the membrane-based carrier is a cell-based carrier (e.g., a mammal such as a human cell). In some embodiments, the membrane-based carrier is a vesicle-based carrier. In some embodiments, the membrane-based carrier comprises one or more vectors (e.g., plasmids, viruses, virus-like particles, or virions) described herein.

[0170] In some embodiments, the agents or pharmaceutical compositions of the present disclosure are formulated to be delivered by one or more liposomes. Liposomes are spherical vesicular structures composed of a single or multiple lipid bilayers surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible and non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB) (see, e.g., Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)).

[0171] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, the teachings of which regarding the preparation of multilamellar vesicle lipids are incorporated herein by reference). Vesicle formation can be spontaneous when lipid membranes are mixed with an aqueous solution, but can also be facilitated by applying force in the form of agitation using a homogenizer, sonicator, or extrusion device (see, e.g., Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)). 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).

[0172] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by lipid nanoparticles (LNPs). In one embodiment, the LNP preparation comprising the agent or pharmaceutical composition of the present disclosure has one or more of the following characteristics: (a) the LNP preparation comprises a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid, and (b) the LNP preparation has an average particle size of 80 nm to 160 nm.

[0173] 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 of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier combining liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs are composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, the two components enhance drug encapsulation efficiency, facilitate surface modification, and prevent the leakage of water-soluble drugs. See, for example, Li et al., Nanomaterials 7(6):122 (2017).

[0174] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by a carbohydrate carrier (e.g., an anhydride-modified phytoglycogen or a glycogen-like material). Non-limiting examples of carbohydrate carriers include octenyl succinic acid phytoglycogen, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin.

[0175] 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.

[0176] 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).

[0177] 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).

[0178] 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.

[0179] 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); U.S. Patent No. 9,644,180; Huang et al., Nature Communications 8:423(2017).

[0180] 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.

[0181] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more anellosomes. The preparation and use of anellosomes for the delivery of therapeutic products are described in U.S. Patent No. 11,166,996, the teachings of which regarding the design, preparation, and use of anellosomes are incorporated herein by reference.

[0182] Method In another aspect, the present disclosure provides a method of detecting cancer in a subject or predicting the likelihood (or risk level) of developing cancer 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 cancer in the subject.

[0183] In another aspect, the present disclosure provides a method of classifying a subject based on the predicted likelihood of developing cancer, the method comprising quantifying the expression or activity of a target protein in a sample derived from the subject; predicting the likelihood of developing cancer based on the expression or activity of the target protein in the sample; and classifying the patient based on the predicted likelihood.

[0184] In another aspect, the present disclosure provides a method of stratifying a set of subjects having cancer, the method comprising quantifying the expression and / or activity of a target protein in a sample derived from an individual subject within the set; and stratifying the set of subjects for treatment according to the level of expression and / or activity of the target protein in the sample for each individual subject.

[0185] In some embodiments, a higher expression or activity level of a target protein in a sample from a subject, as compared to an appropriate control (e.g., a reference standard), indicates cancer or a predisposition to develop cancer. In some embodiments, a lower expression or activity level of a target protein in a sample from a subject, as compared to an appropriate control (e.g., a reference standard), indicates cancer or a predisposition to develop cancer.

[0186] In some embodiments, the method further comprises administering to a subject determined or predicted to have a predisposition to develop cancer (or at risk of developing cancer) an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein.

[0187] In some embodiments, the method further comprises administering to a subject determined or predicted to have a predisposition to develop cancer an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein.

[0188] In another aspect, the disclosure provides a method of preparing a sample useful for detecting a predisposition to develop cancer 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 a predisposition to develop cancer; and c) quantifying the expression or activity of a target protein in the sample prepared in step b). The method is provided.

[0189] In another aspect, the disclosure provides a method of treating cancer 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.

[0190] In another aspect, the present disclosure provides a method of treating cancer 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.

[0191] As used herein, the term “treatment” or “treating” refers to the medical management of a subject with the intent to improve, alleviate, stabilize (i.e., not exacerbate), prevent or cure a disease, condition or disorder. “Treatment” includes curative treatment (treatment directed to improving a disease, condition or disorder), causal treatment (treatment directed to the cause of a related disease, condition or disorder), palliative treatment (treatment designed to relieve symptoms), prophylactic treatment (treatment directed to minimizing or partially or completely inhibiting the onset of a related disease, condition or disorder); and adjuvant treatment (treatment utilized to supplement another therapy). Treatment also 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 as well as those who are at risk of developing or should be prevented from developing the condition or disorder.

[0192] In some embodiments, the subject is an animal. In other embodiments, the subject is a bird, such as a hen, rooster, turkey, or parrot. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. Non-limiting examples of non-human mammals include cows (e.g., dairy cows or beef cattle), sheep, goats, pigs, horses, dogs, cats, mice, rats, and the like. In some embodiments, the subject is a human. In some embodiments, the human is a neonate. In some embodiments, the human is a pediatric patient. In some embodiments, the human is a young person. In some embodiments, the human is an adult. In some embodiments, the human is under 18 years old. In some embodiments, the human is at least 18 years old. In some embodiments, the human is between 18 and 25 years old. In some embodiments, the human is at least 25 years old, such as at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old.

[0193] As used herein, the terms "effective amount", "therapeutically effective amount", or "sufficient amount" refer to an amount sufficient to effect a treatment (e.g., to produce a beneficial or desired result), including effects at the cellular, tissue, or clinical level, when administered to a subject (e.g., a mammal such as a human cancer patient). Thus, the term depends on the context in which it is applied. For example, in the context of treating cancer, it is an amount of 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 a composition 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 a composition 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.

[0194] In some embodiments, the effective amount is sufficient to reduce the growth, proliferation, metastasis, invasion, migration, innervation or combination of the foregoing of cancer (e.g., tumor). 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%.

[0195] In certain embodiments, the effective amount is sufficient to reduce the expression of the target protein. In some 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%.

[0196] In certain embodiments, the effective amount is sufficient to increase the expression of the target protein. In some embodiments, the increase is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is 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%. In some embodiments, the increase is from about 1 to 100-fold, such as from about 1 to 75, 1 to 50, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2-fold.

[0197] In some embodiments, the effective amount is sufficient to prevent the death of the subject, thereby reducing the cancer (e.g., tumor) mortality rate. In certain embodiments, the reduction in cancer (e.g., tumor) mortality rate 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 rate 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%.

[0198] In some embodiments, the effective amount is sufficient to modulate 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.

[0199] 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%.

[0200] In certain embodiments, the effective amount is sufficient to modulate the expression of a target protein in immune cells.

[0201] In some embodiments, the effective amount is sufficient to modulate (e.g., increase) the immune system of the subject against cancer (e.g., modulate the immune response of the subject against cancer). In certain embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease) at least one immune cell-related readout. Non-limiting examples of immune cell-related readouts include immune cell activation, degranulation, maturation, migration, polarization, proliferation and recruitment; lymph node activation, differentiation, egress, homing, innervation and polarization; cytokine production; antigen presentation; antibody-dependent cell cytotoxicity (ADCC); and antibody-dependent cell phagocytosis (ADCP).

[0202] In some embodiments, the effective amount is sufficient to modulate the development of high endothelial venules (HEVs) and / or tertiary lymphoid organs (TLOs); the activation, degranulation, differentiation, maturation, migration, polarization, proliferation and / or recruitment of immune cells; the activation, egress, homing and / or polarization of lymph nodes; cytokine production; antigen presentation; inflammation; autoantibody levels; organ function; the rate and / or number of recurrence and / or relapse; viral load; infection or a combination of the foregoing.

[0203] In certain embodiments, the effective amount is sufficient to inhibit the growth, proliferation, metastasis, invasion or migration of cancer cells or a combination of the foregoing; to promote cancer cell death; to induce cancer cell autophagy or a combination of the foregoing.

[0204] In some embodiments, the effective amount is sufficient to modulate (e.g., increase or decrease) the development of HEVs and / or TLOs, the migration of immune cells (e.g., antigen presenting cells such as dendritic cells and / or macrophages and / or T cells), the proliferation of immune cells, the recruitment of immune cells (e.g., antigen presenting cells such as dendritic cells and / or macrophages, monocytes, T cells and / or B cells), the lymph node homing of immune cells (e.g., dendritic cells and / or T cells), the lymph node egress of immune cells (e.g., dendritic cells and / or T cells), the tumor homing of immune cells (e.g., T cells); to increase the tumor egress of immune cells (e.g., regulatory T cells) or decrease the tumor egress of immune cells (e.g., CD8 + T cells) or a combination of the foregoing.

[0205] In some embodiments, the effective amount is sufficient to modulate the body's response to cancer, for example, by inhibiting cancer growth, reducing cancer malignancy, inhibiting cancer metastasis, promoting remission, modulating (increasing and / or decreasing) the immune-mediated response associated with cancer or a combination of the foregoing.

[0206] 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.

[0207] The therapeutic agents described herein can be administered via a variety of routes of administration, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intraarterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion, and inhalation (e.g., 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.

[0208] 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).

[0209] 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 the use of the therapeutic agents in a sequential manner, either nearly simultaneously or at different times. When two or more therapeutic agents are administered, the therapeutic agents can be administered via the same or different routes of administration.

[0210] In another aspect, the disclosure provides a method of modulating the expression or activity of a target protein or a variant thereof identified in the Sequence Listing, Table A, 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.

[0211] In another aspect, the 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, Table A, or a variant thereof), comprising a) contacting a sample containing a 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 A method is provided that includes comparing with a reference, and a difference in the expression or activity of the target protein contacted with the agent, which indicates that the agent modulates the expression or activity of the target protein.

[0212] In some embodiments, a difference of at least about 10% in the expression or activity of the protein contacted with the agent as compared to the 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.

[0213] In some embodiments, a decrease in the expression or activity of the target protein contacted with the agent as compared to the 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 the protein as compared to the reference indicates that the agent activates the expression or activity of the protein.

[0214] cancer A variety of cancers can be treated according to the methods described herein. In some embodiments, the cancer includes solid tumors (e.g., breast, lung, prostate, colon, bladder, ovary, kidney, stomach, colon, rectum, testis, head and / or neck, pancreas, brain, skin tumors). Thus, in some embodiments, the cancer is a solid tumor cancer. Solid tumor cancers that can be treated according to the methods described herein include breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, gastric cancer, colorectal cancer, rectal cancer, colon-rectal cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, and skin cancer. In some embodiments, the cancer is a blood cancer (e.g., leukemia, lymphoma, myeloma). Blood cancers that can be treated according to the methods described herein include leukemia (e.g., acute leukemia, chronic leukemia), lymphoma (e.g., B-cell lymphoma, T-cell lymphoma), and multiple myeloma.

[0215] Examples of cancers that can be treated according to the methods described in this specification 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 cavity cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides; myelodysplastic syndrome, myelodysplasia / myeloproliferative tumor;Chronic myeloid leukemia (CML); Acute myeloid leukemia (AML); Chronic myeloproliferative neoplasms; Nasal and paranasal sinus cancer; 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 sinus 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.;

[0216] The metastases of the aforementioned cancers can also be treated according to the methods described herein. In some embodiments, the cancer is metastatic cancer.

[0217] 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.

[0218] 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 having 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 representing a substantial difference from what is commonly understood in the art. It will further be understood that terms such as those defined in commonly used dictionaries should be construed to have meanings consistent with the meanings related to the relevant technology and / or as otherwise defined in this specification.

[0219] The terms used in this specification are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0220] As used in this specification, the indefinite articles "a", "an", and "the" are to be understood as including plural references unless the context clearly indicates otherwise.

[0221] 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 understood to mean, for example, including the recited 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 "comprise" can be replaced with the term "contain" or "include".

[0222] 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 connection with an aspect or embodiment of the present disclosure, the terms "comprising", "containing", "including", and "having" may, in some embodiments, be replaced with "consisting of" or "consisting essentially of" in order to change the scope of the disclosure.

[0223] As used herein, the connective term "and / or" between a plurality of recited elements is understood to encompass both individual choices and combinations of 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 ability to apply multiple choices simultaneously also falls within the scope of meaning and is thus understood to meet the requirements of the term "and / or".

[0224] When a list is presented, it should be understood that, unless otherwise stated, each individual element of that list and all combinations of that list are separate embodiments. For example, a list of embodiments presented as "A, B, or C" should be construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

Examples

[0225] Example 1: Verification of a target protein as an oncogene in a cell cycle assay This example demonstrates the validation of the target protein of the present disclosure as a contributing factor to tumor growth and / or acceleration of cell cycle progression.

[0226] Generate stable tumor cell lines with elevated or reduced levels of the target protein or control. Compare the growth rates of the generated cell lines. If the growth rate of the generated cell line correlates with the level of the target protein, the target protein is an oncogene.

[0227] Materials and Methods: Overexpression: Stable overexpressing tumor cell lines are generated by transfecting Jurkat E6-1 (T cell line; ATCC) using Lipofectamine 3000 (ThermoFisher) with pcDNA3.1_Myc plasmid (GenScript Inc) containing 1) no insert, 2) target protein coding sequence, 3) scrambled version of the target protein coding sequence (negative control), or 4) Gfi-1 protein (positive control for cell proliferation; doi.org / 10.1038 / sj.onc.1205216). This vector adds a Myc tag (EQKLISEEDL) to the C-terminus of the expressed protein. After transfection, a stable cell mixed population is selected using neomycin over 6 - 10 days. Expression of the target protein and the scrambled protein is confirmed by immunoblotting of 10 μg of cell lysate using an anti-myc tag polyclonal antibody (Abcam). Clonal cell lines are produced by serial dilution by continuous selection.

[0228] Target protein knockout: Determine the effect of target protein deletion in disease-related tumor cell lines (Jurkat E6-1) by Alt-R CRISPR-Cas9 knockout via RNP particles transfected through electroporation (Idtdna.com). Knock out the target protein as well as the REL protein (positive control; DOI: 10.1016 / j.molimm.2020.06.029) and no-guide (negative control). Neomycin resistance is conferred to the transfected cells without problems. Clone cell lines are generated by serial dilution by continuous selection. The knockout is confirmed by genotyping sequencing.

[0229] Proliferation: Seed clone cell lines overexpressing the target protein or control, wild-type Jurkat E6-1 cells, and clone cell lines knocked out for the endogenous target protein and control at 10,000 or 40,000 cells per well, grow them in standard medium, and then subject them to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT assay (Abcam) is a measure of the metabolic activity of a cell population. The higher the metabolic activity, the higher the growth rate.

[0230] Cell cycle progression: Seed clone cell lines overexpressing the target protein or control, wild-type Jurkat E6-1 cells, and clone cell lines with the endogenous target protein and knocked-out control at 10,000 or 40,000 cells per well, grow them in both standard medium and medium supplemented with a phorbol ester compound (at a level known to induce G1 cell cycle arrest), and then subject them to propidium iodide staining and cell cycle analysis by cytometer (doi.org / 10.1038 / sj.onc.1205216).

[0231] Overexpression of Gfi-1 (positive control) results in increased Jurkat cell proliferation and accelerates S-phase entry of Jurkat cells compared to wild-type and scrambled overexpression. Knockout of the c-REL oncogene results in decreased proliferation compared to the no-guide control. Interestingly, similar to the positive control oncogene, overexpression of the target protein results in increased proliferation, promotes S-phase entry, and knockout of the target protein results in decreased proliferation. Collectively, these experiments demonstrate that the target protein is an oncogene and that targeting this protein may benefit patients suffering from cancer, particularly leukemia.

[0232] Example 2: Validation of the target protein as an oncogene (where the target protein has a gain-of-function driver mutation) This example demonstrates the validation of the target proteins of the present disclosure that contain mutations found in cancer tissues (determined by Genome Wide Association Studies (GWAS) and / or listed in The Cancer Genome Atlas (TCGA)). These mutations can promote cancer progression by activating oncogenic target proteins.

[0233] Generate stable tumor cell lines that overexpress wild-type and cataloged TCGA or GWAS mutations of the target protein. Further, in tumor cell lines expressing the wild-type target protein, use CRISPR-Cas9 to replace the wild-type protein with the mutant target protein. Compare the growth rate and apoptosis rate (basal and induced) of the generated cell lines. If the target protein mutation increases the growth rate and / or slows down the apoptosis rate, the target protein is an oncogene and the mutation is a gain-of-function mutation with respect to the cancer disease phenotype.

[0234] Materials and methods: Overexpression: Stable overexpressing tumor cell lines are generated by transfecting Jurkat E6-1 (T cell line; ATCC) with the pcDNA3.1_Myc plasmid (GenScript Inc) containing 1) no insert, 2) wild-type target protein coding sequence, 3) mutant type of the target protein coding sequence (mutations found in cancer; TCGA and / or GWAS), or 4) cancer protein Gfi-1 protein (positive control for cell proliferation; doi.org / 10.1038 / sj.onc.1205216) using Lipofectamine 3000 (ThermoFisher). This vector adds a Myc tag (EQKLISEEDL) to the C-terminus of the expressed protein. After transfection, a stable cell mixed population is selected using neomycin over 6 - 10 days. The expression of the target protein and mutant protein is confirmed by immunoblotting of 10 μg of cell lysate using an anti-myc tag polyclonal antibody (Abcam). Clonal cell lines are produced by serial dilution by continuous selection.

[0235] Mutant target protein knock-in: The wild-type target protein is expressed in Jurkat E6 cells. To determine whether mutations found in some cancer patients confer disease-related phenotypes to cancer cells, CRISPR-Cas9 is used to replace the wild-type target protein with a mutant type of the target protein. As a control for the CRISPR-Cas9-treated cell lines with increased apoptosis, the FAU tumor suppressor is knocked out separately. Clonal cell lines are produced by serial dilution by continuous selection. Knockout and replacement are confirmed by genotyping sequencing.

[0236] Proliferation: Clonal cell lines are seeded at 10,000 or 40,000 cells per well grown in standard medium and subjected to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay 24 hours later. The MTT assay (Abcam) is a measure of the metabolic activity of a cell population. The higher the metabolic activity, the higher the proliferation rate.

[0237] Apoptosis: Seed clonal cell lines at 10,000 or 40,000 cells / well and grow them in either standard medium or medium supplemented with an apoptosis-inducing drug such as Venetoclax, then subject them to the RealTime-Glo(tm) Annexin 5 Apoptosis and Necrosis Assay (Promega).

[0238] Overexpression of the GFI-1 oncoprotein (positive control) leads to an increase in Jurkat cell proliferation compared to wild-type and scrambled overexpression. Knockout of the FAU tumor suppressor also results in increased proliferation and decreased apoptosis compared to the no-guide control. Interestingly, both overexpression of the mutant target protein and substitution of the wild-type target protein with the mutant target protein lead to increased proliferation and / or decreased apoptosis compared to wild-type Jurkat. Collectively, these experiments may explain why the mutations observed in GWAS / TCGA studies of cancer patients occur; this is because it provides a selective advantage over other tumor cells in terms of showing increased proliferation and decreased apoptosis.

[0239] Example 3: Validation of the target protein as a tumor suppressor in an apoptosis assay This example demonstrates the validation of the target protein of the present disclosure as a tumor suppressor, specifically a protein that induces apoptosis in tumor cells.

[0240] Generate stable tumor cell lines with elevated or reduced levels of the target protein or control. Compare the growth rate and apoptosis rate (basal and induced) of the generated cell lines. If the growth rate decreases and / or the apoptosis rate increases in the generated cell lines with an increase in target protein expression, the target protein is a tumor suppressor, and gain-of-function delivery of the target protein may be beneficial in several types of cancer.

[0241] Materials and methods: Overexpression: Stable overexpressing tumor cell lines are generated by transfection of Jurkat E6-1 (T cell line; ATCC) with Lipofectamine 3000 (ThermoFisher) using the pcDNA3.1_Myc plasmid (GenScript Inc) containing 1) no insert, 2) the target protein coding sequence, 3) a scrambled version of the target protein coding sequence (negative control), or 4) Fau protein (positive control for cell proliferation; doi:10.1016 / j.bbadis.2011.04.009). This vector adds a Myc tag (EQKLISEEDL) (SEQ ID NO: 39018) to the C-terminus of the expressed protein. After transfection, a stable cell mixed population is selected using neomycin over 6 - 10 days. Expression of the target protein and the scrambled protein is confirmed by immunoblot of 10 μg of cell lysate using an anti-myc tag polyclonal antibody (Abcam). Clonal cell lines are generated by serial dilution by successive selection.

[0242] Target protein knockout: The effect of target protein deletion in disease-related tumor cell lines (Jurkat E6-1) is determined by Alt-R CRISPR-Cas9 knockout via transfected RNP particles via electroporation (Idtdna.com). The target protein as well as SODD protein (positive control; DOI dx.doi.org / 10.4238 / 2014.March.24.6) and no guide (negative control) are knocked out. Neomycin resistance is conferred to the successfully transfected cells. Clonal cell lines are generated by serial dilution by successive selection. The knockout is confirmed by genotypic sequencing.

[0243] Proliferation: Clone cell lines overexpressing the target protein or control, wild-type Jurkat E6-1 cells, and clone cell lines with knockout of the endogenous target protein and control are seeded at 10,000 or 40,000 cells per well, grown in standard medium, and then subjected to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT assay (Abcam) is a measure of the metabolic activity of a cell population. The higher the metabolic activity, the higher the growth rate.

[0244] Apoptosis: Clone cell lines overexpressing the target protein or control, wild-type Jurkat E6-1 cells, and clone cell lines with knockout of the endogenous target protein and control are seeded at 10,000 or 40,000 cells per well and grown in either standard medium or medium supplemented with an apoptosis inducer such as Venetoclax, and then subjected to the RealTime-Glo(tm) Annexin 5 Apoptosis and Necrosis Assay (Promega).

[0245] Mouse xenograft model: The overexpression and knockout stable cell lines prepared above are used in a mouse xenograft model to determine the in vivo effect on tumor growth.

[0246] Results: Overexpression of the FAU tumor suppressor (positive control) results in a decrease in Jurkat cell proliferation and an increase in basal apoptosis levels compared to wild-type and scrambled overexpression. Knockout of SODD also results in a decrease in proliferation and an increase in apoptosis compared to the no-guide control. Interestingly, overexpression of the target protein such as the FAU positive control tumor suppressor results in a decrease in proliferation and an increase in apoptosis, and knockout of the target protein results in an increase in proliferation and a decrease in apoptosis. These in vitro changes lead to a decrease in the tumor growth rate in the mouse xenograft model. In summary, these experiments demonstrate that the target protein is a tumor suppressor and that delivery of this protein to tumors may benefit patients suffering from cancer.

[0247] Example 4: Verification of Target Protein as a Tumor Suppressor with Loss-of-Function Mutation This example demonstrates the verification of the target protein of the present disclosure as a tumor suppressor. Specifically, a protein showing a loss-of-function mutation in cancer (determined by Genome Wide Association Studies (GWAS) and / or listed in The Cancer Genome Atlas (TCGA)) that increases tumor cell proliferation and / or apoptosis by inactivating the target protein.

[0248] Generate stable tumor cell lines overexpressing the wild-type and one or more cataloged TCGA or GWAS mutants of the target protein. Further, in the tumor cell line expressing the wild-type target protein, use CRISPR-Cas9 to replace the wild-type protein with the mutant target protein. Compare the growth rate and apoptosis rate (basal and induced) of the generated cell lines. If overexpression of the target protein decreases growth and / or increases apoptosis, and overexpression of the mutated target protein does not show such an effect, the target protein is a tumor suppressor and the cancer-related mutation is a loss-of-function mutation. Similarly, if replacement of the wild-type allele by a cancer-related mutation using CRISPR-Cas9 increases growth and / or decreases apoptosis, the target protein is a tumor suppressor.

[0249] Materials and Methods: Overexpression: Stable overexpressing tumor cell lines are generated by transfecting Jurkat E6-1 (T cell line; ATCC) using Lipofectamine 3000 (ThermoFisher) with a pcDNA3.1_Myc plasmid (GenScript Inc) containing 1) no insert, 2) wild-type target protein coding sequence, 3) a mutant form of the target protein coding sequence (mutations found in cancer; TCGA and / or GWAS), or 4) the tumor suppressor protein FAU (positive control for decreased cell proliferation / increased apoptosis). This vector adds a Myc tag (EQKLISEEDL SEQ ID NO: 39018) to the C-terminus of the expressed protein. After transfection, a stable cell mixed population is selected using neomycin over 6 - 10 days. Expression of the target protein and mutant proteins is confirmed by immunoblot of 10 μg of cell lysate using an anti-myc tag polyclonal antibody (Abcam). Clonal cell lines are produced by serial dilution by continuous selection.

[0250] Mutant target protein knock-in: The wild-type target protein is expressed in Jurkat E6 cells. To determine whether mutations found in some cancer patients confer a disease-related phenotype to cancer cells, CRISPR-Cas9 is used to replace the wild-type target protein with a mutant form of the target protein. As a control, the FAU tumor suppressor protein is knocked out, which should result in increased proliferation and / or decreased apoptosis. Clonal cell lines are produced by serial dilution by continuous selection. Knockout and replacement are confirmed by genotypic sequencing.

[0251] Proliferation: Clonal cell lines are seeded at 10,000 or 40,000 cells per well grown in standard medium and subjected to an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay 24 hours later. The MTT assay (Abcam) is a measure of the metabolic activity of a cell population. The higher the metabolic activity, the higher the proliferation rate.

[0252] Apoptosis: Seed clonal cell lines at 10,000 or 40,000 cells / well and grow them in either standard medium or medium supplemented with an apoptosis-inducing drug such as Venetoclax, and then subject them to the RealTime-Glo(tm) Annexin 5 Apoptosis and Necrosis Assay (Promega).

[0253] Results: Overexpression of the FAU tumor suppressor protein (positive control) results in a decrease in Jurkat cell proliferation and / or an increase in apoptosis compared to wild-type and scrambled overexpression. Knockout of the FAU tumor suppressor results in an increase in proliferation and a decrease in apoptosis compared to the no-guide control. Interestingly, overexpression of the target protein rather than the cancer-associated variant target protein results in a decrease in Jurkat cell proliferation and / or apoptosis. Similarly, replacing the target protein with the cancer-associated variant target protein results in an increase in proliferation and / or a decrease in apoptosis. Collectively, these experiments may explain why the mutations observed in GWAS / TCGA studies of cancer patients occur; this is because it provides a selective advantage over other tumor cells in that it shows an increase in proliferation and a decrease in apoptosis.

[0254] Example 5: Verification of the target protein as an oncogene or tumor suppressor in cancer growth and proliferation This example demonstrates the ability of the target proteins of the present disclosure to function as oncogenes or tumor suppressors in cancer cells. In this example, a library of guide RNAs (gRNAs) encoded by lentiviruses that individually target the proteins of the genes of interest was synthesized (Cellca), yielding a library range of 6 unique gRNAs per gene.

[0255] Materials and methods: Transduce cancer cell lines (dtp.cancer.gov / discovery_development / nci-60 / cell_list.htm) with Cas9 nuclease encoded by lentivirus at a high multiplicity of infection (MOI), and then transduce the gRNA lentivirus library at a low MOI of 0.5 to ensure that each cell receives approximately one gRNA. The gRNA library vector also encodes puromycin resistance. One day after transduction, incubate the cells with puromycin for 4 days to select cells that have successfully undergone transduction.

[0256] After transduction and selection, collect 10×10 6 cells and use them as a "baseline" population to compare the growth effects of individual genes. Seed 10×10 6 cells in the manufacturer's recommended medium, split them twice a week for 4 weeks, and re-seed 10×10 6 cells at each split.

[0257] After 4 weeks of cell / tumor growth, isolate DNA from in vitro samples, lyse it using a DNA extraction kit (Qiagen DNeasy Blood and Tissue Kit), and concentrate it by ethanol precipitation. Amplify the DNA sample by 2 rounds of PCR using the manufacturer's recommended primers and analyze it by next-generation sequencing (BGI America).

[0258] The results of the array determination are analyzed for the called hits using the Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) algorithm as described by Li W et al., Genome Biology 2014 and Li W et al., Genome Biology 2015. Briefly described, sequencing reads are normalized against their median, the variance of the read counts for individual gRNAs is estimated, normalized, and the differences in the individual gRNA read counts are ranked against each other. Target genes are called based on whether there are multiple gRNAs targeting a single gene ranked near the top of the gRNA ranking list. The gRNAs observed in the analysis indicate their corresponding target proteins that act as oncogenes.

[0259] Target genes are quantified along three parameters: β score, essentially the magnitude of the effect (log fold change in the number of gRNAs); p-value; false discovery rate (FDR). A β score less than 0 indicates that six gRNAs targeting a single gene are absent in the late samples compared to the baseline samples, which is a good indicator that the gene is "dropping out" during tumor growth. Both the p-value and FDR reflect the confidence that the results are not artifacts, with lower values indicating higher confidence. The thresholds for calling hits are p-value < 0.1 and FDR < 0.5. gRNAs no longer observed in the analysis (dropout) indicate their corresponding target proteins that act as tumor suppressors.

[0260] Example 6: Verification of Target Proteins as Tumor Suppressors in Apoptosis Assays This example demonstrates the verification of the target protein SEQ ID NO: 37997 of the present disclosure as a secreted tumor suppressor and potential targets for the treatment of multiple cancers. Multiple tumor cell lines were treated with the synthetically produced protein of SEQ ID NO: 37997. After 24 hours, the basal level of apoptosis was measured.

[0261] Materials and Methods: Cell culture: Tumor cell lines HCT-116 (colon cancer cell line, ATCC) and U2OS (osteosarcoma cell line, ATCC) cells were grown adherently at 37 °C (5% CO2) in ATCC-formulated McCoy's 5a medium supplemented with 10% FBS and 1× penicillin-streptomycin. Jurkat cells (acute T cell leukemia cell line, ATCC) were grown as a suspension culture at 37 °C (5% CO2) in ATCC-formulated RPMI-1640 supplemented with 10% FBS and 1× penicillin-streptomycin. HEP-G2 cells (liver cancer cell line, ATCC) were grown adherently at 37 °C (5% CO2) in ATCC-formulated Eagle's minimum essential medium (EMEM) supplemented with 10% FBS and 1× penicillin-streptomycin.

[0262] Peptide treatment: The target protein sequence number 37997 was synthesized by solid-phase peptide synthesis (SPPS) using fluorenylmethyloxycarbonyl (Fmoc) protecting group chemistry. 5 μM of the target peptide and a control peptide were added to 96-well plates containing 10,000 cells (100 μL / well). White-walled 96-well plates suitable for cell culture and compatible with a luminometer were used. (Corning)

[0263] Apoptosis assay: After 24 hours of peptide treatment, an equal volume of the reconstituted Caspase-Glo® 3 / 7 assay reagent mixture (Promega) was added to the cells. The contents of the wells were gently mixed for 30 seconds at 300 - 500 rpm using a plate shaker. The plate was then incubated at room temperature for 3 hours. Subsequently, the treated plate was read for luminescence using a Promega® GloMax® Plate Reader. Background luminescence was measured using a blank reaction containing Caspase-Glo® 3 / 7 reagent, vehicle, and cell culture medium without cells. The blank reaction value was subtracted from the experimental value. The basal caspase activity of the cell culture system was determined, including a negative control containing Caspase-Glo® 3 / 7 reagent and vehicle-treated cells in the medium. The significant difference in apoptosis was determined using ANOVA analysis by comparing the cells treated with the peptide of SEQ ID NO: 37997 with the cells treated with an irrelevant control peptide or vehicle control-treated cells.

[0264] Results: Treatment of multiple cell lines with the peptide of SEQ ID NO: 37997 resulted in an increase in the basal apoptosis level. SEQ ID NO: 37997 is a tumor suppressor and is suitable for the treatment of multiple cancers (Figure 1, Figure 2, Table C).

[0265] [Table C]

[0266] Example 7: Verification of target proteins as tumor suppressors with loss-of-function mutations This example demonstrates the verification of the target proteins of SEQ ID NO: 27301 and SEQ ID NO: 30462 as contributing factors to tumor growth. Stable tumor cell lines with elevated levels of each target protein or control were generated. The growth rates of the generated cell lines were compared. If the growth rate of the generated cell line increased with the increase in the level of the target protein, the target protein is an oncogene.

[0267] Generate stable tumor cell lines that overexpress a target protein with wild-type and one or more cataloged TCGA or GWAS mutations. Further, in tumor cell lines expressing the wild-type target protein, use CRISPR-Cas9 to replace the wild-type protein with the mutant target protein. Compare the growth rates and apoptosis rates (basal and induced) of the generated cell lines. If overexpression of the target protein reduces growth and / or increases apoptosis, and overexpression of the mutated target protein does not show such an effect, the target protein is a tumor suppressor and the cancer-associated mutation is a loss-of-function mutation. Similarly, if replacement of the wild-type allele by a cancer-associated mutation using CRISPR-Cas9 increases growth and / or decreases apoptosis, the target protein is a tumor suppressor.

[0268] Materials and methods: Overexpression: A stable overexpressing tumor cell line was generated by transduction using lentivirus. For virus production, HEK-293T cells were cultured in DMEM containing high glucose and GlutaMAX™ medium (ThermoFisher) supplemented with 10% FBS. After 24 hours, cells were transfected into HCT116 (colon cancer cell line; ATCC) using Lipofectamine 3000 (ThermoFisher) with a Lentiviral packaging plasmid mix (CELLECTA) and pGenLenti plasmid (GenScript Inc) containing 1) without insert, 2) target protein coding sequence, and 3) AP2A1 protein (positive control for cell proliferation; https: / / doi.org / 10.1038 / sj.onc.1205216). A Myc tag (EQKLISEEDL) was added to the C-terminus of the expressed protein. 48 hours after transfection, the culture medium containing the virus was collected, filled into a 10 ml syringe, and filtered through a 0.45 μm filter. For cell transduction, HCT116 colorectal cancer cells were seeded in a 6-well plate and the virus was added to the cells. After transduction, puromycin was used to select a stable cell mixed population for 14 days. The expression of the target protein and the control was confirmed by immunoblotting of 50 μg of cell lysate using an anti-myc tag polyclonal antibody (CST).

[0269] Western blot: 50 μg of cell lysates from control and target protein stable cell lines were separated by NuPAGE 4-12% Bis-Tris gel (invitrogen), transferred to a PVDF membrane (Thermofisher), and subjected to anti-Myc-tag (CST) and anti-GAPDH (CST) primary antibodies, followed by an anti-rabbit HRP secondary antibody (CST). The HRP signal was visualized with an iBright system.

[0270] Proliferation: Polyclonal cell lines overexpressing the target protein or control, wild-type HCT-116 cells were seeded into 96-well plates (10,000 or 5,000 cells / well) in standard medium under both low serum (0.1% FBS) and high serum (10% FBS) conditions and allowed to proliferate. After 48 hours, the cells were subjected to a WST-1 (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The WST-1 assay (Sigma) is a measure of the metabolic activity of a cell population. The higher the metabolic activity, the higher the growth rate. The significant difference in proliferation was determined by using ANOVA analysis to compare the protein-expressing cell lines with the stable cell lines of the empty expression vector (control).

[0271] Maestro Z Impedance-based assay: The Maestro Z platform (Axion Biosystems) uses impedance measurements (ohms, Ω) to quantify the presence of cells on the electrodes. Impedance is non-invasive and label-free, so impedance assays are used to quantify dynamic cell growth over time. For impedance recording, cells were seeded into 96-well assay plates, the plates were docked to the Maestro Z, and the chamber was set to 37°C and 5% CO2 by automatic environmental control. Impedance measurements were recorded every minute for 2 days.

[0272] Results: Overexpression of AP2A (positive control oncogene) results in an increase in HCT-116 cell proliferation compared to control cells. Interestingly, similar to the positive control oncogene, overexpression of the target protein results in an increase in proliferation. These experiments demonstrate that the target protein is an oncogene and that targeting this protein may benefit patients suffering from cancers such as colon cancer (Figures 3, 4, 5, Table D).

[0273]

Table D

[0274] Example 8: Verification of target proteins as cancer genes or tumor suppressors due to cancer growth and proliferation Proliferation: HCT-116 cells were seeded in 96-well plates (5,000 cells / well) supplemented with standard medium containing 10% FBS. After incubation at 37 (5% CO 2 ) for 48 hours, the cells were subjected to a WST-1 (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The WST-1 assay (Sigma) is a measure of the metabolic activity (i.e., proliferation rate) of the cells. The significant difference in proliferation was determined by comparing the cells treated with 10 μM of the protein of SEQ ID NO: 37997 with the cells treated with 10 μM of an irrelevant control peptide or untreated cells using ANOVA analysis (Figure 6, Table E).

[0275]

Table E-1

[0276]

Table E-2

[0277]

Table E-3

[0278]

Table E-4

[0279]

Table E-5

[0280] Example 9: Verification of two target proteins as novel G protein-coupled receptor (GPCR) ligands. This example demonstrates the validation of two novel peptides, SEQ ID NO: 30949 and SEQ ID NO: 34229, which act as GPCR ligands. SEQ ID NO: 30949 blocks CXCR4 and cancer cell migration, making it suitable for the treatment of multiple cancers. On the other hand, SEQ ID NO: 34229 agonizes C3AR1, an important regulator of immune response and inflammation. The target proteins of SEQ ID NO: 30949 and SEQ ID NO: 34229 were treated with gpcrMAX, an inclusive panel that encompasses 168 G protein-coupled receptors (GPCRs) from over 60 different receptor families. This panel utilizes the PathHunter® β-arrestin technology (Eurofins DiscoverX). The PathHunter β-arrestin GPCR cell lines are engineered to co-express a ProLink™ (PK)-tagged GPCR and an enzyme acceptor (EA)-tagged β-arrestin. Activation of GPCR-PK induces the recruitment of β-arrestin-EA, forcing the complementation of two β-galactosidase enzyme fragments (EA and PK). The resulting functional enzyme hydrolyzes the substrate to generate a chemiluminescent signal.

[0281] Materials and Methods: Cell treatment: PathHunter® cell lines were grown from freezer stocks according to standard procedures. Cells were seeded in white-wall 384-well microplates at a total volume of 20 μL and incubated at 37 °C for an appropriate time before testing.

[0282] Peptide processing: The target protein sequence numbers 30949, 34229, and irrelevant peptides were synthesized by solid-phase peptide synthesis (SPPS) using Fluorenylmethyloxycarbonyl (Fmoc) protecting group chemistry. For screening: The target protein sequence numbers 30949, 34229, and eight irrelevant peptides were processed in the gpcrMAX panel at a final top test concentration of 0.12 μM. For hit confirmation, the target protein sequence number 30949 and irrelevant peptides were tested in a CXCR4 human chemokine GPCR cell-based antagonist arrestin assay at the highest test concentrations of 1 μM and 0.3 μM.

[0283] Assay design: Agonist format: For agonist determination, cells were incubated with samples to induce a response. An intermediate dilution of the sample stock was performed to generate 5× samples in assay buffer. 5 μL of the 5× sample was added to the cells and incubated at 37 °C or room temperature for 90 - 180 minutes. The vehicle concentration was 1%. Antagonist format: For antagonist determination, cells were pre-incubated with the antagonist and subsequently challenged with the agonist at the EC80 concentration. An intermediate dilution of the sample stock was performed to generate 5× samples in assay buffer. 5 μL of the 5× sample was added to the cells and incubated at 37 °C or room temperature for 30 minutes. The vehicle concentration was 1%. 5 μL of 6× EC80 agonist in assay buffer was added to the cells and incubated at 37 °C or room temperature for 90 or 180 minutes.

[0284] CXCR4 human chemokine GPCR cell-based antagonist arrestin assay: For hit confirmation, the PathHunter® β-arrestin cell line for the CXCR4 human chemokine GPCR antagonist assay was used with CXCL12 / SDF-1a as the activator and Plerixafor as the inhibitor.

[0285] C3aR Human Complement Peptide GPCR Cell-based Agonist Assay: For hit confirmation, the PathHunter® β-arrestin cell line for the C3AR1 human complement peptide GPCR agonist assay was used with a C3A receptor agonist (Short Fragment) as the control activator.

[0286] Signal Detection: The assay signal was generated by a single addition of 12.5 or 15 μL (50% v / v) of the PathHunter® Detection reagent cocktail, followed by a 1-hour incubation at room temperature. The microplate was read after signal generation using a PerkinElmer Envision® device for chemiluminescence signal detection.

[0287] Data Analysis: Compound activities were analyzed using the CBIS data analysis suite (ChemInnovation, C). For agonist-mode assays, the percentage of activity was calculated using the formula % Activity = 100% × (Average RLU of test sample - Average RLU of vehicle control) / (Average MAX control ligand - Average RLU of vehicle control). For antagonist-mode assays, the % inhibition was calculated using the following formula: % Inhibition = 100% × (1 - (Average RLU of test sample - Average RLU of vehicle control) / (Average RLU of EC80 control - Average RLU of vehicle control)).

[0288] Chemotaxis assay: NAMALWA cells (human Burkitt lymphoma cell line, ATCC) were cultured in serum-free ATCC-formulated RPMI for 24 hours before the assay. After starvation, the cells were harvested and pelleted by centrifugation at 1,000×g for 5 minutes. The cells were resuspended in serum-free medium. Using Abcam's Cell Migration / Chemotaxis Assay Kit (96-well, 8μm), 150 μL of serum-free medium containing the desired chemoattractant was added to the bottom chamber. Then, 50,000 cells and the desired inhibitor (or peptide) were added to each well of the upper chamber. The plate was placed and incubated at 37 °C in a CO2 incubator for 24 hours. After incubation, 110 μL of Cell Dye + Cell Dissociation Solution mixture was added to each bottom well and incubated at 37 °C in a CO2 incubator for 1 hour. After incubation, the upper chamber was removed and the plate was read at Ex / Em = 530 / 590 nm.

[0289] Results: Two target peptides, SEQ ID NO: 30949 and SEQ ID NO: 34229, were identified as novel GPCR ligands using Eurofins' gpcrMAX panel. The target peptide of SEQ ID NO: 34229 is an agonist of C3AR1, an important anaphylatoxin receptor that plays an important role in inflammation. The target peptide, SEQ ID NO: 30949, blocked CXCR4, a chemokine receptor involved in cell migration and homing. Furthermore, since SEQ ID NO: 30949 was able to significantly inhibit the chemotactic migration of human Burkitt lymphoma cells, its potential as a chemokine for treating cancer was suggested (Figures 7, 8, 9, 10).

[0290] The teachings of all patents, published applications, and references cited herein are hereby incorporated by reference in their entirety.

[0291] Although exemplary embodiments have been specifically shown and described, it will be understood by those skilled in the art that various modifications in form and detail may be made 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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