Compositions and methods for treating NRP2-related diseases

Abstract

Provided are therapies for treating neuropilin-2 (NRP2)-related diseases and conditions, including monotherapy and combination therapy, comprising the use of at least one histidyl-tRNA synthetase (HRS) polypeptide.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 703,757, filed July 26, 2018; U.S. Provisional Application No. 62 / 776,208, filed December 6, 2018; U.S. Provisional Application No. 62 / 800,035, filed February 1, 2019; and U.S. Provisional Application No. 62 / 849,440, filed May 17, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Declaration of sequence list

[0004] The sequence list relating to this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference in this specification. The text file containing the sequence list is named ATYR_135_04WO_ST25.txt. The text file is 392KB in size, was created on July 26, 2019, and submitted electronically via EFS-Web. Technical Field

[0005] Embodiments of this disclosure relate to therapies for treating neuropilin-2 (NRP2)-related diseases and symptoms, including monotherapy and combination therapy, said therapies comprising the use of at least one histyl-tRNA synthetase (HRS) peptide. Background Technology

[0006] Recent research advances have shown that tRNA synthases, in addition to their excellent characterization of protein synthesis, also play an important role in cellular responses. Specifically, it is increasingly recognized that tRNA synthases can play a series of previously unknown roles in responding to cellular stress and tissue homeostasis, both intracellularly and extracellularly.

[0007] The resokine protein family (HRS peptides) originates from the histidine tRNA synthetase gene (HARS) through protein hydrolysis or alternative splicing and are important extracellular regulators of cellular activity. Extracellular HARS can be readily detected in the blood circulation of normal healthy volunteers, and an autoantibody against HARS (Jo-1 antibody) has been characterized in some subjects with inflammatory myopathy (IM) and inflammatory lung disease (ILD). Although the role of Jo-1 antibodies in disease progression is unclear, subjects with Jo-1 antibodies tend to be less likely to develop cancer compared to subjects with inflammatory myopathy who do not have Jo-1 antibodies (see, for example, Lu et al., PLOS ONE 9(4)e94128, 2014; Modan et al., Clin. Exp. Dermatol. 34(5)561-565, 2009; and Shi et al., J. Rheum 44(7)doi10.3899 / jrheum.161480).

[0008] Recent advances have been made in elucidating the roles of extracellular HARS-derived proteins, including the identification of a putative cellular receptor—neuropiliin-2 (NRP2 ​​or NRP-2). The interaction between HARS and NRP2 appears to be mediated by the N-terminal region of HARS and may induce important changes in the cellular function of NRP2.

[0009] Therefore, the current discovery of the Reoskine / neurocilione-2 axis represents a previously unknown mechanism that acts as a central regulator of cellular processes directly related to muscle, blood vessels, neurons, bone, and immune homeostasis. Dysregulation of any of these processes can lead to a range of diseases, and novel HRS peptide-based therapies may be able to address these diseases. Summary of the Invention

[0010] Embodiments of this disclosure include methods for treating neuropilin-2 (NRP2)-related diseases or conditions in subjects of need, the methods comprising administering to the subject a therapeutic composition comprising a histidine-tRNA synthetase (HRS) polypeptide.

[0011] In some embodiments, the NRP2-related diseases or conditions are selected from one or more of the following: cancer and cancer-related diseases or pathologies, such as cancer cell growth, cancer initiation, cancer migration, cancer cell adhesion, cancer cell invasion, cancer cell chemoresistance, and cancer cell metastasis; diseases related to inflammation and autoimmunity, optionally inflammatory lung diseases, such as allergic pneumonia and lung inflammation, and diseases related to inappropriate immune cell activation or migration, optionally graft-versus-host disease (GVHD) and rheumatoid arthritis-associated interstitial lung disease (RA-ILD); diseases related to lymphatic development, lymphangiogenesis, and lymphatic damage, optionally edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability; diseases related to infections such as latent infections; and diseases related to allergic conditions and allergic responses, optionally chronic obstructive pulmonary disease. Diseases of the disease (COPD), neutrophilic asthma, systemic vasculitis associated with antineutrophil cytoplasmic antibodies (ANCA), systemic lupus erythematosus, rheumatoid arthritis, one or more inflammasome-related diseases and one or more skin-related neutrophil-mediated diseases, such as pyoderma gangrenosa; diseases associated with granulomatous inflammatory diseases, optionally sarcoidosis, pulmonary granulomatous diseases and non-pulmonary granulomatous diseases; fibrotic diseases, such as endometriosis, fibrosis, endothelial-to-mesenchymal transition (EMT) and wound healing; diseases associated with inappropriate smooth muscle contractility, vascular smooth muscle cell migration and adhesion; diseases associated with inappropriate autophagy, phagocytosis and burial; neuronal diseases, optionally associated with peripheral nervous system remodeling and pain perception; diseases associated with bone development and / or bone remodeling; and diseases associated with inappropriate migratory cell movement.

[0012] In some embodiments, the extracellular fluid level of the subject's bound or free soluble NRP2 peptide (optionally selected from Table N1) is increased relative to the level of healthy controls or matched control criteria or a population consisting of one or more subjects, optionally at a level of about or at least about 10 pM, 20 pM, 30 pM, 50 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1000 pM, 1100 pM, 1200 pM, 1300 pM, 1400 pM, 1500 pM, 1600 pM, 1700 pM, 1800 pM. pM, 1900 pM, 2000 pM, 3000 pM, 4000 pM, 5000 pM, or the level of the soluble NRP2 peptide is about 30-50 pM, 50-100 pM, 100-2000 pM, 200-2000 pM, 300-2000 pM, 400-2000 pM, 500-2000 pM, 600-2000 pM, 700-2000 pM, 800-2000 pM, 900-2000 pM, 1000-2000 pM, 2000-3000 pM, 3000-4000 pM, 4000-5000 pM, and / or the subject is selected for treatment based on the increase.

[0013] In some embodiments, the extracellular fluid level of the subject's NRP2:NRP2 ligand complex (optionally selected from Tables N1 and N2) is increased relative to the level of a healthy control or matched control standard or a population consisting of one or more subjects, optionally at a level about or at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more of the control or reference level, and / or the subject is selected for treatment based on the increase.

[0014] In some embodiments, the extracellular fluid level of the HRS:NRP2 complex (optionally selected from Tables H1 and N1) in the subject is increased relative to the level of a healthy control or matched control standard or a population consisting of one or more subjects, optionally at a level of about or at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more of the control or reference level, and / or the subject is selected for treatment based on the increase.

[0015] In some embodiments, the subject has a single nucleotide polymorphism (SNP) in an NRP2 peptide or a polynucleotide encoding NRP2, and / or the subject is selected for treatment based on having the single nucleotide polymorphism.

[0016] In some embodiments, the subject suffers from a disease associated with an increase in the level or expression of NRP2a and / or NRP2b, or an altered NRP2a:NRP2b expression ratio, relative to the levels or expression of healthy controls, matched controls, or a population of one or more subjects, and / or the subject is selected for treatment based on having said disease. In some embodiments, the level of NRP2b is increased by about or at least about 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% compared to the levels of healthy controls, matched controls, or a population of one or more subjects.

[0017] In some embodiments, the healthy controls or matched control criteria or the population of one or more subjects comprise an average range of age-matched samples of cancerous or non-cancerous cells or tissues of the same type as the cancer, the average range including specific characteristics such as drug resistance, metastatic potential, invasiveness, genetic signature (optionally one or more p53 mutations, PTEN deletion, IGFR expression) and / or expression patterns.

[0018] In some embodiments, the subject in need has an infection, and / or the subject is selected for treatment based on having the infection, and optionally the method further includes administering at least one antimicrobial agent, antifungal agent, and / or antiworm agent to the subject. In some embodiments, (a) the HRS peptide and (b) the antimicrobial agent, antifungal agent, and / or antiworm agent are administered together as part of the same therapeutic composition. In some embodiments, (a) the HRS peptide and (b) the antimicrobial agent, antifungal agent, and / or antiworm agent are administered as separate therapeutic compositions. In some embodiments, the antimicrobial agent, antifungal agent, and / or anti-helmintic agent is selected from one or more of the following: aminoglycosides, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin; carbapenems, such as ertapenem, doripenem, imipenem / cilastatin, and meropenem; cephalosporins, such as cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, and cefalexin. exin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefotoxin The following are listed: ditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, cefepime, ceftarolinefosamil, and ceftobiprole.Glycopeptides, such as teicoplanin, vancomycin, telavancin, dalbavancin, and oritavancin; lincosamines, such as clindamycin and lincomycin; macrolides, such as azithromycin, clarithromycin, erythromycin, and roxithromycin. ycin), telithromycin, and spiramycin; penicillins, such as amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, and penicillin G. G), Temocillin and Ticarcillin; peptides such as Bacitracin, Colistin and Polymyxin B; quinolones / fluoroquinolones such as Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixicacid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin and Temafloxacin;Sulfonamides, such as sulfamidoline (Mafenide), sulfacetamide (Sulfacetamide), sulfadiazine (Sulfadiazine), and silver sulfadiazine (Silver). Sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (Co-trimoxazole) (TMP-SMX), and sulfonamidochrysoidine; tetracyclines, such as demeclocycline, doxycycline, metacycline, minocycline, oxytetracycline, and tetracycline. e) Antibacterial agents, such as clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, and streptomycin; chloramphenicol; metronidazole; mupirocin; tigecycline; tinidazole; and anti-helmintic agents, such as diethylcarbamazine and albendazole.

[0019] It also includes one or more methods selected from the following:

[0020] - A method for improving or restoring lymphatic function in subjects in need;

[0021] - A method for regulating lymphangiogenesis in subjects who require it;

[0022] - A method for treating diseases or conditions related to brachial platelet signaling in subjects in need;

[0023] - A method for modulating vascular endothelial growth factor C (VEGF-C) signaling in subjects in need;

[0024] - A method for modulating integrin signaling in subjects who require it;

[0025] - A method for modulating TGF-β signaling in subjects who require it;

[0026] - A method for modulating autophagy, phagocytosis, or endocytosis in subjects who require it;

[0027] - A method for modulating neuronal development in subjects who require it;

[0028] - A method for reducing the migration or adhesion of lymphoendothelial cells in subjects in need;

[0029] - A method for modulating endothelial-to-mesenchymal transition (EMT) in subjects who require it;

[0030] - A method for regulating bone development in subjects who require it;

[0031] - A method for modulating vascular permeability in subjects who require it;

[0032] - A method for modulating the binding or functional interaction between NRP2 peptides and NRP2 ligands in subjects in need;

[0033] - Methods for inhibiting the activity, migration, or adhesion of immune cells in subjects in need; and

[0034] - A method for reducing tumor cell migration or adhesion in subjects in need.

[0035] The method includes administering a therapeutic composition comprising a histidine-tRNA synthetase (HRS) peptide to the subject in need.

[0036] In some embodiments, the lymphangiogenesis is secondary to cancer, corneal injury, dry eye disease, inflammation, lymphedema, graft rejection, or any combination thereof. In some embodiments, the neuronal development is peripheral neural remodeling associated with inflammatory or autoimmune symptoms. In some embodiments, the NRP2 ligand is selected from VEGF-C, VEGF-D, VEGF-A145, VEGFA165, PIGF-2, brachiopanthenin 3B, 3C, 3D, and 3F, heparin, integrin, and TGF-β. In some embodiments, the NRP2 ligand is selected from VEGF-C, VEGF-D, VEGF-A145, VEGFA165, and PIGF-2. In some embodiments, the NRP2 ligand is selected from brachiopanthenin 3B, 3C, 3D, 3F, and 3G. In some embodiments, the immune cells are selected from myeloid cells, macrophages, neutrophils, eosinophils, granulocytes, dendritic cells, T cells, B cells, and natural killer (NK) cells. In some embodiments, the T cells are T cells. REG Cells, T H1 Cells or T H2 Cells. In some embodiments, the macrophages are M1 or M2 macrophages. In some embodiments, the method includes reducing the migration of the tumor cells within the lymphatic system.

[0037] In some embodiments, the subject suffers from neuropilin-2 (NRP2)-related disease or symptoms, optionally wherein the subject has the following and / or the subject is selected for treatment based on having: increased extracellular fluid levels of soluble NRP2 peptides, increased extracellular fluid levels of NRP2:NRP2 ligand complexes, increased extracellular fluid levels of HRS:NRP2 complexes, and / or single nucleotide polymorphisms (SNPs) in the subject's NRP2 peptides or polynucleotides encoding NRP2.

[0038] In some embodiments, the disease is cancer, for example, wherein the cancer expresses or overexpresses NRP2. In some cases, the cancer exhibits NRP2-dependent growth, NRP2-dependent adhesion, NRP2-dependent migration, NRP2-dependent chemoresistance, and / or NRP2-dependent invasion. In some embodiments, the cancer is primary cancer. In some embodiments, the cancer is metastatic cancer, optionally, metastatic cancer expressing NRP2a and / or NRP2b.

[0039] In some embodiments, the cancer is chemoresistant to at least one cancer therapy (e.g., cancer immunotherapy agents, chemotherapy agents, hormone therapy agents, and / or kinase inhibitors). In some cases, the method includes selecting subjects with cancer that is chemoresistant to at least one cancer therapy prior to administration of the HRS peptide.

[0040] In some embodiments, the HRS peptide regulates autophagy, burial, or phagocytic cell maturation in cancer cells or cancer-associated macrophages. In a specific embodiment, the HRS peptide regulates autophagy in the cancer cells.

[0041] In some embodiments, the cancer is selected from one or more of the following: melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neuroblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0042] In some embodiments, the metastatic cancer is selected from one or more of the following:

[0043] (a) Bladder cancer that has metastasized to the bone, liver and / or lungs;

[0044] (b) Breast cancer that has metastasized to the bone, brain, liver and / or lungs;

[0045] (c) Colorectal cancer that has metastasized to the liver, lungs and / or peritoneum;

[0046] (d) Kidney cancer that has metastasized to the adrenal glands, bone, brain, liver and / or lungs;

[0047] (e) Lung cancer that has metastasized to the adrenal glands, bone, brain, liver and / or other lung sites;

[0048] (f) Melanoma that has metastasized to bone, brain, liver, lungs and / or skin / muscle;

[0049] (g) Ovarian cancer that has metastasized to the liver, lungs and / or peritoneum;

[0050] (h) Pancreatic cancer that has metastasized to the liver, lungs and / or peritoneum;

[0051] (i) Prostate cancer that has metastasized to the adrenal glands, bones, liver and / or lungs;

[0052] (j) Gastric cancer that has metastasized to the liver, lungs and / or peritoneum;

[0053] (l) Thyroid cancer that has metastasized to the bone, liver, and / or lung; and

[0054] (m) Uterine cancer that has metastasized to the bone, liver, lungs, peritoneum and / or vagina.

[0055] Some embodiments involve administering to the subject at least one additional agent selected from one or more of the following: cancer immunotherapy agents, chemotherapy agents, hormone therapy agents, and kinase inhibitors. In some embodiments, at least one HRS peptide and at least one agent are administered alone as a separate composition. In some embodiments, at least one HRS peptide and at least one agent are administered together as part of the same therapeutic composition.

[0056] In some embodiments, the cancer immunotherapy agent is selected from one or more of the following: immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies. In some embodiments, the immune checkpoint modulator is a peptide, optionally an antibody or its antigen-binding fragment or ligand or small molecule. In some embodiments, the immune checkpoint modulator includes:

[0057] (a) Antagonists of inhibitory immune checkpoint molecules; or

[0058] (b) An agonist of an immune checkpoint molecule, for example, wherein the immune checkpoint modulator specifically binds to the immune checkpoint molecule.

[0059] In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of the following: programmed death-ligand 1 (PD-L1), programmed death-1 (PD-1), programmed death-ligand 2 (PD-L2), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), T-cell activation V-domain Ig inhibitor (VISTA), B and T lymphocyte attenuating factor (BTLA), CD160, herpesvirus entry mediator (HVEM), and T-cell immune receptor having Ig and ITIM domains (TIGIT).

[0060] In some embodiments, the antagonist is optionally selected from one or more of the following PD-L1 and / or PD-L2 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and / or PD-L2, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), wherein the cancer is optionally selected from one or more of the following: colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma;

[0061] The antagonist is optionally selected from one or more of the following PD-1 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001, and pidilizumab, wherein the PD-1 antagonist is optionally nivolumab, and the cancer is optionally selected from one or more of the following: Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer;

[0062] The PD-1 antagonist is pembrolizumab, and the cancer is optionally selected from one or more of the following: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma;

[0063] The antagonist is optionally selected from one or more of the following CTLA-4 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4, ipilimumab, and tremelimumab, wherein the cancer is optionally selected from one or more of the following: melanoma, prostate cancer, lung cancer, and bladder cancer.

[0064] The antagonist is optionally selected from one or more of the following IDO antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinica acid, and epacadostat, and wherein the cancer is optionally selected from one or more of the following: metastatic breast cancer and brain cancer, optionally glioblastoma multiforme, glioma, gliosarcoma, or malignant brain tumor;

[0065] The antagonist is optionally a TDO antagonist selected from one or more of the following: antibodies or antigen-binding fragments or small molecules that specifically bind to TDO, 680C91, and LM10;

[0066] The antagonist is optionally selected from one or more of the following TIM-3 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3;

[0067] The antagonist is optionally selected from one or more of the following LAG-3 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and BMS-986016;

[0068] The antagonist is optionally selected from one or more of the following VISTA antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA;

[0069] The antagonist is optionally selected from one or more of the following BTLA, CD160 and / or HVEM antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA, CD160 and / or HVEM;

[0070] The antagonist is optionally selected from one or more of the following TIGIT antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT.

[0071] In some embodiments, the stimulating immune checkpoint molecule is selected from one or more of the following: OX40, CD40, glucocorticoid-induced TNFR family-associated gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0072] In some embodiments, the agonist is optionally selected from one or more of the following OX40 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40, OX86, Fc-OX40L and GSK3174998;

[0073] The agonist is optionally selected from one or more of the following CD40 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD40, CP-870, 893, dacetuzumab, Chi Lob7 / 4, ADC-1013 and rhCD40L, and the cancer is optionally selected from one or more of the following: melanoma, pancreatic cancer, mesothelioma and hematologic malignancies, optionally lymphoma, such as non-Hodgkin's lymphoma;

[0074] The agonist is optionally a GITR agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1, and MEDI1873;

[0075] The agonist is optionally selected from one or more of the following CD137 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137, utomilumab, and 4-1BB ligands;

[0076] The agonist is optionally selected from one or more of the following CD27 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27, varlilumab, and CDX-1127(1F5);

[0077] The agonist is optionally selected from one or more of the following CD28 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and TAB08; and / or

[0078] The agonist is optionally selected from one or more of the following HVEM agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM.

[0079] In some embodiments, the cancer vaccine is selected from one or more of the following: Oncophage; human papillomavirus (HPV) vaccine, optionally Gardasil or Cervarix; hepatitis B vaccine, optionally Engerix-B, Recombivax HB, or Twinrix; and sipuleucel-T (Provenge), or the cancer vaccine includes one or more cancer antigens selected from the following: human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor (VEGF) (e.g., V... EGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tendinin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclization Enzyme C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activating protein α (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7) The subjects included EGP40 pan-cancer antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialyl ganglioside expressed on neuroectodermal tumors), phosphatidylinositol proteoglycan-3 (GPC3), and mesothelin, optionally wherein the subject had or was at risk of having cancer, the cancer including the corresponding cancer antigen.

[0080] In some embodiments, the oncolytic virus is selected from one or more of the following: talimogenelaherparepvec (T-VEC), Coxsackievirus A21 (CAVATAK). TM Oncorine (H101), Pelareorip Seneca Valley virus (NTX-010), Seneca virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0081] In some embodiments, the cytokines are selected from one or more of the following: interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0082] In some embodiments, the cell-based immunotherapeutic agent comprises cancer antigen-specific T cells, optionally ex vivo T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR) modified T cells and T cell receptor (TCR) modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0083] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of the following: alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or II), and antimicrotubule agents.

[0084] In some embodiments, the alkylating agent is selected from one or more of the following: nitrogen mustard (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosourea (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCN)). U), fotemustine and streptozotocin), tetrazine (optionally dacarbazine, mitozolomide and temozolomide), aziridine (optionally thiotepa, mytomycin and diaziquone (AZQ)), cisplatin and its derivatives (optionally carboplatin and oxaliplatin) and atypical alkylating agents (optionally procarbazine and hexamethylmelamine));

[0085] The antimetabolite is selected from one or more of the following: antifolate agents (optionally methotrexate and pemetrexed)), fluoropyrimidines (optionally 5-fluorouracil and capecitabine)), deoxynucleoside analogs (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin) and thiopurines (optionally thioguanine and mecaptopurine));

[0086] The cytotoxic antibiotics are selected from one or more of the following: anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin;

[0087] The topoisomerase inhibitor is selected from one or more of the following: camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and azorubicin; and / or

[0088] The antimicrotubule agent is selected from one or more of the following: taxanes (optionally paclitaxel and docetaxel) and vinblastine alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0089] In some embodiments, the at least one hormone therapeutic agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of the following: progestins (progesterone), corticosteroids (optionally prednisolone, methylprednisolone, or dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factors (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galactagogue, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, androgens, estrogens, and somatostatin analogs.In some embodiments, the hormone antagonist is selected from one or more of the following: hormone synthesis inhibitors, optionally aromatase inhibitors or gonadotropin-releasing hormone (GnRH) or analogues thereof; and hormone receptor antagonists, optionally selective estrogen receptor modulators (SERMs) or anti-androgens; or antibodies against hormone receptors, optionally cixutumab, dalotuzumab, figitumab, ganitumab, isitiratumab, robatumab, alacizumab. pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine), matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab, olaratumab, or tovetumab.

[0090] In some embodiments, the kinase inhibitor is selected from one or more of the following: adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, etc. Imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib. In some embodiments, the kinase inhibitor is a PI3 kinase inhibitor selected from one or more of the following: alpelisib, buparlisib, copanlisib, CUDC-907, dactolisib, duvelisib, GNE-477, idelasib, IPI-549, LY294002, ME-401, perifosine, PI-103, pictilisib, PWT33597, RP6503, taselisib, umbralisib, voxtalisib, wortmannin, and XL147.

[0091] It also includes a method for treating an inflammatory lung disease in a subject of need, the method comprising administering to the subject a therapeutic composition comprising a histyl-tRNA synthetase (HRS) peptide. In some embodiments, the inflammatory lung disease is a neuropilin-2 (NRP2)-related disease or condition, such as RA-ILD, chronic allergic pneumonia, lung inflammation, pulmonary granulomatosis, or sarcoidosis. In some embodiments, as described herein, the subject has and / or is selected for treatment based on having: increased extracellular fluid levels of soluble NRP2 peptides, increased extracellular fluid levels of NRP2:NRP2 ligand complexes, increased extracellular fluid levels of HRS:NRP2 complexes, and / or single nucleotide polymorphisms (SNPs) in the subject's NRP2 peptides or polynucleotides encoding NRP2.

[0092] Some embodiments include a method for treating lymphedema in a subject of need, the method comprising administering to the subject a therapeutic composition comprising a histyl-tRNA synthetase (HRS) peptide. In some embodiments, the subject has lymphedema selected from the stages of: stage 1, stage 2, stage 3, stage 4, stage 5, stage 6, and stage 7, and / or the subject is selected for treatment based on having said stage of lymphedema. In some embodiments, the subject has lymphedema selected from the grades of: grade 1 (mild edema), grade 2 (moderate edema), grade 3a (severe edema), grade 3b (very severe edema), and grade 4 (extremely severe edema), and / or the subject is selected for treatment based on having said grade of lymphedema. In some embodiments, the subject has lymphedema-associated fibrosis and / or the subject is selected for treatment based on having said lymphedema-associated fibrosis. In some embodiments, the subject has lymphedema and / or the subject is selected for treatment based on having said lymphedema, which is secondary to cancer (optionally breast cancer), surgery (optionally cancer surgery, optional breast cancer surgery), radiation therapy, obesity, congestive heart failure, hypertension, peripheral vascular / venous disease, or any combination thereof.

[0093] Some embodiments include measuring a reduction in at least one symptom of lymphedema in the subject. In some embodiments, the at least one symptom of lymphedema is selected from swelling, thickened skin, hardened skin, fullness, pain, discomfort, limited range of motion, and any combination thereof.

[0094] In some embodiments, the lymphedema is a neuropilin-2 (NRP2)-related disease or symptom. In some embodiments, the subject is selected for treatment based on having: increased extracellular fluid levels of soluble NRP2 peptides, increased extracellular fluid levels of NRP2:NRP2 ligand complexes, increased extracellular fluid levels of HRS:NRP2 complexes, and / or single nucleotide polymorphisms (SNPs) in the subject's NRP2 peptides or polynucleotides encoding NRP2.

[0095] In some embodiments, the subject in need suffers from a lymphedema-associated infection, optionally erysipelas, cellulitis, lymphangitis, and / or sepsis, and / or the subject is selected for treatment based on having the lymphedema-associated infection, and optionally the method further comprises administering at least one antibacterial agent, antifungal agent, and / or antiworm agent to the subject. In some embodiments, (a) the HRS peptide and (b) the antibacterial agent, antifungal agent, and / or antiworm agent are administered together as part of the same therapeutic composition. In some embodiments, (a) the HRS peptide and (b) the antibacterial agent, antifungal agent, and / or antiworm agent are administered as separate therapeutic compositions.In some embodiments, the antimicrobial agent, antifungal agent, and / or anti-helmintic agent is selected from one or more of the following: aminoglycosides, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin; carbapenems, such as ertapenem, donipenem, imipenem / cilastatin, and meropenem; cephalosporins, such as cefadroxil, cefazolin, cefradine, cefepime, cefotaxime, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefnicillin, cefaclor, cefuroxime, cefuroxime, cefprozil, cefuroxime. Cefixime, cefdinir, ceftoran, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cefbufen, cefazolin, latamoxef, cefepime, cefuroxime, and cefepime; glycopeptides, such as teicoplanin, vancomycin, teilavancin, dabavancin, and orivancin; lincosamides, such as clindamycin and lincomycin; macrolides, such as azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, and spiramycin; penicillins, such as amoxicillin, ampicillin, azoxicillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, ethoxynaphthylpenicillin, and benzylpenicillin. Zocillin, penicillin G, penicillin V, piperacillin, penicillin G, temoxicillin, and ticarcillin; polypeptides such as bacitracin, colistin, and polymyxin B; quinolones / fluoroquinolones such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, naflufloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grefloxacin, sparfloxacin, and temafloxacin; sulfonamides such as sulfamilon, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadisin, sulfamethoxazole, sulfamethoxazole, sulfonamides, sulfasalazine, and sulfisoxazole. Trimethoprim-sulfamethoxazole (TMP-SMX) and sulfamethoxazole; tetracyclines, such as desmochlortetracycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline; antimycobacterial agents, such as clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, and streptomycin; chloramphenicol; metronidazole; mupirocin; tigecycline; tinidazole; and anti-helmintic agents, such as diethylcarbazine and albendazole.

[0096] In some embodiments, the HRS peptide comprises SEQ ID NO:156(Fc-HRS(2-60) or HRS FC1The HRS polypeptide comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences selected from Tables H1, H2, and H4. In some embodiments, the HRS polypeptide comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences selected from Tables H1, H2, and H4. In some embodiments, the HRS polypeptide is 500-506 amino acids in length and is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7 (HRS(1-506)) or SEQ ID NO:8 (HRS(2-506)), but lacks residues 507-509 of SEQ ID NO:1.

[0097] In some embodiments, the HRS peptide is fused with a heterologous peptide. In some embodiments, the heterologous peptide includes an Fc region to form an HRS-Fc fusion peptide, optionally wherein the HRS-Fc fusion peptide includes, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table H8. In some embodiments, the heterologous peptide includes a chondroitin oligomeric protein (COMP) peptide, optionally a COMP pentameric domain peptide, to form an HRS-COMP fusion peptide, optionally wherein the HRS-COMP fusion peptide includes, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table H9.

[0098] It also includes a therapeutic composition comprising:

[0099] (a) Histidine-tRNA synthetase (HRS) polypeptide; and

[0100] (b) At least one other agent selected from antibacterial agents, antifungal agents, anti-worm agents, cancer immunotherapy agents, chemotherapy agents, hormone therapy agents and kinase inhibitors.

[0101] In some embodiments, the HRS peptide comprises SEQ ID NO:156(Fc-HRS(2-60) or HRS FC1The HRS polypeptide comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences selected from Tables H1, H2, and H4. In some embodiments, the HRS polypeptide comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences selected from Tables H1, H2, and H4. In some embodiments, the HRS polypeptide is 500-506 amino acids in length and is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7 (HRS(1-506)) or SEQ ID NO:8 (HRS(2-506)), but lacks residues 507-509 of SEQ ID NO:1.

[0102] In some embodiments, the HRS peptide is fused with a heterologous peptide. In some embodiments, the heterologous peptide includes an Fc region to form an HRS-Fc fusion peptide, optionally wherein the HRS-Fc fusion peptide includes, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table H8. In some embodiments, the heterologous peptide includes a chondroitin oligomeric protein (COMP) peptide, optionally a COMP pentameric domain peptide, to form an HRS-COMP fusion peptide, optionally wherein the HRS-COMP fusion peptide includes, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table H9.

[0103] In some embodiments, the antimicrobial agent, antifungal agent, and / or anti-helmintic agent is selected from one or more of the following: aminoglycosides, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin; carbapenems, such as ertapenem, donipenem, imipenem / cilastatin, and meropenem; cephalosporins, such as cefadroxil, cefazolin, cefradine, cefepime, cefotaxime, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefnicillin, cefaclor, cefuroxime, cefuroxime, cefprozil, cefuroxime. Cefixime, cefdinir, ceftoran, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cefbufen, cefazolin, latamoxef, cefepime, cefuroxime, and cefepime; glycopeptides, such as teicoplanin, vancomycin, teilavancin, dabavancin, and orivancin; lincosamides, such as clindamycin and lincomycin; macrolides, such as azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, and spiramycin; penicillins, such as amoxicillin, ampicillin, azoxicillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, ethoxynaphthylpenicillin, and benzylpenicillin. Zocillin, penicillin G, penicillin V, piperacillin, penicillin G, temoxicillin, and ticarcillin; polypeptides such as bacitracin, colistin, and polymyxin B; quinolones / fluoroquinolones such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, naflufloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grefloxacin, sparfloxacin, and temafloxacin; sulfonamides such as sulfamilon, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadisin, sulfamethoxazole, sulfamethoxazole, sulfonamides, sulfasalazine, and sulfisoxazole. Trimethoprim-sulfamethoxazole (TMP-SMX) and sulfamethoxazole; tetracyclines, such as desmochlortetracycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline; antimycobacterial agents, such as clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, and streptomycin; chloramphenicol; metronidazole; mupirocin; tigecycline; tinidazole; and anti-helmintic agents, such as diethylcarbazine and albendazole.

[0104] In some embodiments, the cancer immunotherapy agent is selected from one or more of the following: immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies.

[0105] In some embodiments, the immune checkpoint modulator is a polypeptide, optionally an antibody or its antigen-binding fragment or ligand or small molecule. In some embodiments, the immune checkpoint modulator comprises:

[0106] (a) Antagonists of inhibitory immune checkpoint molecules; or

[0107] (b) An agonist of an immune checkpoint molecule, for example, wherein the immune checkpoint modulator specifically binds to the immune checkpoint molecule.

[0108] In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of the following: programmed death-ligand 1 (PD-L1), programmed death-1 (PD-1), programmed death-ligand 2 (PD-L2), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), T-cell activation V-domain Ig inhibitor (VISTA), B and T lymphocyte attenuating factor (BTLA), CD160, herpesvirus entry mediator (HVEM), and T-cell immune receptor having Ig and ITIM domains (TIGIT).

[0109] In some embodiments, the antagonist is optionally selected from one or more of the following PD-L1 and / or PD-L2 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and / or PD-L2, atezolizumab (MPDL3280A), avelumumab (MSB0010718C), and durvalumab (MEDI4736);

[0110] The antagonist is optionally selected from one or more of the following PD-1 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514, PDR001 and pedilizumab.

[0111] The antagonist is optionally selected from one or more of the following CTLA-4 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4, ipilimumab, and tramemumab;

[0112] The antagonist is optionally selected from one or more of the following IDO antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to IDO, indomod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norhalman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and icardol.

[0113] The antagonist is optionally a TDO antagonist selected from one or more of the following: antibodies or antigen-binding fragments or small molecules that specifically bind to TDO, 680C91, and LM10;

[0114] The antagonist is optionally selected from one or more of the following TIM-3 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3;

[0115] The antagonist is optionally selected from one or more of the following LAG-3 antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and BMS-986016;

[0116] The antagonist is optionally selected from one or more of the following VISTA antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA;

[0117] The antagonist is optionally selected from one or more of the following BTLA, CD160, and / or HVEM antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA, CD160, and / or HVEM; and / or

[0118] The antagonist is optionally selected from one or more of the following TIGIT antagonists: antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT.

[0119] In some embodiments, the stimulating immune checkpoint molecule is selected from one or more of the following: OX40, CD40, glucocorticoid-induced TNFR family-associated gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0120] In some embodiments, the agonist is optionally selected from one or more of the following OX40 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40, OX86, Fc-OX40L and GSK3174998;

[0121] The agonist is optionally selected from one or more of the following CD40 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD40, CP-870, 893, dasizumab, Chi Lob 7 / 4, ADC-1013 and rhCD40L;

[0122] The agonist is optionally a GITR agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1, and MEDI1873;

[0123] The agonist is optionally selected from one or more of the following CD137 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137, utomilumumab, and 4-1BB ligand;

[0124] The agonist is optionally selected from one or more of the following CD27 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27, valilumab, and CDX-1127(1F5);

[0125] The agonist is optionally selected from one or more of the following CD28 agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and TAB08; and / or

[0126] The agonist is optionally selected from one or more of the following HVEM agonists: antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM.

[0127] In some embodiments, the cancer vaccine is selected from one or more of the following: Oncophage; human papillomavirus (HPV) vaccine, optionally Degardex or Cervarix; hepatitis B vaccine, optionally Desmodium-B, Recombivax HB, or Twinrix; and Ciproluca-T (Provinci), or the cancer vaccine includes one or more cancer antigens selected from the following: human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor (VEGF) (e.g., VEGF-A), VE GFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tendinin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA) Guanylate cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activating protein α (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R) 2) SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialyl ganglioside expressed on neuroectodermal tumors), phosphatidylinositol proteoglycan-3 (GPC3), and mesothelin.

[0128] In some embodiments, the oncolytic virus is selected from one or more of the following: Talilavi (T-VEC), Coxsackievirus A21 (CAVATAK). TM ), Ancore (H101), Pera Leopard Seneca Valley Virus (NTX-010), Seneca Virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0129] In some embodiments, the cytokines are selected from one or more of the following: interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0130] In some embodiments, the cell-based immunotherapeutic agent comprises cancer antigen-specific T cells, optionally ex vivo T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR) modified T cells and T cell receptor (TCR) modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0131] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of the following: alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or II), and antimicrotubule agents.

[0132] In some embodiments, the alkylating agent is selected from one or more of the following: nitrogen mustard (optionally dichloromethyldiethylamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosourea (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), formustine, and streptozotocin), tetrazine (optionally dacarbazine, mitozopranilamide, and temozolomide), aziridine (optionally thiotepa, mitomycin, and diazinon (AZQ)), cisplatin and its derivatives (optionally carboplatin and oxaliplatin), and atypical alkylating agents (optionally diprocarbazine and hexamethylmelamine);

[0133] The antimetabolite is selected from one or more of the following: antifolate (optionally methotrexate and pemetrexed), fluoropyrimidine (optionally 5-fluorouracil and capecitabine), deoxynucleoside analogues (optionally descitabine, enoxabin, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelabine, cladribine, clofarapine, fludarabine and pentostatin), and thiopurine (optionally thioguanine and mercaptopurine);

[0134] The cytotoxic antibiotics are selected from one or more of the following: anthracyclines (optionally dioxin, daunorubicin, epirubicin, idarubicin, pirarubicin, arubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin;

[0135] The topoisomerase inhibitor is selected from one or more of the following: camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, neomycin, meparone, and azorubicin; and / or

[0136] The antimicrotubule agent is selected from one or more of the following: taxanes (optionally paclitaxel and docetaxel) and vinblastine alkaloids (optionally vincristine, vinblastine, vindesine, vinorelbine).

[0137] In some embodiments, the at least one hormone therapeutic agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of the following: progestins (progesterone), corticosteroids (optionally prednisolone, methylprednisolone, or dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factors (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galactagogue, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, androgens, estrogens, and somatostatin analogs. In some embodiments, the hormone antagonist is selected from one or more of the following: hormone synthesis inhibitors, optionally aromatase inhibitors or gonadotropin-releasing hormone (GnRH) or analogues thereof; and hormone receptor antagonists, optionally selective estrogen receptor modulators (SERMs) or anti-androgens; or antibodies against hormone receptors, optionally cetuximab, dotuzumab, fentuximab, ganituzumab, isotuximab, rotuximab. Peaceptizumab, bevacizumab, irukumab, ramucirumab, fusumumab, metemumab, nalcituzumab, cetuximab, mofotinide pertoxiclomab, vortoxiclomab, imatroxumab, entrinatatoxiclomab, matezumab, motuximab, nalcituzumab, nitoxiclomab, panitumumab, toltuximab, zalumumab, ixadodine aprastatumab, bemaritoxiclomab, olarumab, or tovertumab.

[0138] In some embodiments, the kinase inhibitor is selected from one or more of the following: adastotinib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobitinib, crizotinib, dasatinib, entrectinib, erdatinib, erlotinib, fortaminophen, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mulitinib, nilotinib, panitumumab, pazopanib, pilgatanib, panatinib, lanibimumab, regorafenib, ruxotinib, sorafenib, sunitinib, SU6656, tofatinib, trastuzumab, vandetanib, and vemurafenib. In some embodiments, the kinase inhibitor is a PI3 kinase inhibitor selected from one or more of the following: apeleliximab, bupaliximab, cupanliximab, CUDC-907, dattaliximab, daviliximab, GNE-477, edaraximab, IPI-549, LY294002, ME-401, perifoxine, PI-103, picricillin, PWT33597, RP6503, taseleximab, umbaliximab, votaliximab, wouma penicillin, and XL147.

[0139] In some embodiments, the therapeutic composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% relative to the HRS peptide on a protein basis and is substantially free of aggregates. In some embodiments, the therapeutic composition is substantially free of endotoxins. In some embodiments, the therapeutic composition is optionally a sterile injectable solution suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration.

[0140] It also includes a patient care kit, which comprises:

[0141] (a) the histidine-tRNA synthetase (HRS) polypeptide as described herein; and

[0142] (b) At least one other agent as described herein, wherein the at least one other agent is selected from antimicrobial agents, antifungal agents, anti-helmintic agents, cancer immunotherapy agents, chemotherapy agents, hormone therapy agents and kinase inhibitors.

[0143] In some embodiments, (a) and (b) are in separate therapeutic compositions. In some embodiments, (a) and (b) are in the same therapeutic composition.

[0144] Some embodiments include isolated fusion proteins comprising histidine-tRNA synthetase peptides fused with chondrocyte oligomeric protein (COMP) peptides (optionally COMP pentameric domain peptides) to form HRS-COMP fusion peptides, wherein optionally the HRS-COMP fusion peptide comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table H9.

[0145] Some embodiments include therapeutic compositions comprising the HRS-COMP fusion protein described herein. In some embodiments, the therapeutic composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% relative to the HRS-COMP fusion peptide on a protein basis and is substantially free of aggregates. In certain embodiments, the therapeutic composition is substantially free of endotoxins. In some embodiments, the therapeutic composition is optionally a sterile injectable solution suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration. Attached Figure Description

[0146] Figure 1A-1B The general domain structure of neurocilia is shown (1A) and the exemplary neurocilia coreceptor function is shown (1B).

[0147] Figure 2 The structures of some NRP2 isotype domains and NRP2 ligand-binding domains are shown.

[0148] Figure 3 The binding of human NRP2 to Fc-HRS(2-60) on an SPR chip is shown. 50 nM NRP2 (solid black line), NRP1 (solid gray line), and mouse plexin A1 (dotted line) were flowed as analytes onto an SPR chip coated with immobilized Fc-HRS(2-60).

[0149] Figures 4A-4B The binding of NRP2 from humans, mice, and rats to Fc-HRS(2-60) without binding to the truncated form of Fc-HRS(2-11) is shown. 50 nM of human NRP2 (solid black line), mouse NRP2 (dash line), rat NRP2 (solid gray line), or NRP1 (dotted line) were flowed as analytes onto an SPR chip coated with a fixed full-length Fc-HRS(2-60) (4A) or a truncated form of Fc-HRS(2-11) (4B) with 49 amino acids missing at the C-terminus.

[0150] Figures 5A-5DThe binding of human NRP2 to Fc-HRS (2-60) and t-RNA synthetase is shown, the t-RNA synthetase including a domain sharing homology with the WHEP domain of Fc-HRS (2-60). 20 nM NRP2 was used as an analyte and flowed onto the surface of an SPR chip coated with immobilized Fc-HRS (2-60) (5A), GARS Fc-WHEP (5B), MARS Fc-WHEP (5C), or WARS WHEP (5D).

[0151] Figures 6A-6B The binding of human NRP2 to Fc-HRS(2-60) coated with immobilized Fc-HRS(2-60) on an SPR chip is shown in the presence and absence of divalent cations. The run buffer for this experiment was 50 mM HEPES, 300 mM NaCl, and 0.005% Tween 20 (pH 7.4). For each analyte, 20 nM NRP2 was prepared in a run buffer supplemented with 5 mM CaCl2, EDTA (6A) or MgCl2, MgCl2+CaCl2 or ZnCl2 (6B).

[0152] Figures 7A-7B The binding of a pre-formed complex of Fc-HRS(2-60) and NRP2 to the 4D4 monoclonal antibody is shown, but not to the 1C8 monoclonal antibody. Monoclonal antibodies against Fc-HRS(2-60) (monoclonal antibody clones 1C8(7A) and 4D4(7B)) were immobilized on an SPR chip. The analytes consisted of: 200 nM NRP2 (dotted line), 100 nM Fc-HRS(2-60) (solid black line), a mixture of 100 nM Fc-HRS(2-60) and 200 nM NRP2 (solid gray line), or a mixture of 100 nM Fc-HRS(2-60) and 200 nM 1C8 mAb (dashed line).

[0153] Figures 8A-8DThe binding of NRP2 to Fc-HRS(2-60) captured by some monoclonal antibodies against Fc-HRS(2-60), rather than other monoclonal antibodies, is shown. Monoclonal antibodies against Fc-HRS(2-60) (monoclonal antibody clones 12H6(8A), 1C8(8B), 4D4(8C), and 13E9(8D)) were immobilized on an SPR chip. Co-injection was then performed, in which one analyte was injected, followed immediately by a second analyte. The timing of the two injections is indicated by arrows. In each of the above groups, 2000 nM Fc-HRS(2-60) was injected as the first analyte to saturate the antibody surface, followed by injection of another Fc-HRS(2-60) (solid gray line) or 200 nM NRP2 (solid black line). To rule out nonspecific binding of NRP2 to the antibody surface, co-injection of buffer followed by 200 nM NRP2 was also performed (dotted line).

[0154] Figures 9A-9B The dose-dependent binding of Fc-HRS(2-60) to cells expressing the NRP2a-GFP fusion protein is shown. Staining intensity (9A) and staining intensity CV (9B) of the Fc-HRS(2-60) / anti-Fc-PE complex on HEK293T cells overexpressing NRP2v2-GFP are quantified. Intensity values ​​are derived from cells gated based on high NRP2 expression (bright GFP). Fc-HRS(2-60) was titrated in a 2-fold step and then combined with 87.5 nM of anti-Fc-PE. As a specificity control, 175 nM Fc-HRS(2-11) / anti-Fc-PE was used.

[0155] Figure 10 The binding inhibition of Fc-HRS(2-60) to cells expressing the NRP2a-GFP fusion protein is shown in the presence of anti-HRS antibody clone 1C8. The staining intensity of the Fc-HRS(2-60) / anti-Fc-PE complex pre-incubated with either the allotype antibody control or anti-HRS (WHEP) clone 1C8 on HEK293T cells overexpressing NRP2v2-GFP was quantified. Intensity values ​​were derived from cells gated based on high NRP2 expression (bright GFP). 175 nM of Fc-HRS(2-60) / anti-Fc-PE was used. As a specific control, 175 nM of Fc-HRS(2-11) / anti-Fc-PE was used.

[0156] Figure 11A-11BThis study demonstrates that anti-HRS antibodies from the KL31 series blocked the binding of Fc-HRS(2-60) to NRP2 in a concentration-dependent manner, while other antibodies from the AB04 and AB13 series did not exhibit significant blocking properties in this assay. Quantitative staining of stably Expi293-NRP2 cells with biotinylated Fc-HRS-streptavidin-PE was performed using flow cytometry in the presence of various concentrations of anti-HRS antibodies. Data are from two experiments using different antibodies. Figure 11A shows control human IgG1 (solid circle), KL31-467 (solid triangle), KL31-356 (partially solid triangle), and mouse clone 13C8 (cross shape), and Figure 11B shows control human IgG1 (solid circle), AB04-425 (hollow triangle), AB13-288 (partially solid square), and KL31-478 (solid triangle), which show the percentage of streptavidin-PE+ / NRP2+ cells in viable singlet gate.

[0157] Figure 12A-12B The binding inhibition of Fc-HRS(2-60) to cells expressing the NRP2a-GFP fusion protein in the presence of VEGF-C is shown. The staining intensity of Fc-HRS(2-60) / anti-Fc-PE complexes pre-incubated with different doses of VEGF-C on HEK293T cells overexpressing NRP2v2-GFP was quantified. Intensity values ​​were derived from cells gated according to high NRP2 expression (bright GFP). 175 nM Fc-HRS(2-60) / anti-Fc-PE was used. As a specific control, 175 nM Fc-HRS(2-11) / anti-Fc-PE was used.

[0158] Figure 13 The quantification of circulating NRP2 levels in serum and plasma from normal healthy donors is shown. Serum and plasma from normal healthy volunteers (n=72) were separated, and the circulating levels of NRP-2 in said serum and plasma were quantified. Serum (black circles) and plasma (hollow squares) samples were tested in an ELISA specific for human NRP-2. The average serum level (16.3 pM) and average plasma level (15.6 pM) are shown for all 72 samples. The limit of quantification for NRP2 ELISA is 1.5 pM.

[0159] Figure 14A comparison of circulating HRS and NRP2 levels is shown. Serum HRS levels (black circle) show a wide circulating range among the 72 healthy volunteers tested. Matched serum NRP2 levels from the same donor are superimposed on the same axis. Donors with low HRS levels showed undetectable levels of soluble NRP2 (limit of quantification = 1.5 pM). Those donors with elevated HRS levels generally had correspondingly increased circulating NRP2 levels.

[0160] Figure 15 This illustrates HRS N-terminal interference in human serum from healthy donors. Normal serum from healthy donors was measured in two separate HARS ELISAs. Samples were measured in an ELISA for detecting full-length HARS (HARS_FL; black circle) and an ELISA specifically targeting the N-terminus (HARS_NT; hollow square). The lack of correlation between the two ELISAs with increasing full-length HARS levels is termed N-terminal interference and can indicate the presence of HRS cofactors, binding couplers, or soluble receptors.

[0161] Figure 16 The correlation between HARS N-terminal interference and soluble NRP2 levels is illustrated. Differences in detection using two HARS ELISA methods in normal healthy serum were analyzed and compared with circulating NRP2 levels. The difference in detected levels between the full-length HARS ELISA and the N-terminal HARS ELISA is termed HARS N-terminal interference units. These interference units are plotted against soluble NRP2 levels. The results show the relationship between increased N-terminal interference and soluble NRP2 in normal serum.

[0162] Figure 17 The detection of endogenous HRS and NRP2 soluble complexes is illustrated. Serum samples from normal healthy donors were analyzed in several HRS and NRP-2 complex ELISA assays. These assays utilized the capture of circulating HRS (HARS_NT or HARS_CT) and detection in the presence of NRP2 monoclonal antibodies. Similarly, the opposite assays were used to capture circulating NRP2 and observe detection in the presence of anti-HRS antibodies. In both assays, the signal was elevated in high-interference samples compared to low-interference serum samples.

[0163] Figure 18The presence of complexed HRS and NRP2 in highly interfering samples demonstrates how they can hinder the detection of site-specific HRS antibodies. Serum samples from low and high HRS N-terminal interference samples were measured in an HRS and NRP2 complex ELISA. Serum samples were captured with an NRP2 monoclonal antibody and detected with either of two distinct HRS N-terminal monoclonal antibodies. When detected with HRS_NT (black bar), highly interfering samples showed complex formation, but this signal was completely blocked by the N-terminal anti-HRS antibody (HRS blocking antibody; gray bar).

[0164] Figures 19A-19C The activity of Fc-HRS(2-60) on skin markers is shown in a mouse model of scleroderma-like chronic graft-versus-host disease. Figure 19A shows the effect of treatment with the mediator, Fc-HRS(2-60), or nintedanib initiated on day 7 (7D) or day 21 (21D) after allogeneic transplantation on dermal thickness. Figure 19B shows the effect of treatment with the mediator, Fc-HRS(2-60), or nintedanib initiated on day 7 (7D) or day 21 (21D) after allogeneic transplantation on the number of myofibroblasts counted in skin sections. Figure 19C shows the effect of treatment with the mediator, Fc-HRS(2-60), or nintedanib initiated on day 7 (7D) or day 21 (21D) after allogeneic transplantation on hydroxyproline content (an indicator or collagen content) in the skin. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Kruskal-Wallis nonparametric ANOVA was performed on the euthanized mediator animals (group 3 from the left) 8 weeks after allogeneic transplantation, followed by Dunn's multiple comparison test.

[0165] Figure 20A-20DThe activity of Fc-HRS(2-60) for lung markers is shown in a mouse model of scleroderma-like chronic graft-versus-host disease. Figure 20A shows the effect of treatment with the mediator, Fc-HRS(2-60), or nintedanib initiated on day 7 (7D) or day 21 (21D) after allogeneic transplantation on the Ashcroft score. Figure 20B shows the effect of treatment with the mediator, Fc-HRS(2-60), or nintedanib initiated on day 7 (7D) or day 21 (21D) after allogeneic transplantation on the percentage of tissue stained with Sirius red (a staining agent specifically for collagen fibers) in each section. Figure 20C shows the effect of treatment with the mediator, Fc-HRS(2-60), or nintedanib initiated on day 7 (7D) or day 21 (21D) after allogeneic transplantation on the number of myofibroblasts counted in lung sections. Figure 20D shows the effect of treatment with mediator, Fc-HRS (2-60), or nintedanib initiated on day 7 (7D) or day 21 (21D) after allogeneic transplantation on hydroxyproline content (indicator or collagen content) in the skin. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Kruskal-Wallis nonparametric ANOVA was performed on mediator animals (group 3 from the left) that were euthanized 8 weeks after allogeneic transplantation, followed by Dunn's multiple comparison test.

[0166] Figures 21A-21C The effect of protein-containing HRS(2-60) on LPS-induced leukocyte infiltration into mouse lungs is shown. Mice were treated intravenously with designated doses of Fc-HRS(2-60) or HRS(2-60)-COMP. The following day, saline or LPS (10 μg / mouse) was administered orally via airway instillation, and infiltrating cells were collected by bronchoalveolar lavage 24 hours later and analyzed by flow cytometry. Groups receiving LPS are delineated by level bars. Data from individual animals (circles) are shown along with mean and SEM. An asterisk indicates a significant difference (p < 0.05) between the LPS / carrier groups as determined by ANOVA and subsequent Dunnett's post-hoc test.

[0167] Figures 22A-22B This study demonstrated that incubating bone marrow-derived macrophages with 100 nM or 200 nM Fc-HRS(2-60) (Imod) instead of the Fc control compound N15 for 5 days during monocyte differentiation resulted in profound inhibition of macrophage maturation, as indicated by the pH-sensitive fluorescent dye pH rhodo. TMThe report reveals a significant reduction in spectral shift.

[0168] Figures 23A-23B This study demonstrated that incubating bone marrow-derived macrophages with 100 nM HRS(2-60)-COMP (rather than the control compound COMP) for 5 days during monocyte differentiation resulted in a profound inhibition of cytotoxicity, as indicated by the pH-sensitive fluorescent dye pHrhodo. TM The report reveals a significant reduction in spectral shift.

[0169] Figures 24A-24B The model induction was demonstrated by histological confirmation of increased inflammation (H&E) and fibrosis (Masson's Trichrome) in mice receiving Propionibacterium acnes (Group 3 and 4) compared to mice that did not receive Propionibacterium acnes (Group 2).

[0170] Figures 25A-25B The results of lung inflammation (25A) and fibrosis (25B) measurements at the end of the study are shown.

[0171] Figures 26A-26H As indicated, in response to treatment with 3 mg / kg Fc-HRS(2-60), several profibrotic cytokines in the lungs were reduced.

[0172] Figure 27A shows that mice exposed to *S. rectivirgula* in the control groups (groups 3 and 4) had refractory and consistent multifocal chronic pneumonia compared to mice exposed to PBS in group 2. Figure 27B shows the histopathological scores determined by a veterinary pathologist. Figure 27C shows the reduction in individual BALT area in the Fc-HRS(2-60) 3 mg / kg group after in-depth analysis of H&E-stained lung tissue sections using the HALO platform.

[0173] Figure 28A-28G The results showed that in the presence of Fc-HRS(2-60) at 0.4 mg / kg and 3 mg / kg, several pro-inflammatory cytokines and chemokines were significantly reduced.

[0174] Figures 29A-29E The results showed that matrix metalloproteinases (MMPs) were significantly reduced in the presence of 0.4 mg / kg and 3 mg / kg Fc-HRS(2-60).

[0175] Figures 30A-30H The study showed that inflammatory arthritis was successfully induced in SKG mice after administration of 5 mg of yeast polysaccharide, and that Fc-HRS(2-60) reduced the number of specific immune cells in the lungs of SKG mice, most notably B cells and T cells.

[0176] Figures 31A-31D The overall clinical arthritis scores are shown on days 35, 42, 49, and 56. Detailed Implementation

[0177] Unless otherwise indicated, the practice of this invention will take place using conventional methods of molecular biology and recombinant DNA technology within the scope of the art, many of which are described below for illustrative purposes. Such techniques are well explained in the literature. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual* (3rd edition, 2000); *DNA Cloning: A Practical Approach*, Volumes I and II (edited by D. Glover); *Oligonucleotide Synthesis* (edited by N. Gait, 1984); *Oligonucleotide Synthesis: Methods and Applications* (edited by P. Herdewijn, 2004); *Nucleic Acid Hybridization* (edited by B. Hames and S. Higgins, 1985); *Nucleic Acid Hybridization: Modern Applications* (edited by Buzdin and Lukyanov, 2009); *Transcription and Translation* Translation) (B. Hames and S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, RI (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th ed., Hoboken NJ, John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd ed., 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd ed., 2005)."Poly(ethyleneglycol), Chemistry and Biological Applications", American Chemical Society (ACS), Washington, 1997; Veronese, F. and J.M. Harris, eds., "Peptide and protein PEGylation", Advanced Drug Delivery Reviews, 54(4)453-609 (2002); Zalipsky, S. et al., "Use of functionalized Poly(EthyleneGlycols) for modification of polypeptides" in "Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications".

[0178] definition

[0179] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While methods, materials, compositions, reagents, and cells similar to or equivalent to those described herein may be used in practice or testing of the subject matter of this disclosure, preferred methods and materials are described. All publications and references cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety as if each individual publication or reference were specifically and individually indicated as fully elucidated and incorporated herein by reference. Any patent application claiming priority to this application is also incorporated herein by reference in its entirety as described above regarding the publications and references.

[0180] For the purposes of this disclosure, the following terms are defined as follows.

[0181] This article uses the article “a / an” to refer to one or more (i.e., at least one) grammatical objects of the article. By way of example, “element” means one or more elements.

[0182] "Approximately" means that a quantity, level, value, number, frequency, percentage, size, weight, or length changes by as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, size, weight, or length.

[0183] An "antagonist" or "inhibitor" is a biological or chemical agent that interferes with or otherwise reduces the physiological effects of another drug or molecule. In some cases, antagonists bind specifically to another drug or molecule. They include complete antagonists and partial antagonists.

[0184] An "agonist" is a biological or chemical agent that increases or enhances the physiological effects of another drug or molecule. In some cases, agonists specifically bind to another drug or molecule. They include full agonists and partial agonists.

[0185] The term "unresponsiveness" refers to the functional inactivation of the response of T cells or B cells to restimulation of antigens.

[0186] As used herein, the term "amino acid" is intended to mean naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimics. For example, naturally occurring amino acids include 20(L)-amino acids used in protein biosynthesis, as well as other amino acids such as 4-hydroxyproline, hydroxylysine, desmodium, isodesmodium, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, leucine, valine, p-fluorophenylalanine, ethionine, etc., known to those skilled in the art. Amino acid analogs comprise modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and portions on the amino acid, or derivatization of the amino acid. Amino acid mimics comprise organic structures that exhibit functionally similar properties, such as charge and charge spacing characteristics, to a reference amino acid. For example, an organic structure mimicking arginine (Arg or R) would have a positively charged portion located in a similar molecular space and having the same mobility of the e-amino group of the side chain as the naturally occurring Arg amino acid. Mimics also comprise constrained structures to maintain optimal spacing and charge interactions between amino acids or amino acid functional groups. Those skilled in the art know or can identify which structurally and functionally equivalent amino acid analogs and amino acid mimics exist.

[0187] As used herein, a subject at "risk" for developing a disease or experiencing an adverse reaction may or may not have a detectable disease or disease symptoms, and may or may not have exhibited a detectable disease or disease symptoms prior to the treatments described herein. "At risk" means that the subject has one or more risk factors, which are measurable parameters associated with the development of a disease as described herein and known in the art. A subject with one or more of these risk factors is more likely to develop a disease or experience an adverse reaction than a subject without one or more of these risk factors.

[0188] A “coding sequence” refers to any nucleic acid sequence that contributes to the encoding of the polypeptide product of a gene. Conversely, the term “non-coding sequence” refers to any nucleic acid sequence that does not directly contribute to the encoding of the polypeptide product of a gene.

[0189] The term "association" refers to the direct association between two molecules resulting from interactions such as covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding (including interactions such as salt bridges and water bridges).

[0190] The term "clonal deletion" refers to the absence (e.g., loss or death) of self-reactive T cells. Clonal deletion can be concentrated in the thymus, or in the periphery, or both.

[0191] The term "chemoresistance" refers to the change in the treatment sensitivity of a cancer cell population over time after exposure to cancer therapies, including resistance to at least one of cancer immunotherapies, chemotherapy agents, hormone therapy agents, and / or kinase inhibitors. Ultimately, chemoresistance leads to cancer recurrence and / or metastasis and poses a challenge to improving clinical outcomes for cancer patients. It remains a major obstacle to long-term successful cancer therapies. For example, approximately 30% of women diagnosed with early-stage breast cancer develop chemoresistance and eventually progress to metastatic breast cancer. The molecular mechanisms of chemoresistance include transport pump induction, oncogenes, tumor suppressor genes, mitochondrial alterations, DNA repair, autophagy, epithelial-mesenchymal transition (EMT), cancer stem cell characteristics, and exogenous body generation. These mechanisms can operate through different mechanisms but ultimately work together to prevent cell death in response to chemotherapy agents. For example, the protein encoded by an oncogene (EGFR-Akt-NF-κB) can regulate the expression of apoptosis-related genes and thereby contribute to EMT, stem cell characteristics, and autophagy. Autophagic cells are characterized by resistance to apoptosis during chemoresistance. Therefore, agents that reduce chemoresistant cancers, including those that regulate autophagy, endosome maturation, phagocytosis, and / or burial, can be used to treat or alleviate chemoresistant cancers.

[0192] Throughout this disclosure, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of the stated steps or elements or groups of steps or elements, but do not exclude any other steps or elements or groups of steps or elements.

[0193] "Comprising..." means including and limited to anything following the phrase "comprising...". Therefore, the phrase "comprising..." indicates that the listed element is necessary or mandatory, and other elements may be absent. "Substantially comprising..." means including any element listed following the phrase, and is limited to other elements that do not interfere with or facilitate the activities or actions specified for the listed elements in this disclosure. Therefore, the phrase "substantially comprising..." indicates that the listed element is necessary or mandatory, but other elements are optional and may be present or absent depending on whether they substantially affect the activities or actions of the listed elements.

[0194] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or blood vessels containing at most trace amounts (e.g., amounts that have no clinically adverse physiological effects on the subject) of endotoxins, and preferably undetectable amounts. Endotoxins are toxins associated with certain microorganisms, such as bacteria (usually Gram-negative bacteria), but can also be found in Gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in the outer membranes of various Gram-negative bacteria, where LPS and LOS represent central pathogenic features in the ability of these bacteria to cause disease. Small amounts of endotoxins in the human body may cause fever, hypotension, inflammation, coagulation activation, and other adverse physiological effects.

[0195] Therefore, in pharmaceutical manufacturing, it is often desirable to remove most or all trace amounts of endotoxins from pharmaceutical products and / or containers, as even small amounts can have adverse effects on humans. Pyrogen-free ovens can be used for this purpose, as temperatures exceeding 300°C are typically required to decompose most endotoxins. For example, based on primary packaging materials such as syringes or vials, a glass temperature of 250°C and a holding time of 30 minutes are often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for removing endotoxins are envisioned, including, for example, chromatography and filtration methods as described herein and known in the art.

[0196] Endotoxins can be detected using conventional techniques known in the art. For example, the assay using limulus amoebocyte lysate from horseshoe crab blood is a very sensitive assay for detecting the presence of endotoxins. In this test, very low LPS levels can cause detectable agglutination of the lysate due to the amplification of this reaction by a strong enzymatic cascade. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, endotoxin levels can be below about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1–10 EU.

[0197] As used herein, the terms “contacting cells,” “introduction,” or “delivery” include delivering or administering the agents described herein (e.g., peptide agents, polynucleotide agents) to cells or to a subject by methods conventional in the art (e.g., transfection (e.g., liposomes, calcium phosphate, polyethyleneimine), electroporation (e.g., nuclear transfection), microinjection).

[0198] The terms "cell-penetrating peptide" (CPP) or "peptide moiety that enhances cellular uptake" are used interchangeably and refer to a cationic cell-penetrating peptide, also known as a "transport peptide," "carrier peptide," or "peptide transduction domain." In some embodiments, the peptide has the ability to induce cell (e.g., muscle cells) penetration into about or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the intracellular domain in a given cell culture population, and allows macromolecules to translocate within multiple tissues (e.g., muscle tissue) in vivo following systemic or other forms of administration. In some embodiments, the CPP has the formula - [(C(O)CHR'NH) m R”, ​​where R’ is a side chain of a naturally occurring amino acid or a homologue of one or two carbons thereof, R” is selected from hydrogen or acyl, and m is an integer of up to 50. Other CPPs are well known in the art and are disclosed, for example, in U.S. Application No. 2010 / 0016215, which is incorporated herein by reference in its entirety. In some embodiments, m is an integer selected from 1 to 50, where when m is 1, the portion is a single amino acid or a derivative thereof. Any polynucleotide agent described herein (e.g., antisense, RNAi agent) may be conjugated to a CPP, for example, to increase uptake into target cells (e.g., muscle cells).

[0199] The term "half-maximum effective concentration" or "EC50" refers to the concentration of a drug (e.g., an HRS peptide or other drug) that induces a response between baseline and maximum after a specified exposure time, as described herein; thus, the EC50 of a graded dose-response curve represents the concentration at which the compound is observed to have 50% of its maximum effect. EC50 also represents the plasma concentration required to achieve 50% of the maximum effect in vivo. Similarly, "EC90" refers to the concentration at which a drug or composition is observed to have 90% of its maximum effect. "EC90" can be calculated from "EC50" and the Hill slope, or it can be determined directly from data using conventional knowledge in the art. In some embodiments, the EC50 of the agent is less than about 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, or 500 nM. In some embodiments, the EC50 value of the biotherapy composition will be about 1 nM or less.

[0200] "Homology" refers to the percentage of identical or conserved amino acids. Homology can be determined using sequence comparison procedures such as GAP (Deveraux et al., 1984, Nucleic Acids Research, 12, 387-395). In this way, sequences similar in length or substantially different from those cited herein can be compared by inserting gaps in the alignment; such gaps are determined, for example, by the comparison algorithm used by GAP.

[0201] The term “innate immune response” refers to the response of immune cells (including myeloid-derived cells such as macrophages, neutrophils, eosinophils, granulocytes, and natural killer (NK) cells), as well as the related mechanisms that regulate cytokine expression and release (e.g., interferon and interferon signaling), induce cell death, and inhibit protein synthesis, which protect the host from pathogen infection.

[0202] "Isolation" means a substance that is substantially or substantially free of the components that normally accompany it in its natural state. For example, "isolated polynucleotide," "isolated oligonucleotide," or "isolated oligonucleotide" as used herein can refer to a polynucleotide that has been purified or removed from a sequence flanking it in its natural state, such as a fragment removed from a sequence adjacent to a DNA segment in the genome. When it is in the context of cells, the term "isolation" refers to the purification of cells (e.g., fibroblasts, lymphocytes) from a source subject (e.g., a subject with a polynucleotide duplication disease). In the context of mRNA or protein, "isolation" refers to the recovery of mRNA or protein from a source (e.g., cells).

[0203] The term "wandering cell" refers to a cell that can move from one place to another in response to a stimulus. Exemplary wandering cells include immune cells such as monocytes; natural killer (NK) cells; dendritic cells (immature or mature); dendritic cell subsets including myeloid cells, plasmacytoid cells (also called lymphoid cells), and Langerhans cells; and macrophages such as tissue cells, such as Kupffer's cells. Tissue-resident macrophages, microglia in the CNS, alveolar macrophages, and peritoneal macrophages; macrophage subtypes, such as M0, M1, Mox, M2a, M2b, and M2c macrophages; neutrophils; eosinophils; mast cells; basophils; B cells, including plasma B cells, memory B cells, B-1 cells, and B-2 cells; CD45RO (natural T) cells; CD45RA (memory T) cells; CD4 helper T cells, including Th1, Th2, and Tr1 / Th3 cells; CD8 cytotoxic T cells; regulatory T cells; γδ T cells; and thymocytes. Other examples of wandering cells include fibroblasts, fibroblasts, tumor cells, and stem cells. Therefore, the term "cell migration" refers to the movement of wandering cells, and the term "regulation of cell migration" refers to the regulation of the movement of any such wandering cells.

[0204] The term "modulation" includes optionally "increasing" or "decreasing" one or more quantifiable parameters in a defined and / or statistically significant amount. "Increase" or "increasing," "enhance" or "enhancing," or "stimulate" generally refers to the ability of one or more agents or compositions to produce or evoke a physiological response (i.e., a downstream effect) in cells or a subject that is greater than the response induced by the absence of the agent / compound or control compound. The associated physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and may include an increase in the desired tissue or skeletal muscle mass of the subject. The “increased” or “enhanced” amount is generally a “statistically significant” amount and may include 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500 times, 1000 times) the amount produced by the absence of the agent / compound (in the absence of the agent) or the control compound, including all integers and decimals greater than 1 (e.g., 1.5, 1.6, 1.7, 1.8). The terms “reduced” or “inhibited” can generally refer to the ability of one or more agents or compositions to “reduce” a relevant physiological or cellular response (such as the expression of a target gene or symptoms of the disease or symptom described herein), as measured according to conventional techniques in the diagnostic field. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction or improvement in the symptoms or pathology of lung inflammation or ILD as described herein. The “reduction” in response compared to the response produced by no agent or composition or a control agent or composition can be “statistically significant” and can include reductions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all integers in between.

[0205] In some embodiments, the “purity” of any given agent in the composition can be specifically defined. For example, some compositions may include agents with a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, including all decimal places therein, and are, for example and by no means limited, as measured by high-performance liquid chromatography (HPLC), a well-known form of column chromatography commonly used in biochemistry and analytical chemistry for the separation, identification, and quantification of compounds.

[0206] "Lipid nanoparticles" or "solid lipid nanoparticles" refer to one or more spherical nanoparticles with an average diameter between about 10 nanometers and about 1000 nanometers and comprising a solid lipid core matrix capable of solubilizing lipophilic molecules. The lipid core is stabilized by a surfactant (e.g., an emulsifier) ​​and may comprise one or more of triglycerides (e.g., tristearate), diglycerides (e.g., behenate), monoglycerides (e.g., monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), including combinations thereof. For example, lipid nanoparticles are described in the following literature: Petrilli et al., *Curr PharmBiotechnol.* 15:847-55, 2014; and U.S. Patents 6,217,912; 6,881,421; 7,402,573; 7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750; 8,871,509; 9,017,726; 9,173,853; 9,220,779; 9,227,917; and 9,278,130, which are incorporated herein by reference in their entirety.

[0207] The term "neurociliin 2-related disease" or "NRP2-related disease" refers to diseases and conditions in which NRP2 activity, expression, and / or spatial distribution play a role in the pathophysiology of the disease or condition. In some cases, NRP2-related diseases are modulated by the HRS peptides of this disclosure, for example, by altering the interaction of NRP2 with at least one NRP2 ligand to affect NRP2 activity, signal transduction, expression, and / or spatial distribution. Exemplary NRP2-related diseases and conditions include, but are not limited to, cancer and cancer-related diseases or pathologies, including cancer cell growth, cancer initiation, cancer migration, cancer cell adhesion, cancer cell invasion, cancer cell chemoresistance, and cancer cell metastasis. Diseases related to inflammation and autoimmunity are also included, including inflammatory lung diseases such as chronic allergic pneumonia, lung inflammation, and related inflammatory diseases. Diseases associated with inappropriate immune cell activation or migration are also included, such as graft-versus-host disease (GVHD) and rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Other examples include diseases related to lymphatic development, lymphangiogenesis, and lymphatic damage, including edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability. It also includes diseases related to infection (including latent infection) and diseases related to allergic conditions / diseases and allergic responses, including chronic obstructive pulmonary disease (COPD), neutrophilic asthma, systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA), systemic lupus erythematosus, rheumatoid arthritis, one or more inflammasome-related diseases, and one or more skin-related neutrophil-mediated diseases, such as pyoderma gangrene. Other examples include diseases related to granulomatous inflammatory diseases, including sarcoidosis and other pulmonary granulomatous diseases, as well as non-pulmonary granulomatous diseases. It also includes fibrotic diseases such as endometriosis, fibrosis, endothelial-to-mesenchymal transition (EMT), and wound healing. It also includes diseases associated with inappropriate smooth muscle contractility and vascular smooth muscle cell migration and / or adhesion, as well as diseases associated with inappropriate autophagy, phagocytosis, and burial. Additional examples include neuronal diseases, including those related to peripheral nervous system remodeling and pain perception. It also includes diseases related to bone development and / or bone remodeling, as well as diseases associated with inappropriate migratory cell movement.

[0208] As used herein, the terms “nucleobase” (Nu), “base pairing portion”, or “base” are used interchangeably to refer to purine or pyrimidine bases (uracil, thymine, adenine, cytosine, and guanine) found in natural DNA or RNA, as well as naturally occurring purine and pyrimidine analogs that impart improved properties, such as binding affinity to oligonucleotides. Exemplary analogs include inosine (the base component of nucleoside inosine); 2,6-diaminopurine; 5-methylcytosine; C5-propynyl-modified pyrimidines; 9-(aminoethoxy)phenoxazine (G-clamp), etc.

[0209] Other examples of base-pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine, and hypoxanthine, whose corresponding amino groups are protected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs (such as pseudoisocytosine and pseudouracil), and other modified nucleobases, such as 8-substituted purines, xanthines, or hypoxanthines (the latter two being natural degradation products). Modified nucleobases have also been envisioned in Chiu and Rana, RNA, 2003, 9, 1034-1048; Limbach et al., Nucleic Acid Research, 1994, 22, 2183-2196; and Revankar and Rao, Comprehensive Natural Products Chemistry, Vol. 7, 313.

[0210] Further examples of base-pairing moieties include, but are not limited to, nucleobases with one or more enlarged benzene rings added therein. Nucleobase substitutions described in the following sources are considered useful for the synthesis of the oligonucleotides described herein: Glen Research Catalogue (www.glenresearch.com); Krueger AT et al., *Acc. Chem. Res.*, 2007, 40, 141-150; Kool, ET., *Acc. Chem. Res.*, 2002, 35, 936-943; Benner SA et al., *Nat. Rev. Genet.*, 2005, 6, 553-543; Romesberg, FE et al., *Curr. Opin. Chem. Biol.*, 2003, 7, 723-733; Hirao, I., *Curr. Opin. Chem. Biol.*, 2006, 10, 622-627. Examples of enlarged nucleobases are shown below:

[0211]

[0212] Nucleobases covalently linked to ribose, sugar analogs, or morpholino groups include nucleosides. A "nucleotide" consists of a nucleoside and a phosphate group. The phosphate group covalently links adjacent nucleotides together to form oligonucleotides.

[0213] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers containing amino acid residues and their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers, where one or more amino acid residues are synthetic, non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers.

[0214] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. These terms generally refer to a polymeric form of nucleotides with a length of at least 10 bases, either ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotide. The terms include single-stranded and double-stranded forms of DNA. The terms "isolated DNA," "isolated polynucleotide," and "isolated nucleic acid" refer to molecules that have been isolated from the total genomic DNA of a specific species. Therefore, an isolated DNA fragment encoding a polypeptide refers to a DNA fragment containing one or more coding sequences but substantially isolated from or purified from the total genomic DNA of the species from which the DNA fragment was obtained. It also includes non-coding polynucleotides that do not encode polypeptides (e.g., primers, probes, oligonucleotides). It also includes recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, phage particles, bacteriophages, viruses, etc.

[0215] The polynucleotides described herein may, but need not, contain additional coding or non-coding sequences, and the polynucleotides may, but need not, be linked to other molecules and / or supporting materials. Therefore, polynucleotides, or expressible polynucleotides, regardless of the length of the coding sequence itself, can be combined with other sequences, such as expression control sequences.

[0216] "Expression control sequences" contain regulatory sequences of nucleic acids or corresponding amino acids, such as promoters, precursors, enhancers, introns, recognition motifs of RNA or DNA-binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals, etc., which can affect the transcription or translation or subcellular or cellular localization of coding sequences in host cells. An exemplary expression control sequence is described in the following literature: Goeddel; *Gene Expression Technology: Methods in Enzymology*, 185, Academic Press, San Diego, California, 1990.

[0217] A promoter is a DNA regulatory region that binds to RNA polymerase in a cell and initiates transcription of a downstream (3' direction) coding sequence. As used herein, the promoter sequence is defined at its 3' end by a transcription start site and extends upstream (5' direction) to contain the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. The transcription start site can be found within the promoter sequence and the protein-binding domain (common sequence) responsible for RNA polymerase binding (conveniently defined by mapping with nuclease S1). Eukaryotic promoters typically contain, but do not always contain, a TATA box and a CAT box. Prokaryotic promoters contain a Shine-Dalgarno sequence in addition to the -10 and -35 common sequences.

[0218] A wide variety of promoters (including constitutive, inducible, and repressive promoters) from diverse sources are well known in the art. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available or can be synthesized based on sequences publicly available online or from depositories such as ATCC and other commercial or personal sources. Promoters can be unidirectional (i.e., initiating transcription in one direction) or bidirectional (i.e., initiating transcription in the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include the Tet system (US Patents 5,464,758 and 5,814,618), the ecdysone inducible system (No et al., Proceedings of the National Academy of Sciences (NAACAS) (1996) 93(8):3346-3351; and the T-REx™ system (Invitrogen Carlsbad, CA)). (Stratagene, San Diego, CA) and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nucleic Acid Research (1999) 27(22):4324-4327; Nucleic Acid Research (2000) 28(23):e99; U.S. Patent No. 7,112,715; and Kramer and Fussenegger, Methods in Molecular Biology (2005) 308:123-144) or any promoter known in the art suitable for expression in the desired cells.

[0219] "Expressible polynucleotides" include cDNA, RNA, mRNA or other polynucleotides that include at least one coding sequence and optionally at least one expression control sequence (e.g., transcription and / or translation regulatory elements) and can express an encoded polypeptide (e.g., HRS polypeptide) when introduced into a cell (e.g., a subject's cell).

[0220] In some embodiments, the expressible polynucleotide is modified RNA or modified mRNA polynucleotide, such as a non-naturally occurring RNA analog. In some embodiments, the modified RNA or mRNA polypeptide comprises one or more modified or non-natural bases, such as nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). In some embodiments, the modified mRNA comprises one or more modified or non-natural internucleotide linkages. Expressible RNA polynucleotides for delivering encoded therapeutic polypeptides are described, for example, in the following documents: Kormann et al., Nature Biotechnology 29:154-7, 2011; and U.S. Applications No. 2015 / 0111248; No. 2014 / 0243399; No. 2014 / 0147454; and No. 2013 / 0245104, all of which are incorporated herein by reference in their entirety.

[0221] In some embodiments, various viral vectors that can be used to deliver expressible polynucleotides include adenovirus vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retroviral vectors. In some cases, the retroviral vector is a derivative of a mouse or avian retrovirus, or a lentiviral vector. Examples of retroviral vectors in which a single exogenous gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Many other retroviral vectors can bind multiple genes. All of these vectors can transfer or bind genes for selectable markers, enabling the identification and generation of transduced cells. Target specificity can be achieved by, for example, inserting the polypeptide sequence of interest along with another gene encoding a ligand for a receptor on a specific target cell into the viral vector. Retroviral vectors can be made target-specific by inserting, for example, polynucleotides that encode proteins. Illustrative targeting can be achieved by targeting the retroviral vector with antibodies. Those skilled in the art will understand, or can readily determine, specific polynucleotide sequences that can be inserted into the retroviral genome to allow for target-specific delivery of the retroviral vector without excessive experimentation.

[0222] In some cases, the expressible polynucleotides described herein are engineered for intracellular localization, potentially located in specific compartments such as the cell nucleus, or engineered for secretion from the cell or translocation to the cell membrane. In an exemplary embodiment, the expressible polynucleotide is engineered for nuclear localization.

[0223] It also includes bioactive “variants” and “fragments” of the polypeptide described herein, as well as the polynucleotides encoding them. A “variant” contains one or more substitutions, additions, deletions, and / or insertions relative to a reference polypeptide or polynucleotide (see, for example, the tables and sequence listings). Variant polypeptides or polynucleotides comprise amino acid or polynucleotide sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, similarity, or homology to the reference sequence described herein, and substantially retain the activity of the reference sequence. It also includes sequences consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides, or sequences differing from a reference sequence by the addition, deletion, insertion or substitution of said amino acids or nucleotides, and substantially retaining the activity of said reference sequence. In some embodiments, additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0224] As used herein, the term “sequence identity” or, for example, including “a sequence that is 50% identical to…”, refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. Therefore, the “sequence identity percentage” can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence identity percentage. The optimal alignment of sequences for the comparison window can be achieved through a computerized implementation of the algorithm (GAP, BESTFIT, FASTA, and TFASTA in version 7.0 of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA), or by examining and determining the best alignment produced by any of the chosen methods (i.e., producing the highest percentage of homology within the comparison window). Reference can also be made to the BLAST family of programs, such as those disclosed, for example, in Altschul et al., Nucleic Acid Research 25:3389, 1997.

[0225] "Statistically significant" means that the result is unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Commonly used significance measures include the p-value, which is the frequency or probability of the observed event occurring if the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is limited to a p-value of 0.05 or less.

[0226] The term "solubility" refers to the property of a pharmaceutical preparation provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, which is the mass of solute per unit volume of solvent (grams of solute per kilogram of solvent, g / dL (100 mL), mg / ml, etc.), molar concentration, mass molar concentration, mole fraction, or other similar concentration descriptions. The maximum equilibrium amount of solute that a unit volume of solvent can dissolve is the solubility of the solute in the solvent under specified conditions including temperature, pressure, pH, and solvent properties. In some embodiments, solubility is measured at physiological pH or other pH values, such as pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In some embodiments, solubility is measured in water or physiological buffers such as PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively low pH values ​​(e.g., pH 6.0) and relatively high salt concentrations (e.g., 500 mM NaCl and 10 mM NaP). In some embodiments, solubility is measured in biological fluids (solvents) such as blood or serum. In some embodiments, the temperature may be approximately room temperature (e.g., about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C) or approximately body temperature (37°C). In some embodiments, the agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37°C.

[0227] "Subject" or "subject in need" includes mammalian subjects, such as human subjects.

[0228] "Basically" or "substantially" means almost entirely or completely, for example, 95% or more of a given amount.

[0229] "Therapeutic response" refers to symptom improvement (whether or not it is sustained) based on the application of a therapeutic response.

[0230] As used herein, the term "target" refers to RNA, and more specifically, to the RNA region of the target gene described herein. Targets may include coding and noncoding sequences, 5' upstream sequences, 3' downstream sequences, and other RNA sequences described herein.

[0231] The term "target sequence" refers to a portion of the target RNA that an antisense or RNAi agent targets. For example, it is the sequence that an antisense oligonucleotide will hybridize with through Watson-Crick base pairing of complementary sequences, or the sequence of the sense strand corresponding to an RNAi agent.

[0232] As used herein, the term "quantifying" or other related terms refers to determining the quantity, mass, or concentration per unit volume of nucleic acids, polynucleotides, oligonucleotides, peptides, polypeptides, or proteins.

[0233] As used herein, the terms “therapeutic effective amount,” “therapeutic dose,” “prophylactic effective amount,” or “diagnostic effective amount” refer to the amount of a drug agent required to elicit a desired biological response after administration. Similarly, the terms “antisense therapy” or “RNAi therapy” encompass therapies that maintain the mean steady-state concentration of an antisense or RNAi drug agent in a patient’s plasma or other tissue compartment (e.g., muscle tissue) above the minimum effective therapeutic level.

[0234] As used herein, “treatment” of a subject (e.g., a mammal, such as a human) or cell is any type of intervention intended to alter the natural course of a subject or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be administered preventively or after the onset of a pathological event or after exposure to a pathogen. It also includes “preventive” treatment, which may be designed to reduce the rate of progression of the treated disease or symptom, delay the onset of said disease or symptom, or reduce the severity of its onset. “Treatment” or “prevention” does not necessarily imply the complete eradication, cure, or prevention of a disease or symptom or its associated symptoms.

[0235] The term "wild type" refers to the gene or gene product (e.g., polypeptide) that is most commonly observed in a population and is therefore arbitrarily engineered to be in the "normal" or "wild type" form of the gene.

[0236] Histyl-tRNA synthetase (HRS) peptides and polynucleotides

[0237] Some embodiments comprise a histyl-tRNA synthetase polypeptide (“HRS” or “HisRS” polypeptide), including conjugates (e.g., fusion proteins, Fc conjugates, Fc fusion proteins, COMP conjugates, COMP fusion proteins), variants thereof, and fragments thereof, as well as an expressible polynucleotide encoding the HRS polypeptide. Histyl-tRNA synthetases belong to the class II tRNA synthetase family, which has three highly conserved sequence motifs. Class I and II tRNA synthetases are widely considered to be responsible for the specific attachment of amino acids to their associated tRNAs in a two-step reaction: the amino acid (AA) is first activated by ATP to form AA-AMP and then translocated to the acceptor terminal of the tRNA. Full-length histyl-tRNA synthetases typically exist as cytoplasmic homodimers or, alternatively, spliced ​​mitochondrial forms.

[0238] Certain biological fragments or alternatively spliced ​​isoforms of eukaryotic histyl-tRNA synthetases, or in some cases the complete full-length synthetase, regulate certain treatment-related cellular signaling pathways, bind one or more neurociliary peptides (see, for example, Table N1), and / or possess anti-inflammatory properties. These activities, distinct from the classic role of tRNA synthetases in protein synthesis, are referred to herein as “atypical activities.” For example, as presented herein, HRS peptides such as the N-terminal regions of histyl-tRNA synthetases (e.g., HRS 1-48, HRS 1-60) are particularly capable of binding neurociliary peptides and thereby regulating the migration, activation, and / or differentiation of inflammatory or migratory cells, and treating neurociliary-related diseases. Furthermore, certain fragments, splice variants, mutations, and / or deletions relative to the full-length HRS peptide sequence (e.g., HRS 1-60) confer increased activity and / or improved pharmacological properties. Sequences of some exemplary HRS peptides are provided in Table H1 below.

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251] Therefore, in some embodiments, the HRS polypeptide comprises, consists of, or is substantially composed of the mammalian HRS amino acid sequence in Table H1 (e.g., SEQ ID NO: 1-116 and 172) or its active variants or fragments. In some embodiments, the HRS polypeptide comprises, consists of, or is substantially composed of the human HRS amino acid sequence in Table H1 (e.g., SEQ ID NO: 1-108 and 172) or its active variants or fragments. In some embodiments, an expressible polynucleotide encodes an HRS polypeptide comprising, consists of, or is substantially composed of the amino acid sequence in Table H1 (e.g., SEQ ID NO: 1-116 and 172) (e.g., the human HRS sequences SEQ ID NO: 1-108 and 172 in Table H1) or its active variants or fragments.

[0252] As described herein, HRS peptides can be altered in a variety of ways, including amino acid substitution, deletion, truncation, addition, and insertion. Methods for such manipulation are well known in the art. For example, amino acid sequence variants of HRS reference peptides can be prepared by mutation of DNA. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Kunkel (1985, Proceedings of the National Academy of Sciences 82:488-492); Kunkel et al. (1987, Methods in Enzymol, 154:367-382); U.S. Patent No. 4,873,192; Watson, JD et al. (Molecular Biology of the Gene, 4th Edition, Benjamin / Cummings, Menlo Park, California, 1987), and references cited herein. Guidelines on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978), Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington, DC).

[0253] Compared to the reference HRS amino acid residues, bioactive truncated and / or variant HRS peptides may contain conserved amino acid substitutions at different positions along their sequence. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are defined in the art and can generally be subdivided as follows:

[0254] Acidity: Due to the loss of H ions at physiological pH, the residues are negatively charged and are attracted to aqueous solutions to seek surface positions in the peptide conformation when the peptide containing these residues is in an aqueous medium at physiological pH. Amino acids with acidic side chains include glutamic acid and aspartic acid.

[0255] Basicity: Residues are positively charged due to association with H ions at physiological pH or within one or two pH units (e.g., histidine), and are attracted to aqueous solutions to seek surface sites in the conformation of the peptide when the peptide containing these residues is in an aqueous medium at physiological pH. Amino acids with basic side chains include arginine, lysine, and histidine.

[0256] Charged: The residues are charged at physiological pH and therefore contain amino acids with acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine, and histidine).

[0257] Hydrophobicity: The residues are uncharged at physiological pH and are repelled by aqueous solutions so that they seek an internal position in the conformation of the peptide when the peptide containing the residues is in an aqueous medium. Amino acids with hydrophobic side chains include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine, and tryptophan.

[0258] Neutral / Polar: The residues are uncharged at physiological pH, but are not sufficiently repelled by aqueous solutions. When the peptide containing the residues is in an aqueous medium, the residues will seek an internal position within the conformation of the peptide. Amino acids with neutral / polar side chains include asparagine, glutamine, cysteine, histidine, serine, and threonine.

[0259] This description also characterizes certain amino acids as “small” because their side chains are not large enough to impart hydrophobicity even in the absence of polar groups. Apart from proline, “small” amino acids are those with four or fewer carbons when there is at least one polar group on the side chain, and three or fewer carbons when there is no polar group on the side chain. Amino acids with small side chains include glycine, serine, alanine, and threonine. The genetically encoded secondary amino acid proline is a special case due to its known influence on the secondary conformation of the peptide chain. The structure of proline differs from all other naturally occurring amino acids because its side chain is bound to both the nitrogen of the α-amino group and the α-carbon. Several amino acid similarity matrices are known in the art (see, for example, the PAM120 and PAM250 matrices disclosed by Dayhoff et al., 1978, “A model of evolutionary change in proteins”). However, “Matrices for determining distance relationships,” MO Dayhoff (ed.), *Atlas of Protein Sequence and Structure*, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington, D.C.; and Gonnet et al. (*Science*, 256:14430-1445, 1992) include proline in the same group as glycine, serine, alanine, and threonine. Therefore, proline is classified as a “small” amino acid.

[0260] The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary, and therefore, the amino acids specifically contemplated in this invention are classified as one or another. Most unnamed amino acids can be classified based on known behavior.

[0261] Amino acid residues can be further subdivided into cyclic or acyclic, aromatic or non-aromatic, categories relative to the side chain substituents of the residue, and small or large. A residue is considered small if it contains four or fewer carbon atoms in total (including the carboxyl carbon), provided there are additional polar substituents; if there are no additional polar substituents, then a residue is considered small if it contains three or fewer carbon atoms in total (including the carboxyl carbon). Of course, small residues are always non-aromatic. Based on their structural properties, amino acid residues can be divided into two or more classes. Table A presents the subclasses according to this scheme for naturally occurring protein amino acids.

[0262]

[0263] Conservative amino acid substitutions also include groupings based on side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that substituting leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similarly substituting amino acids with structurally related amino acids, will not have a major impact on the properties of the resulting variant peptides. Whether amino acid changes result in functionally truncated and / or variant HRS peptides can be readily determined by measuring their atypical activities, as described herein. Conservative substitutions are shown under the headings of exemplary substitutions in Table B. Amino acid substitutions falling within the scope of this invention are typically achieved by selecting substitutions that do not significantly differ in maintaining the following effects: (a) the structure of the peptide backbone in the substituted region, (b) the charge or hydrophobicity of the molecule at the target site, (c) the volume of the side chain, or (d) biological function. After the substitution is introduced, the biological activity of the variants is screened.

[0264]

[0265]

[0266] Alternatively, similar amino acids used for conserved substitution can be grouped into three categories based on the properties of their side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, and histidine, all of which have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, and asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, and methionine, as described in Zubay, G., *Biochemistry*, 3rd ed., Wm.C. Brown Publishers (1993).

[0267] In some embodiments, the HRS peptide has one or more cysteine ​​insertions or substitutions, for example, wherein one or more non-cysteine ​​residues are substituted with cysteine ​​residues (e.g., to alter stability, to facilitate thiol-based conjugation of the Fc fragment, to facilitate thiol-based attachment of PEG or other molecules). In some embodiments, one or more cysteine ​​residues substitute near the N-terminus and / or C-terminus of the HRS peptide or other surface-exposed regions of the HRS peptide. Specific embodiments include one or more residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids relative to the N-terminus and / or C-terminus of the HRS peptide being substituted with cysteine ​​residues. In some embodiments, cysteine ​​residues can be added to the HRS peptide by generating an N-terminal or C-terminal fusion protein. Such fusion proteins can be of any length, but are typically about 1-5, or about 5-10, about 10 to 20, or about 20 to 30 amino acids.

[0268] Table H2 shows specific examples of cysteine-modified proteins based on HRS peptides HRS(1-60). This approach can be applied to the HRS peptides in Table H1 and other HRS peptides described herein.

[0269]

[0270] Therefore, in some embodiments, the HRS polypeptide comprises, consists of, or is substantially composed of the amino acid sequence (SEQ ID NO: 117-119) in Table H2 or its active variants or fragments. In some embodiments, a polynucleotide encoding an HRS polypeptide may be expressed, the HRS polypeptide comprising, consists of, or is substantially composed of the amino acid sequence (e.g., SEQ ID NO: 117-119) in Table H2 or its active variants or fragments.

[0271] In some embodiments, the HRS peptide has a mutation in which an endogenous or naturally occurring cysteine ​​residue is mutated to a substitute amino acid or deleted. In some embodiments, the insertion or substitution of one or more cysteine ​​residues into the HRS peptide is combined with the elimination of other surface-exposed reactive cysteine ​​residues. Thus, in some embodiments, the HRS peptide includes one or more substitutions and / or deletions at any one or more of Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, Cys455, Cys507 and / or Cys509 (as defined in SEQ ID NO:1), for example to remove naturally occurring cysteine ​​residues, including combinations thereof.

[0272] Specific embodiments include the HRS polypeptides of Table H1, which have mutations or deletions of one or more of Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, and Cys455, or deletions of Cys507 and Cys509, for example, through the deletion of three amino acids at the C-terminus (Δ507-509). Exemplary mutations at these positions include, for example, cysteine ​​mutations to serine, alanine, leucine, valine, or glycine. In some embodiments, the amino acid residues used for a particular cysteine ​​substitution may be selected from naturally occurring substitutions found in HRS orthologs from other species and organisms. Exemplary substitutions of this type are presented in Table H3.

[0273]

[0274]

[0275] In some embodiments, the selection of naturally occurring cysteine ​​residues for mutagenesis is based on their surface exposure. Thus, in one aspect, the cysteine ​​residues selected for substitution are selected from Cys224, Cys235, Cys507, and Cys509. In some embodiments, the last three (C-terminal) residues of SEQ ID NO:1 are deleted to remove residues 507 to 509. In some embodiments, cysteine ​​residues are selected for mutation or deletion to eliminate intramolecular cysteine ​​pairs, such as Cys174 and Cys191.

[0276] Specific examples of cysteine ​​mutations / substitutions (indicated by bold underline) used to reduce the number of cysteine ​​residues exposed on the surface include those listed in Table H4 below.

[0277]

[0278]

[0279]

[0280] Therefore, in some embodiments, the HRS polypeptide comprises, consists of, or is substantially composed of the amino acid sequence (SEQ ID NO: 120-126) in Table H4 or its active variants or fragments. In some embodiments, a polynucleotide encoding an HRS polypeptide may be expressed, the HRS polypeptide comprising, consists of, or is substantially composed of the amino acid sequence (e.g., SEQ ID NO: 120-126) in Table H4 or its active variants or fragments.

[0281] In some embodiments, such cysteine-substituted mutants are modified to engineer, insert, or otherwise introduce new surface-exposed cysteine ​​residues at defined surface-exposed sites, wherein the introduced residues substantially do not interfere with the atypical activity of the HRS peptide. Specific examples include, for example, inserting (or reinserting) additional cysteine ​​residues at the N-terminus or C-terminus of any of the aforementioned cysteine-reduced HRS peptides. In some embodiments, such insertion of N-terminal or C-terminal surface-exposed cysteine ​​involves reinserting the last one, last two, or last three naturally occurring C-terminal amino acids of the full-length human HRS into a cysteine-reduced variant of the HRS peptide, such as reinserting all or part of the CIC (CysIle Cys) sequence. Exemplary cysteine-reducing mutants include any combination of mutations (or deletions) at residues Cys174, Cys191, Cys224 and Cys235 and / or deletions or substitutions of Cys507 and Cys509 (based on the numbering of the full-length human cytoplasmic HRS (SEQ ID NO:1) in any HRS polypeptide in Table H1).

[0282] For some types of site-specific conjugation or attachment to heterologous molecules (such as Fc regions or PEG or other heterologous molecules), HRS peptides may have one or more glutamine substitutions, wherein one or more naturally occurring (non-glutamine) residues are substituted with glutamine, for example, to facilitate transglutaminase-catalyzed attachment of one or more molecules to the amide group of glutamine. In some embodiments, glutamine substitution is introduced near the N-terminus and / or C-terminus of the HRS peptide. Specific embodiments include one or more residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids relative to the N-terminus and / or C-terminus of the HRS peptide being substituted with glutamine residues. If desired, these and associated HRS peptides may also contain substitutions (e.g., conserved substitutions) to remove any naturally occurring glutamine residues, thereby modulating the degree of site-specific conjugation or attachment.

[0283] For certain types of site-specific conjugation or attachment to heterologous molecules (such as Fc regions or PEG or other heterologous molecules), HRS peptides may have one or more lysine substitutions, wherein one or more naturally occurring (non-lysine) residues are replaced with lysine, for example, to facilitate acylation- or alkylation-based attachment of one or more molecules to lysine amino groups. These methods typically also result in attachment of one or more molecules to N-terminal residues. In some embodiments, the lysine substitution is located near the N-terminus and / or C-terminus of the HRS peptide. Specific embodiments include one or more residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids relative to the N-terminus and / or C-terminus of the HRS peptide being replaced with lysine residues. If desired, these and associated HRS peptides may also contain substitutions (e.g., conserved substitutions) to remove any naturally occurring lysine residues, thereby modulating the degree of site-specific conjugation or attachment.

[0284] Site-specific conjugation with the HRS peptide can also be performed by substituting one or more solvent-accessible surface amino acids. For example, suitable solvent-accessible amino acids can be determined based on solvent accessibility predicted using the SPIDDER server (http: / / sppider.cchmc.org / ) and the published crystal structures of exemplary HRS peptides (see Xu et al., Structure. 20:1470-7, 2012; and U.S. Application No. 61 / 674,639). Based on this analysis, several amino acids on the surface can potentially be used as mutation sites to introduce functional groups suitable for conjugation or attachment. A surface accessibility score for the amino acids based on the crystal structure can be calculated, where a higher score indicates better accessibility. In certain embodiments, higher scores (e.g., >40) are preferred. Thus, in some embodiments, amino acid positions with surface accessibility scores greater than 40 can be used to introduce cysteine, lysine, glutamine, or other non-naturally occurring amino acids.

[0285] In a particular embodiment, the solvent-accessible surface amino acids are selected from the group consisting of alanine, glycine, and serine, and may be replaced by naturally occurring amino acids or by non-naturally occurring amino acids optimized for site-specific conjugation or attachment, wherein the naturally occurring amino acids include, but are not limited to, cysteine, glutamine, or lysine.

[0286] Some embodiments involve site-specific conjugation or attachment at any amino acid position to the HRS peptide by means of substituting non-naturally present amino acids, including functional groups, which will form covalent bonds with functional groups attached to heterologous molecules (such as Fc regions or PEG or other heterologous molecules). Non-natural amino acids may be inserted or substituted at one or more residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids relative to the N-terminus and / or C-terminus of the HRS peptide described herein, at the N-terminus and / or C-terminus of the HRS peptide, or at solvent-accessible surface amino acid residues of the HRS peptide.

[0287] In certain embodiments, non-naturally occurring amino acids include, but are not limited to, any amino acid, modified amino acid, or amino acid analogue other than selenocysteine ​​and the following twenty genetically encoded α-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The general structure of an α-amino acid is shown by the following formula:

[0288]

[0289] Non-natural amino acids are generally any structure having the aforementioned formula, where the R group is any substituent other than those used in the twenty natural amino acids. For the structures of the twenty natural amino acids, see, for example, a biochemistry textbook, such as L. Stryer's *Biochemistry*, 3rd edition, 1988, Freeman and Company, New York. It should be noted that the non-natural amino acids disclosed herein can be naturally occurring compounds other than the twenty α-amino acids mentioned above. Because the non-natural amino acids disclosed herein generally differ from natural amino acids only in their side chains, non-natural amino acids form amide bonds with other amino acids (e.g., natural or non-natural) in the same way that amide bonds are formed in naturally occurring proteins. However, non-natural amino acids have side chain groups that distinguish them from natural amino acids. For example, R in the above formula optionally includes alkyl-, aryl-, aryl halogen, vinyl halogen, alkyl halogen, acetyl, ketone, aziridine, nitrile, halide, acyl-, ketol-, azide-, hydroxyl-, hydrazine, cyano-, halogen-, acylhydrazine, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boric acid, borate ester, borane, phenylboronic acid, thiol, selenyl-, sulfonyl-, borate ester, borate, phosphine, phosphine carboxyl, phosphine, heterocyclic group, nitrophenyl, naphthyl, benzophenone, restricted ring such as cyclooctyne, thioester, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid, α-ketocarboxylic acid, α or β unsaturated acid and amide, acetaldehyde amide or organosilicon group, etc., or any combination thereof.

[0290] Specific examples of non-natural amino acids include, but are not limited to: p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, β-O-GlcNAc-L-serine, tri-O-acetyl-GalNAc-α-threonine, α-GalNAc-L-threonine, levodopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonyltyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and those listed below or elsewhere in this document.

[0291] Therefore, non-naturally occurring amino acids can be selected, including functional groups that form covalent bonds with any preferred functional group of the desired molecule (e.g., Fc region, PEG). Once selected, the non-natural amino acids can be purchased from a supplier or chemically synthesized. Any number of non-natural amino acids can be bound to the target molecule and can vary depending on the number of molecules to be attached. The molecule can be attached to all or only some of the non-natural amino acids. Further, depending on the desired result, the same or different non-natural amino acids can be bound to the HRS peptide. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-natural amino acids are bound to the HRS peptide, wherein any or all of the said non-natural amino acids can be conjugated to a molecule including the desired functional group.

[0292] In some respects, the use of non-natural amino acids can be used to modify (e.g., increase) selected atypical activities of HRS peptides or to alter the in vivo or in vitro half-life of proteins. As described herein, non-natural amino acids can also be used to promote (selective) chemical modifications of HRS peptides (e.g., PEGylation). For example, certain non-natural amino acids allow polymers such as Fc regions or PEG to selectively attach to a given protein, thereby improving its pharmacokinetic properties.

[0293] Specific examples of amino acid analogs and simulants can be found, for example, described in: Roberts and Vellaccio, *The Peptides: Analysis, Synthesis, Biology*, edited by Gross and Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, NY (1983), the entire contents of which are incorporated herein by reference. Other examples include fully alkylated amino acids, and particularly fully methylated amino acids. See, for example, *Combinatorial Chemistry*, edited by Wilson and Czarnik, Chapter 11, p. 235, John Willie & Son, New York, NY (1997), the entire contents of which are incorporated herein by reference. Still other examples include amino acids whose amide moiety (and thus the amide backbone of the resulting peptide) has been replaced by, for example, a sugar ring, steroid, benzodiazepine, or carbocyclic ring. See, for example, Burger, *Medicinal Chemistry and Drug Discovery*, edited by Manfred E. Wolff, Chapter 15, pp. 619-620, John Willie & Son, New York, NY (1995), the entire contents of which are incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptide mimics, and proteins are well known in the art (see, for example, U.S. Patent No. 5,420,109; M. Bodanzsky, *Principles of Peptide Synthesis* (1st and 2nd revised editions), Springer-Verlag, New York, NY (1984 & 1993), see Chapter 7; Stewart and Young, *Solid Phase Peptide Synthesis*, (2nd edition), Pierce Chemical Co., Rockford, Ill. (1984), each of which is incorporated herein by reference). Therefore, HRS peptides can be composed of naturally occurring and non-natural amino acids, as well as amino acid analogs and mimics.

[0294] In some embodiments, the HRS polypeptide comprises, consists of, or is substantially composed of a minimal active fragment of a full-length HRS polypeptide capable of modulating anti-inflammatory activity in vivo or having neurocilia peptide-binding activity. In some embodiments, such a minimal active fragment comprises, consists of, or is substantially composed of a WHEP domain (e.g., about amino acids 1-43 of SEQ ID NO:1) or an active variant or fragment thereof.

[0295] In some embodiments, the length of the HRS peptide is about, at least about and / or at most about 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, 5 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, or 509 amino acids (inclusive of all integer ranges in between), and including, consisting of, or substantially consisting of the amino acid sequences in Table H1, Table H2, or Table H4.

[0296] In some embodiments, the HRS peptide has at least one atypical activity, such as anti-inflammatory activity or binding to neurocilia peptides, examples of which are described herein. Assays for determining anti-inflammatory activity or binding of neurocilia peptides or receptors, including routine measurements of cytokine release based on in vitro cells and animal studies, are well established in the field (see, for example, Wittmann et al., *Journal of Visual Experiments* (65):e4203.doi:10.3791 / 4203, 2012; Feldman et al., *Molecular Cell* 47:585-95, 2012; Clutterbuck et al., *Journal of Proteomics* 74:704-15, 2011; Giddings and Maitra, *Journal of Biomol Screen* 15:1204-10, 2010; Wijnhoven et al., *Journal of Glycoconjugates* 25:177-85, 2008; and Frow et al., *Med Research Review*). (Revised) 24:276-98, 2004) and can be readily used to analyze and optimize anti-inflammatory activity. An exemplary in vivo experimental system is also described in the accompanying examples.

[0297] It should be understood that in any HRS polypeptide, the N-terminal amino acid (e.g., N-terminal Met) of the HRS polypeptide may be missing or replaced by different amino acids.

[0298] In some embodiments, the fusion protein further comprises an HRS peptide with other (non-HARS) proteins (e.g., heterologous proteins or peptides), and these fusion proteins can modulate the biological activity, secretion, antigenicity, targeting, biological lifespan, ability to penetrate cell membranes or the blood-brain barrier, or pharmacokinetic properties of the HRS peptide. Examples of fusion proteins (“PK modulators”) that improve pharmacokinetic properties include, but are not limited to, fusions with: human albumin (Osborn et al.: European Journal of Pharmacology 456(1-3):149-158, (2002)), antibody Fc domains, polyglutamic or polyasp sequences, and transferrin. Additionally, fusions with amino acids Pro, Ala, and Ser (“PASylation”) or hydroxyethyl starch (as marketed) are also included. The fusion of conformally disordered polypeptide sequences (for sale) provides a simple method for increasing the hydrodynamic volume of HRS polypeptides. This additional extension employs a large-volume random structure, which significantly increases the size of the resulting fusion protein. In this way, the typical rapid clearance of smaller HRS polypeptides by renal filtration is delayed by several orders of magnitude. In addition, it has been shown that the use of IgG fusion proteins enables some fusion proteins to cross the blood-brain barrier (Fu et al., (2010) Brain Research 1352:208-13).

[0299] Examples of fusion proteins that regulate the antigenicity or other properties of HRS peptides include fusion with a T-cell binding ligand comprising, for example, MHC class I and II proteins, β-2 microglobulin, a moiety of LFA-3, a portion of its heavy chain Fc region, and conjugates and derivatives thereof. Examples of such fusion proteins are described in: EP 1 964854; U.S. Patent Nos. 5,468,481, 5,130,297, 5,635,363, and 6,451,314; and U.S. Application No. 2009 / 0280135.

[0300] In some embodiments, the HRS peptide may include a synthetic or naturally occurring secretion signal sequence derived from other well-characterized secretory proteins. In some embodiments, such proteins may be processed by proteolytic cleavage to form the HRS peptide in situ. In some embodiments, the HRS peptide may include a heterologous proteolytic cleavage site to enable in situ expression and production of the HRS peptide at intracellular or extracellular sites. Other fusion proteins may also include, for example, fusion of the HRS peptide with ubiquitin to provide a novel N-terminal amino acid, or use of a secretion signal to mediate high-level secretion of the HRS peptide into the extracellular medium, or N-terminal or C-terminal epitope tags to improve purification or detection, and fusion with cell-penetrating peptides.

[0301] In some respects, the use of non-natural amino acids can be used to modify (e.g., increase) selected atypical activities of HRS peptides or to alter the in vivo or in vitro half-life of proteins. As described elsewhere in this document, non-natural amino acids can also be used to facilitate (selective) chemical modifications of HRS proteins (e.g., PEGylation). For example, certain non-natural amino acids allow polymers such as PEG to selectively attach to a given protein, thereby improving its pharmacokinetic properties.

[0302] Some embodiments comprise HRS-Fc conjugates, which include at least one Fc region covalently attached to one or more HRS peptides. Examples of HRS-Fc conjugates include fusion proteins and various forms of chemically cross-linked proteins. A wide variety of Fc region sequences can be employed in HRS-Fc conjugates, including wild-type sequences from any number of species, as well as their variants, fragments, hybrids, and chemically modified forms. HRS-Fc peptides may also (optionally) include one or more linkers that typically separate one or more Fc regions from one or more HRS peptides, including peptide linkers and chemical linkers as described herein and known in the art. It should be understood that in any of these HRS-Fc conjugates, the native N-terminal or C-terminal amino acid of the HRS peptide, or the native N or C amino acid in the Fc domain, may be missing and / or substituted with one or more non-natural amino acids, for example, to facilitate expression and / or cloning or as a linker sequence between two proteins.

[0303] HRS-Fc conjugated peptides offer several advantages over unconjugated or unmodified HRS peptides (e.g., those without the same or similar sequences of one or more Fc regions to which they are attached). Simply by way of illustration, covalent attachment of one or more Fc regions to an unmodified HRS peptide with the same or similar sequences can alter (e.g., increase, decrease) the solubility, half-life (e.g., in serum, in selected tissues, or in test tubes under storage conditions (e.g., at room temperature or under refrigeration) of the HRS peptide, dimerization or multimerization, one or more biological activities, for example, by providing Fc region-associated effector functions (e.g., activation of the classical complement cascade, interaction with immune effector cells via Fc receptors (FcRs), compartmentalization of immunoglobulins), cellular uptake, intracellular transport, tissue distribution, and / or bioavailability. In some respects, the Fc region can confer effector functions associated with complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cell-mediated phagocytosis (ADCP), which are believed to play a role in clearing specific target cells such as tumor cells and infected cells.

[0304] Some embodiments employ an HRS-Fc fusion protein. The term "fusion protein" is defined elsewhere herein and is well known in the art, as are methods for preparing fusion proteins (for general disclosures and methods relating to Fc fusion proteins, see, for example, U.S. Patent Nos. 5,116,964; 5,428,130; 5,455,165; 5,514,582; 6,406,697; 6,291,212; and 6,300,099). In an HRS-Fc fusion protein, the Fc region may be fused to the N-terminus, C-terminus, or both of the HRS polypeptide. In some embodiments, one or more Fc regions may be internally fused relative to the HRS sequence, for example, by placing the Fc region between a first HRS sequence (e.g., a domain) and a second HRS sequence (e.g., a domain), wherein the first HRS sequence is fused to the N-terminus of the Fc region and the second HRS sequence is fused to the C-terminus of the Fc region. In a particular embodiment, the first and second HRS sequences are identical. In some embodiments, the first and second HRS sequences are different (e.g., they contain different functional domains of the HRS polypeptide). Some HRS-Fc fusion proteins may also contain additional heterologous protein sequences, i.e., non-Fc regions and non-HRS polypeptide sequences.

[0305] The term "HRS-Fc" can indicate, but does not necessarily indicate, the attachment of the Fc region to the N-terminus or C-terminus of the HRS peptide. For example, in some cases, the term "Fc-HRS" indicates the fusion of the Fc region to the N-terminus of the HRS peptide, and the term "HRS-Fc" indicates the fusion of the Fc region to the C-terminus of the HRS peptide. However, either term can more generally refer to any fusion protein or conjugate of the Fc region and the HRS peptide.

[0306] In some embodiments, the HRS-Fc fusion protein may include a tandem repeat copy of an HRS polypeptide coupled to a single Fc domain, the tandem repeat copy optionally being separated by a linker peptide. Exemplary tandem repeat HRS-Fc fusion proteins are provided in Table H5. The preparation and sequence of specific tandem repeat HRS-Fc conjugates are shown in the examples.

[0307]

[0308] Some embodiments relate to HRS-Fc conjugates, wherein, for example, one or more Fc regions are chemically conjugated or crosslinked with one or more HRS peptides. In these and related aspects, the Fc regions may be conjugated with the HRS peptide at the N-terminal region (e.g., within the first 10, 20, 30, 40, 50, 60, 70, 80, 90, or approximately 100 amino acids), the internal region (between the N-terminal and C-terminal regions), and / or the C-terminal region (e.g., within the last 10, 20, 30, 40, 50, 60, 70, 80, 90, or approximately 100 amino acids). According to various conventional techniques in the art, the peptide may be conjugated or crosslinked with other peptides. For example, some techniques employ a carboxyl-reactive carbodiimide crosslinking agent EDC (or EDAC), which is covalently attached via D-terminal, E-terminal, and C-terminal carboxyl groups. Other techniques employ activated EDC, which is covalently attached via K-terminal and N-terminal amino groups. Other techniques still employ m-maleimide benzoyl-N-hydroxysuccinimide (MBS) or sulfonyl-MBS, which are covalently attached via thiol groups of cysteine ​​residues (see also U.S. Application No. 2007 / 0092940 for Ig regions engineered from cysteine ​​that can be used for thiol conjugation). Such cross-linked proteins may also include linkers comprising cleavable or otherwise releasable linkers (e.g., enzymatically cleavable linkers, hydrolyzable linkers) and non-cleavable linkers (i.e., physiologically stable linkers). Some embodiments may employ non-peptide polymers (e.g., PEG polymers; HRS-N-PEG-N-Fc conjugates) as cross-linking agents between one or more Fc regions and one or more HRS peptides, as described, for example, in U.S. Application No. 2006 / 0269553. For an exemplary description of Fc region conjugation sites, see also U.S. Application No. 2007 / 0269369.

[0309] In some embodiments discussed in more detail below, variant or otherwise modified Fc regions may be employed, including those that have altered properties or biological activities relative to one or more wild-type Fc regions. Examples of modified Fc regions include: Fc regions having mutated sequences, for example, by substitution, insertion, deletion, or truncation of one or more amino acids relative to the wild-type sequence; hybrid Fc polypeptides composed of domains from different immunoglobulin classes / subclasses; Fc polypeptides with altered glycosylation / sialylation patterns; and Fc polypeptides modified or derivatized, for example, by biotinylation (see, for example, U.S. Application No. 2010 / 0209424), phosphorylation, sulfation, etc.; or any combination of the foregoing. This modification, relative to the corresponding wild-type Fc sequence, can alter (e.g., increase or decrease) the binding properties of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn), and their pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, C0). max t max C min (variation), its immunogenicity, its complement binding or activation and / or CDC / ADCC / ADCP-related activity in the Fc region, and other properties described herein.

[0310] The "Fc region" of the HRS-Fc conjugates described in this article typically originates from the heavy chain of an immunoglobulin (Ig) molecule. A typical Ig molecule consists of two heavy chains and two light chains. The heavy chain can be divided into at least three functional regions: the Fd region, the Fc region (fragment crystallizable region), and the hinge region, the latter found only in IgG, IgA, and IgD immunoglobulins. The Fd region includes the variable (V) region of the heavy chain. H ) and constant (CH1) structural domains, and with variable (V) domains of the light chain. L ) and constant (C L The ) domains together form antigen-binding fragments or Fab regions.

[0311] The Fc regions of IgG, IgA, and IgD immunoglobulins include heavy chain constant domains 2 and 3, designated CH2 and CH3, respectively; and the Fc regions of IgE and IgM immunoglobulins include heavy chain constant domains 2, 3, and 4, designated CH2, CH3, and CH4, respectively. The Fc regions are primarily responsible for immunoglobulin effector functions, including, for example, complement binding and binding to homologous Fc receptors on effector cells.

[0312] Hinge regions (found in IgG, IgA, and IgD) act as flexible spacers, allowing the Fab moiety to move freely in space relative to the Fc region. Unlike constant regions, hinge regions are structurally diverse, differing in both sequence and length across immunoglobulin classes and subclasses. Hinge regions may also contain one or more glycosylation sites, encompassing many structurally different types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of its hinge region, conferring significant resistance to intestinal proteases to hinge region peptides. Residues in the proximal region of the CH2 domain hinge can also affect the specificity of interactions between immunoglobulins and their corresponding one or more Fc receptors (see, for example, Shin et al., *International Review of Immunology* 10:177-186, 1993).

[0313] Therefore, as used herein, the terms "Fc region" or "Fc fragment" or "Fc" refer to a protein containing one or more of the CH2, CH3, and / or CH4 regions of one or more selected immunoglobulins, including fragments and variants thereof, as well as combinations thereof. An "Fc region" may also contain one or more hinge regions of the heavy chain constant region of an immunoglobulin. In some embodiments, the Fc region does not contain the CH1, CH3, or CH4 regions of an immunoglobulin. L V L and / or V H One or more in the district.

[0314] The Fc region can be derived from the CH2, CH3, CH4 regions and / or one or more hinge regions of any one or more immunoglobulin classes, including but not limited to IgA, IgD, IgE, IgG, and IgM, and including subclasses and combinations thereof. In some embodiments, the Fc region is derived from IgA immunoglobulin, including subclasses IgA1 and / or IgA2. In some embodiments, the Fc region is derived from IgD immunoglobulin. In certain embodiments, the Fc region is derived from IgE immunoglobulin. In some embodiments, the Fc region is derived from IgG immunoglobulin, including subclasses IgG1, IgG2, IgG3, and / or IgG4. In some embodiments, the Fc region is derived from IgM immunoglobulin.

[0315] Certain Fc regions exhibit specific binding to one or more Fc receptors (FcRs). Examples of Fc receptor classes include Fcγ receptors (FcγR), Fcα receptors (FcαR), Fcε receptors (FcεR), and neonatal Fc receptors (FcRn). For example, some Fc regions exhibit increased binding (or affinity) to one or more FcγRs relative to FcαR, FcεR, and / or FcRn. In some embodiments, Fc regions exhibit increased binding to FcαRs relative to one or more FcγRs, FcεRs, and / or FcRn. In some embodiments, Fc regions exhibit increased binding to FcεRs (e.g., FcαRI) relative to one or more FcγRs, FcαRs, and / or FcRn. In specific embodiments, Fc regions exhibit increased binding to FcRn relative to one or more FcγRs, FcαRs, and / or FcεRs. In some embodiments, the binding (or affinity) of the Fc region to one or more selected FcRs is increased relative to its binding (or affinity) to one or more different FcRs, typically by about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times or more (inclusive of all integers therein).

[0316] Examples of FcγR include FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. FcγRI (CD64) is expressed on macrophages and dendritic cells and plays a role in phagocytosis, respiratory burst, cytokine stimulation, and dendritic cell endocytosis. FcγRI expression is upregulated by GM-CSF and γ-IFN and downregulated by interleukin-4 (IL-4). FcγRIIa is expressed on polymorphonuclear leukocytes (PMNs), macrophages, dendritic cells, and mast cells. FcγRIIa plays a role in phagocytosis, respiratory burst, and cytokine stimulation. FcγRIIa expression is upregulated by GM-CSF and γ-IFN and downregulated by IL-4. FcγIIb is expressed on B cells, PMNs, macrophages, and mast cells. FcγIIb inhibits the immune receptor tyrosine activation motif (ITAM)-mediated response and is therefore an inhibitory receptor. FcγRIIc expression is upregulated by intravenously injected immunoglobulin (IVIG) and IL-4 and downregulated by γ-IFN. FcγRIIc is expressed on NK cells. FcγRIIIa is expressed on natural killer (NK) cells, macrophages, mast cells, and platelets. This receptor is involved in phagocytosis, respiratory burst, cytokine stimulation, platelet aggregation and degranulation, and NK-mediated ADCC. FcγRIII expression is upregulated by C5a, TGF-β, and γ-IFN and downregulated by IL-4. FcγRIIIb is a GPI-linked receptor expressed on PMNs.

[0317] Compared to FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and / or FcγRIIIb, the binding of certain Fc regions to FcγRI is increased. Compared to FcγRI, FcγRIIb, FcγRIIc, FcγRIIIa and / or FcγRIIIb, some embodiments show increased binding to FcγRIIa. Compared to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb, specific Fc regions show increased binding to FcγRIIb. Compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and / or FcγRIIIb, some Fc regions show increased binding to FcγRIIc. Compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc and / or FcγRIIIb, the binding of certain Fc regions to FcγRIIIa is increased. Compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc and / or FcγRIIIa, the binding of certain Fc regions to FcγRIIIb is increased.

[0318] FcαR comprises FcαRI (CD89). FcαRI is found on the surface of neutrophils, eosinophils, monocytes, certain macrophages (e.g., Kupffer cells), and certain dendritic cells. FcαRI consists of two extracellular Ig-like domains, is a member of the immunoglobulin superfamily and the multi-chain immune recognition receptor (MIRR) family, and signals by associating with two FcRγ signaling chains.

[0319] FcεR comprises FcεRI and FcεRII. The high-affinity receptor FcεRI is a member of the immunoglobulin superfamily, expressed on epidermal Langerhans cells, eosinophils, mast cells, and basophils, and plays a major role in controlling allergic responses. FcεRI is also expressed on antigen-presenting cells and regulates the production of pro-inflammatory cytokines. The low-affinity receptor FcεRII (CD23) is a C-type lectin that can function as a membrane-bound or soluble receptor. FcεRII regulates B cell growth and differentiation and blocks IgE binding on eosinophils, monocytes, and basophils. Binding to FcεRII is increased in certain Fc regions relative to FcεRII. Binding to FcεRII is increased in other Fc regions relative to FcεRII.

[0320] Table H6 below summarizes some characteristics of FcR.

[0321]

[0322]

[0323] The Fc region can be derived from immunoglobulin molecules of any animal, including vertebrates such as mammals, such as cattle, goats, pigs, dogs, mice, rabbits, hamsters, rats, guinea pigs, non-human primates, and humans. Table H7 shows the amino acid sequences of the CH2, CH3, CH4, and hinge regions of exemplary wild-type human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM immunoglobulins.

[0324]

[0325]

[0326]

[0327] Therefore, the Fc region of the HRS-Fc conjugate may include one or more, constitute or substantially constitute one of the amino acid sequences of the human Fc region in Table H7, including its variants, fragments, homologs, orthologs, paralogs and combinations thereof. Some illustrative embodiments include Fc regions of a size ranging from about 20-50, 20-100, 20-150, 20-200, 20-250, 20-300, 20-400, 50-100, 50-150, 50-200, 50-250, 50-300, 50-400, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 200-250, 200-300, 200-350, or 200-400 amino acid lengths, and optionally include, constitute, or substantially constitute any one or more sequences in Table H7. Some embodiments include an Fc region of up to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400 or more amino acids, which optionally includes, constitutes or is substantially constituted by any one or more amino acid sequences of the amino acid sequences in Table H7.

[0328] Certain Fc regions include, consist of, or are substantially composed of the human IgA1 sequences from Table H7 (including combinations thereof, as well as variants and fragments thereof) in any order read from the N-end to the C-end.

[0329] Some Fc regions include, consist of, or are substantially composed of the human IgA2 sequence from Table H7 (including combinations thereof, as well as variants and fragments thereof) in any order read from the N-end to the C-end.

[0330] Certain Fc regions, read in any order from N-terminus to C-terminus, include, consist of, or are substantially composed of the human IgD sequence (including combinations thereof, as well as variants and fragments of such sequences and combinations) from Table H7. Certain Fc regions, read in any order from N-terminus to C-terminus, include, consist of, or are substantially composed of the human IgE sequence (including combinations thereof, as well as variants and fragments of such sequences and combinations) from Table H7. Certain Fc regions, read in any order from N-terminus to C-terminus, include, consist of, or are substantially composed of the human IgG1 sequence (including combinations thereof, as well as variants and fragments of such sequences and combinations) from Table H7. Certain Fc regions, read in any order from N-terminus to C-terminus, include, consist of, or are substantially composed of the human IgG2 sequence (including combinations thereof) from Table H7. Certain Fc regions, read in any order from N-terminus to C-terminus, include, consist of, or are substantially composed of the human IgG3 sequence (including combinations thereof) from Table H7. Certain Fc regions include, consist of, or are substantially composed of the human IgG4 sequence from Table H7, in any order read from the N-terminus to the C-terminus. Certain Fc regions include, consist of, or are substantially composed of the human IgM sequence from Table H7, in any order read from the N-terminus to the C-terminus.

[0331] Table H8 below provides an example HRS-Fc fusion conjugate.

[0332]

[0333]

[0334]

[0335] Therefore, in some embodiments, the HRS peptide is fused to or otherwise conjugated to the Fc region and comprises, consists of, or substantially comprises the amino acid sequence in Table H8 (SEQ ID NO: 156-171) or its active variants or fragments. In some embodiments, a polynucleotide encoding the HRS peptide may be expressed, the HRS peptide comprising, consists of, or substantially comprises the amino acid sequence in Table H8 (e.g., SEQ ID NO: 156-171) or its active variants or fragments.

[0336] As described above, some embodiments employ variants, fragments, hybrids, and / or other modifications of the Fc region as described herein and known in the art. Variants include those with one or more amino acid substitutions, insertions, deletions, and / or truncations relative to a reference sequence (such as any one or more reference sequences in Table H7 or Table H8). Peptide and polynucleotide variants are described elsewhere herein.

[0337] It also includes hybrid Fc regions, such as Fc regions comprising combinations of Fc domains (e.g., hinge, CH2, CH3, CH4) of immunoglobulins from different species, different Ig classes, and / or different Ig subclasses. Typical examples include hybrid Fc regions comprising, consisting of, or substantially consisting of the following combinations of CH2 / CH3 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, Ig... D / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgE / IgA1, IgE / IgA2, IgE / IgD, IgE / IgE, IgE / IgG1, IgE / IgG2, IgE / IgG3, IgE / IgG4, IgE / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3 , IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG 3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / Ig D, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM, IgM / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or fragments or variants thereof), and optionally comprising a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4 and / or a CH4 domain from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0338] Other examples include hybridization Fc regions consisting of, or substantially consisting of, the following combinations of CH2 / CH4 domains: IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 The IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof) may optionally include a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 and / or a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0339] Certain instances contain hybridization Fc regions comprising, consisting of, or substantially consisting of the following combinations of CH3 / CH4 domains: IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 The IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof) may optionally include a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 and / or a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM. In a specific embodiment, the hinge, CH2, CH3 and CH4 domains are derived from human Ig.

[0340] Specific examples include hybridization Fc regions comprising, consisting of, or substantially consisting of the following combinations of hinge / CH2 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / Ig G3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / I The IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), and optionally include one or more CH3 domains from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM and / or CH4 domains from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0341] Certain instances contain hybridization Fc regions comprising, consisting of, or substantially consisting of the following hinge / CH3 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / Ig G3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / I The IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), and optionally include a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM and / or a CH4 domain from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0342] Some examples contain hybridization Fc regions comprising, consisting of, or substantially consisting of the following hinge / CH4 domains: IgA1 / IgE, IgA1 / IgM, IgA2 / IgE, IgA2 / IgM, IgD / IgE, IgD / IgM, IgG1 / IgE, IgG1 / IgM, IgG2 / IgE, IgG2 / IgM, IgG3 / IgE, IgG3 / IgM, IgG4 / IgE, IgG4 / IgM (or fragments or variants thereof), and optionally containing one or more CH2 domains from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM and / or one or more CH3 domains from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM.

[0343] Specific examples of hybrid Fc regions can be found, for example, in WO 2008 / 147143, where the hybrid Fc region is derived from a combination of IgG subclasses or a combination of human IgD and IgG.

[0344] It also includes derived or otherwise modified Fc regions. In some aspects, Fc regions can be modified relative to wild-type or naturally occurring Fc regions by, for example, phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, acetylation, amidation, etc. In some embodiments, the Fc region may include wild-type or natural glycosylation patterns, or alternatively, its glycosylation may be increased or decreased relative to the natural form, or it may be completely deglycosylated. As an example of a modified Fc glycoform, reduced glycosylation of the Fc region reduces binding to the C1q region of the first complement component C1, reduces ADCC-related activity, and / or reduces CDC-related activity. Therefore, some embodiments employ deglycosylated or glycosyl-free Fc regions. For examples of the generation of glycosyl-free Fc regions, see, for example, WO 2005 / 047337. Another example of an Fc region glycoform can be generated by substituting a cysteine ​​residue at position Q295 according to the numbering system of Kabat et al. (see, for example, U.S. Application No. 2010 / 0080794). Some embodiments may include an Fc region in which approximately 80-100% of the glycoprotein comprises a mature core carbohydrate structure lacking fructose (see, for example, U.S. Application No. 2010 / 0255013). Some embodiments may include an Fc region optimized by substitution or deletion to reduce fucosylation levels, such as increasing affinity for FcγRI, FcγRIa, or FcγRIIIa and / or improving phagocytosis by FcγRIIa-expressing cells (see U.S. Applications No. 2010 / 0249382 and 2007 / 0148170).

[0345] As another example of a modified Fc glycoform, the Fc region may include oligomannose-type N-glycans and optionally have one or more of the following: increased ADCC activity, increased binding affinity to FcγRIIIA (and certain other FcRs) relative to the corresponding Fc region containing a complex-type N-glycan or HRS-Fc conjugate, similar or increased binding specificity to targets of HRS peptides, similar or higher binding affinity to targets of HRS peptides, and / or similar or lower binding affinity to mannose receptors (see, for example, U.S. Application No. 2007 / 0092521 and U.S. Patent No. 7,700,321). As another example, engineered glycoforms produced by expressing antibodies in engineered or variant cell lines have been used to enhance the affinity of the Fc region for FcγR (see, for example, Umana et al., Nature Biotechnology 17:176-180, 1999; Davies et al., Biotechnology and Bioengineering 74:288-294, 2001; Shields et al., Journal of Biochemistry 277:26733-26740, 2002; Shinkawa et al., Journal of Biochemistry 278:3466-3473, 2003; and U.S. Application No. 2007 / 0111281). Some Fc region glycoforms include an increased proportion of N-glycosidic complex glycans that lack the first position of fucose-6-binding to N-acetylglucosamine at the reducing end of the glycan (see, for example, U.S. Application No. 2010 / 0092997). Certain embodiments may include an IgG Fc region glycosylated with at least one galactose moiety linked via an α-2,6 bond to a corresponding terminal sialic acid moiety, optionally wherein the Fc region exhibits higher anti-inflammatory activity relative to the corresponding wild-type Fc region (see U.S. Application No. 2008 / 0206246). These and related methods of altering glycosylation significantly enhance the ability of the Fc region to selectively bind FcRs (such as FcγRIII) to mediate ADCC and modify other properties of the Fc region as described herein.

[0346] The binding of certain variant Fc regions, fragment Fc regions, hybrid Fc regions, or otherwise modified Fc regions to one or more Fc receptors may be altered relative to the corresponding wild-type Fc sequence (e.g., within the same species, the same Ig class, or the same Ig subclass). For example, the binding of such Fc regions to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor may be increased relative to the corresponding wild-type Fc sequence. In some embodiments, the binding of variant Fc regions, fragment Fc regions, hybrid Fc regions, or modified Fc regions to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor may be reduced relative to the corresponding wild-type Fc sequence. Specific FcRs are described elsewhere in this document.

[0347] Specific examples of Fc variants with altered (e.g., increased, decreased) FcR binding can be found, for example, in the following literature: U.S. Patents 5,624,821 and 7,425,619; U.S. Applications 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046; and WO 2000 / 42072 and WO 2004 / 016750. Some examples contain human Fc regions with one or more substitutions at positions 298, 333, and / or 334, such as S298A, E333A, and / or K334A (based on EU index numbering by Kabat et al.), which have been shown to increase binding to the activating receptor FcγRIIIa and decrease binding to the repressive receptor FcγRIIb. These mutations can be combined to obtain double and triple mutant variants that further improve FcR binding. Some embodiments include the S298A / E333A / K334A triple mutant, which exhibits increased binding to FcγRIIIa, decreased binding to FcγRIIb, and increased ADCC (see, for example, Shields et al., Journal of Biochemistry 276:6591-6604, 2001; and Presta et al., Proceedings of the Biochem SocTrans. 30:487-490, 2002). Also see engineered Fc glycoforms with increased binding to FcR, such as those disclosed in Umana et al., ibid.; and U.S. Patent No. 7,662,925. Some embodiments include an Fc region comprising one or more substitutions selected from 434S, 252Y / 428L, 252Y / 434S, and 428L / 434S based on the EU index of Kabat et al. (see U.S. Applications No. 2009 / 0163699 and No. 20060173170).

[0348] The effector functions of certain variant Fc regions, fragment Fc regions, hybrid Fc regions, or modified Fc regions may be altered relative to the corresponding wild-type Fc sequence. For example, complement binding or activation, Clq binding affinity, CDC-related activity, ADCC-related activity, and / or ADCP-related activity may be increased relative to the corresponding wild-type Fc sequence. In some embodiments, complement binding or activation, Clq binding affinity, CDC-related activity, ADCC-related activity, and / or ADCP-related activity may be decreased relative to the corresponding wild-type Fc sequence. As an illustrative example only, the Fc region may include deletions or substitutions in complement binding sites such as the C1q binding site and / or deletions or substitutions in ADCC sites. Examples of such deletions / substitutions are described, for example, in U.S. Patent No. 7,030,226. Many Fc effector functions, such as ADCC, can be determined according to conventional techniques in the art. (See, for example, Zuckerman et al., CRC Crit RevMicrobiol. 7:1-26, 1978). Useful effector cells for this assay include, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMCs). Alternatively or additionally, certain Fc effector functions can be assessed in vivo, for example, using animal models described in Clynes et al., Proceedings of the National Academy of Sciences (PNAS) 95:652-656, 1998.

[0349] The stability or half-life of certain variant Fc regions, hybrid Fc regions, or modified Fc regions may be altered relative to the corresponding wild-type Fc sequence. In some embodiments, the half-life of such an Fc region may be increased relative to the corresponding wild-type Fc sequence. In some embodiments, the half-life of a variant Fc region, hybrid Fc region, or modified Fc region may be decreased relative to the corresponding wild-type Fc sequence. The half-life can be measured in vitro (e.g., under physiological conditions) or in vivo using conventional techniques in the art (e.g., radiolabeling, ELISA, or other methods). In vivo measurements of stability or half-life can be measured in one or more bodily fluids or given tissues, such as blood, serum, plasma, urine, or cerebrospinal fluid, and the given tissues such as liver, kidney, muscle, central nervous system tissue, bone, etc. As an example, modifications that alter the binding affinity of the Fc region to FcRn can change its in vivo half-life. For example, non-limiting examples of Fc modifications for measuring in vivo pharmacokinetic properties (e.g., mean elimination half-life) and altering the binding of Fc to FcRn are described in the following documents: U.S. Patent Nos. 7,217,797 and 7,732,570; and U.S. Applications Nos. 2010 / 0143254 and 2010 / 0143254.

[0350] Further non-limiting examples of modifications that alter stability or half-life include substitutions / deletions at one or more amino acid residues selected from amino acid residues 251-256, 285-290, and 308-314 in the CH2 domain and 385-389 and 428-436 in the CH3 domain, according to the numbering system of Kabat et al. See U.S. Application No. 2003 / 0190311. Specific examples include: substitution at position 251 with leucine; at position 252 with tyrosine, tryptophan, or phenylalanine; at position 254 with threonine or serine; at position 255 with arginine; at position 256 with glutamine, arginine, serine, threonine, or glutamic acid; at position 308 with threonine; at position 309 with proline; at position 311 with serine; at position 312 with aspartic acid; and at position 314 with leucine. The following substitutions are made: arginine, aspartic acid, or serine at position 385; threonine or proline at position 386; arginine or proline at position 387; proline, asparagine, or serine at position 389; methionine or threonine at position 428; tyrosine or phenylalanine at position 434; histidine, arginine, lysine, or serine at position 433; and / or histidine, tyrosine, arginine, or threonine at position 436, including any combination thereof. This modification optionally increases the affinity of the Fc region for FcRn relative to the corresponding wild-type Fc region and thereby increases the half-life.

[0351] The solubility of certain variant Fc regions, hybrid Fc regions, or modified Fc regions may differ relative to the corresponding wild-type Fc sequence. In some embodiments, the solubility of such Fc regions may increase relative to the corresponding wild-type Fc sequence. In some embodiments, the solubility of variant Fc regions, hybrid Fc regions, or modified Fc regions may decrease relative to the corresponding wild-type Fc sequence. Solubility can be measured in vitro (e.g., under physiological conditions), for example, according to conventional techniques in the art. Exemplary solubility measurements are described elsewhere herein.

[0352] Further examples of variants include IgG Fc regions with conserved or non-conserved substitutions (as described elsewhere herein) at one or more of positions 250, 314, or 428 of the heavy chain, or any combination thereof (e.g., at positions 250 and 428, or at positions 250 and 314, or at positions 314 and 428, or at positions 250, 314, and 428) (see, for example, U.S. Application No. 2011 / 0183412). In specific embodiments, the residue at position 250 is substituted with glutamic acid or glutamine, and / or the residue at position 428 is substituted with leucine or phenylalanine. As another illustrative example of an IgG Fc variant, any one or more amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, and / or 327 to 331 can be used as suitable targets for modification (e.g., conserved or non-conserved substitutions, deletions). In certain embodiments, the CH2 domain of the IgG Fc variant contains amino acid substitutions at positions 228, 234, 235, and / or 331 (e.g., human IgG4 with the Ser228Pro and Leu235Ala mutations) to attenuate effector function of the Fc region (see U.S. Patent No. 7,030,226). Here, the residues in the heavy chain are numbered according to the EU index (see Kabat et al., “Sequences of Proteins of Immunological Interest”, 5th edition, National Institutes of Health, Bethesda, Md. (1991)). The FcRn binding and / or serum half-life are altered (e.g., increased, decreased) in some embodiments of these and related embodiments, while effector function, optionally such as ADCC or CDC-related activity, is not reduced.

[0353] Other examples include variant Fc regions that include one or more amino acid substitutions or any combination thereof at positions 279, 341, 343, or 373 of the wild-type Fc region (see, for example, U.S. Application No. 2007 / 0224188). The wild-type amino acid residues at these positions in human IgG are valine (279), glycine (341), proline (343), and tyrosine (373). The one or more substitutions may be conserved or non-conserved, or may contain non-naturally occurring amino acids or mimics as described herein. In some embodiments, either alone or in combination with these substitutions, a variant Fc region may also be employed, the variant Fc region comprising at least one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions selected from the following: 235G, 235R, 236F, 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 245R, 247A, 247D, 247E, 247F, 247M, 247N. , 247Q, 247R, 247S, 247T, 247W, 247Y, 248F, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, 251H, 251I, 251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P, 254Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 2 56V, 256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 27 1T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279 Y, 280T, 283F, 283G, 283H, 283I, 283K, 283L, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G , 292I, 292L, 293S, 293V, 301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L, 315P, 315R, 316F, 316K, 317P, 317T, 318N, 318P,318T, 332F, 332G, 332L, 332M, 332S, 332V, 332W, 339D, 339E, 339F, 339G, 339H, 339I, 339K, 339L, 339M, 339N, 339Q, 339R, 339S, 339 W, 339Y, 341D, 341E, 341F, 341H, 341I, 341K, 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343E, 34 3F, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, 3 73L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E, 376F, 376G, 376H, 376I, 376L, 376M, 376N, 376P, 376Q, 376R, 376S 376T, 376V, 376W, 376Y, 377G, 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 380T, 382D, 382F, 382H, 382I, 382K, 382 L、382M、382N、382P、382Q、382R、382S、382T、382V、382W、382Y、38 5E、385P、386K、423N、424H、424M、424V、426D、426L、427N、429A、42 9F, 429M, 430A, 430D, 430F, 430G, 430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431K, 4 31P、432R、432S、438G、438K、438L、438T、438W、439E、439H、439Q、 440D、440E、440F、440G、440H、440I、440K、440L、440M、440Q、440T、440V or 442K. As mentioned above, the residues in the heavy chain are numbered according to the EU index (see Kabat et al., ibid.). This variant Fc region typically alters the effector function or serum half-life of the variant Fc region and the HRS peptide to which it is operatively attached. Preferably, the effector function alteration is, compared to the corresponding Fc region lacking this one or more such amino acid substitutions, an increase in ADCC, a decrease in ADCC, an increase in CDC, a decrease in CDC, an increase in Clq binding affinity, a decrease in Clq binding affinity, an increase in FcR (preferably FcRn) binding affinity, or a decrease in FcR (preferably FcRn) binding affinity.

[0354] Other examples include variant Fc regions that include amino acid substitutions at one or more of the following positions: 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 27 3, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 and / or 428 (see, for example, U.S. Patent No. 7,662,925). In specific embodiments, the variant Fc region includes at least one amino acid substitution selected from the group consisting of: P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E. K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y. In other specific embodiments, the variant Fc region includes at least one amino acid substitution selected from the group consisting of: V264I, F243L / V264I, L328M, I332E, L328M / I332E, V264I / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, A330Y, I332D, L328I / I332E, L328Q / I332E, V264T, V240I, V266I, S239D, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q, S239E / I332D.S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332 E. V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I33 2E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S 239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E, V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, S239D / V264I / A330L / I332E, S239D / I332 E / A330I, P230A, P230A / E233D / I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S32 4D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L 235D, S239D / A330Y / I332E / V240I, S239D / A330Y / I332E / V264T, S239D / A330Y / I332E / K326E and S239D / A330Y / I332E / K326T. In a more specific embodiment, the variant Fc region includes a series of substitutions selected from the group consisting of: N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E, S239D / N297D / I332E, S239E / N297D / I332E, S239D / D265Y / N297D / I332E, S239D / D265H / N297D / I332E, V264E / N297D / I332E.Y296N / N297D / I332E, N297D / A330Y / I332E, S239D / D265V / N297D / I332E, S239D / D265I / N297D / I332E, and N297D / S298A / A330Y / I332E. In specific embodiments, the variant Fc region includes an amino acid substitution at position 332 (using EU index numbering, Kabat et al., ibid.). Examples of substitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, and 332Y. The residues in the Fc region are numbered according to the EU index of Kabat et al. Among other properties described herein, this variant Fc region may exhibit increased affinity for FcγR, increased stability, and / or increased solubility compared to the corresponding wild-type Fc region.

[0355] Other examples include variant Fc regions comprising one or more of the following amino acid substitutions: 224N / Y, 225A, 228L, 230S, 239P, 240A, 241L, 243S / L / G / H / I, 244L, 246E, 247L / A, 252T, 254T / P, 258K, 261Y, 265V, 266A, 267G / N, 268N, 269K / G, 273A, 27 6D, 278H, 279M, 280N, 283G, 285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 30 5A, 306I / F, 311R, 312N, 315D / K / S, 320R, 322E, 323A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338 R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S, 352A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 3 65Q / P, 370R, 372L, 377V, 378T, 383N, 389S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F / P, 428T, 430K, 431S, 432P, 433P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, 447E, optionally wherein the variants have altered recognition of Fc ligands and / or their effector functions compared to the parental Fc polypeptide, and wherein the residues are numbered according to the EU index as described in Kabat et al.Specific examples of these and related embodiments include variant Fc regions comprising or consisting of the following substitution groups: (1) N276D, R292Q, V305A, I377V, T394A, V412A, and K439E; (2) P244L, K246E, D399G, and K409R; (3) S304G, K320R, S324T, K326E, and M358T; (4) F243S, P247L, D265V, V266A, S383N, and T411I; (5) H224N, F243L, T393A, and H433P; (6) V240A, S267 (7) G, G341E and E356G; (8) M252T, P291L, P352A, R355W, N390D, S408G, S426F and A431S; (9) P228L, T289A, L365Q, N389S and 5440G; (10) F241L, V273A, K340Q and L441F; (11) F241L, T299A, I332T and M428T; (12) E269K, Y300H, Q342R, V422I and G446A; (13) T225A, R301c, S304G, D312N, N315D, L351 S and N421S; (13) S254T, L306I, K326R and Q362L; (14) H224Y, P230S, V323A, E333D, K338R and S364C; (15) T335I, K414M and P445R; (16) T335I and K414M; (17) P247A, E258K, D280N, K288R, N297D, T299A, K322E, Q342R, S354A and L365P; (18) H268N, V279M, A339T, N361D and S426P; (19) C261Y, K29 (20) E283G, N315K, E333G, R344Q, L365P, and S442T; (21) Q347R, N361Y, and K439R; (22) S239P, S254P, S267N, H285R, N315S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E; and (23) E269G, Y278H, N325S, and K370R, wherein the residues are numbered as in the EU index as in Kabat et al. (See, for example, U.S. Application No. 2010 / 0184959).

[0356] Further specific examples of Fc variants include the Fc sequences in Table H7, where Xaa at position 1 is Ala or deleted; Xaa at position 16 is Pro or Glu; Xaa at position 17 is Phe, Val, or Ala; Xaa at position 18 is Leu, Glu, or Ala; Xaa at position 80 is Asn or Ala; and / or Xaa at position 230 is Lys or deleted (see, for example, U.S. Application No. 2007 / 0253966). These Fc regions and certain Fc regions and HRS-Fc conjugates exhibit increased half-lives, decreased effector activity, and / or significantly lower immunogenicity than wild-type Fc sequences.

[0357] The variant Fc region may also have one or more mutant hinge regions, such as those described in U.S. Application No. 2003 / 0118592. For example, one or more cysteine ​​residues in the hinge region may be missing or substituted with different amino acids. The mutant hinge region may not include cysteine ​​residues, or it may include one, two, or three fewer cysteine ​​residues than the corresponding wild-type hinge region. In some embodiments, the Fc region having this type of mutant hinge region exhibits reduced dimerization capacity relative to the wild-type Ig hinge region.

[0358] As mentioned above, the pharmacokinetic properties of HRS-Fc conjugates, such as HRS-Fc fusion proteins, are typically altered (e.g., improved, increased, or decreased) relative to their corresponding HRS peptides. Examples of pharmacokinetic properties include: stability or half-life, bioavailability (the fraction of drug absorbed), tissue distribution, volume of distribution (the apparent volume of the drug immediately after intravenous injection and after reaching equilibrium between plasma and surrounding tissues), concentration (the initial or steady-state concentration of the drug in plasma), elimination rate constant (the rate at which the drug is removed from the body), elimination rate (the rate of infusion required for equilibrium elimination), area under the curve (AUC or exposure; the integral of the concentration-time curve after a single dose or at steady state), clearance (the volume of plasma from which the drug is cleared per unit time), C max (Peak plasma concentration of the drug after oral administration), t max (reaching C) max (time) C min (The lowest concentration the drug reaches before the next administration) and fluctuations (peak-to-trough fluctuations within a single dosing interval under steady-state conditions). In some respects, these improved properties are achieved without significantly altering the secondary structure of the HRS peptide and / or reducing its atypical biological activity. In fact, some HRS-Fc conjugates exhibit increased atypical biological activity.

[0359] Therefore, in some embodiments, when administered to mammals under the same or equivalent conditions, the plasma or serum pharmacokinetic AUC curves of the HRS-Fc conjugate or HRS-Fc fusion peptide are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, or 500 times larger than those of the corresponding unmodified or differently modified HRS peptides. In some embodiments, when compared at room temperature under similar conditions, such as approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or approximately 1, 2, 3, 4 weeks in PBS at pH 7.4, the stability (e.g., measured by half-life) of the HRS-Fc conjugate or HRS-Fc fusion peptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater than that of the corresponding unmodified or differently modified HRS peptides.

[0360] In certain embodiments, the biological half-life of the HRS-Fc conjugate or HRS-Fc fusion peptide at pH 7.4, 25°C (e.g., physiological pH), and human body temperature (e.g., in vivo, in serum, in a given tissue, or in a given species such as rat, mouse, monkey, or human) is about or at least about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about 144 hours or longer, or any intermediate half-life, is as follows:

[0361] In some embodiments, following subcutaneous (SC) administration, the HRS-Fc conjugate or HRS-Fc fusion peptide exhibits higher bioavailability compared to the corresponding unmodified HRS-peptide. In some embodiments, the HRS-Fc conjugate or HRS-Fc fusion peptide has at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more bioavailability compared to the corresponding unmodified HRS peptide.

[0362] In some embodiments, as determined by UV circular dichroism analysis, the HRS-Fc fusion peptide has substantially the same secondary structure as the corresponding unmodified or differently modified HRS peptide. In some embodiments, in anti-inflammatory activity assays, the HRS-Fc fusion peptide has substantially the same activity as the corresponding unmodified or differently modified HRS peptide. In some embodiments, in anti-inflammatory activity assays, the activity of the HRS-Fc fusion peptide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times greater than the activity of the corresponding unmodified or differently modified HRS peptide.

[0363] Examples of HRS fusion proteins that regulate HRS peptide oligomerization include the fusion of HRS with cartilage oligomeric matrix protein (COMP). Therefore, some embodiments include fusion proteins comprising HRS peptides fused herein with a COMP (e.g., a pentameric domain from COMP) consisting of approximately 28-73 residues of COMP (see, for example, Prodeus et al., JCI Insight, 2017; 2(18):e94308; and Kim et al., Biochim Biophys Acta., May 2009; 1793(5):772-80). Exemplary HRS-COMP fusion proteins are provided in Table H9 below.

[0364]

[0365] Therefore, in some embodiments, the HRS peptide is fused with or otherwise conjugated to a COMP or COMP pentameric domain and comprises, consists of, or is substantially composed of the amino acid sequence in Table H9 or an active variant or fragment thereof. In some embodiments, a polynucleotide encoding an HRS peptide may be expressed, the peptide comprising, consists of, or is substantially composed of the amino acid sequence in Table H9 (e.g., SEQ ID NO: 173-175) or an active variant or fragment thereof.

[0366] In some embodiments, peptide linker sequences may be employed to separate one or more HRS peptides from one or more Fc regions, one or more PEGs, or one or more other fusion couplers to a distance sufficient to ensure that each peptide folds to its desired secondary and tertiary structures. Such peptide linker sequences can be incorporated into conjugates or fusion proteins using standard techniques well-known in the art.

[0367] Certain peptide linker sequences may be selected based on the following exemplary factors: (1) their ability to adopt flexible extended conformations; (2) their inability to adopt secondary structures that can interact with functional epitopes on the first and second peptides; (3) their physiological stability; and (4) their lack of hydrophobic or charged residues that can react with functional epitopes or other features of the peptide. See, for example, George and Heringa, J., *Protein Engineering* 15:871-879, 2002.

[0368] The length of the linker sequence can typically range from 1 to approximately 200 amino acids. Specific linkers can have the following total amino acid lengths: approximately 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1... 1, 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, 60, 70, 80, 90, 100 or more amino acids.

[0369] Peptide linkers can be any one or more naturally occurring amino acids, one or more non-naturally occurring amino acids, amino acid analogs, and / or amino acid mimics as described elsewhere herein and known in the art. Certain amino acid sequences that can be effectively used as linkers include those disclosed in: Maratea et al., *Gene* 40:39-46, 1985; Murphy et al., *Proceedings of the National Academy of Sciences of the United States of America (PNAS USA)* 83:8258-8262, 1986; U.S. Patent Nos. 4,935,233 and 4,751,180. Certain peptide linker sequences contain Gly residues, Ser residues, and / or Asn residues. Other near-neutral amino acids, such as Thr and Ala, may also be used in the peptide linker sequence if desired.

[0370] Some exemplary connectors include connectors containing Gly, Ser, and / or Asn as follows: [G] x [S] x [N]x [GS] x [GGS] x [GSS] x [GSGS] x (SEQ ID NO:201), [GGSG] x (SEQ ID NO:202), [GGGS] x (SEQ ID NO:203), [GGGGS] x (SEQ ID NO:204), [GN] x [GGN] x [GNN] x [GNGN] x (SEQ ID NO:205), [GGNG] x (SEQ ID NO:206), [GGGN] x (SEQ ID NO:207), [GGGGN] x (SEQ ID NO:208) connector, wherein x The amino acids may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to those skilled in the art.

[0371] Additional examples of linker peptides include, but are not limited to, the following amino acid sequence: Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser- (SEQ ID NO: 209); Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 210); Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 211); Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala-Ala-Ala-Arg-Asp-Ala-Ala-Ala-Lys-(SEQ ID NO:212); and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(SEQ ID NO:213).

[0372] Other non-limiting examples of adaptor peptides include DGGGS (SEQ ID NO:214); TGEKP (SEQ ID NO:215) (see, for example, Liu et al., Proceedings of the National Academy of Sciences, 94:5525-5530, 1997); GGRR (SEQ ID NO:216) (Pomerantz et al., 1995); (GGGGS) n(SEQ ID NO:204)(Kim et al., Proceedings of the National Academy of Sciences, 93:1156-1160, 1996); EGKSSGSGSESKVD (SEQ ID NO:217)(Chaudhary et al., Proceedings of the National Academy of Sciences, 87:1066-1070, 1990); KESGSVSSEQLAQFRSLD (SEQ ID NO:218)(Bird et al., Science, 242:423-426, 1988); GGRRGGGS (SEQ ID NO:219); LRQRDGERP (SEQ ID NO:220); LRQKDGGGSERP (SEQ ID NO:221); LRQKd(GGGS)2ERP (SEQ ID NO:222). In a particular embodiment, the linker sequence includes a Gly3 linker sequence containing three glycine residues. In certain embodiments, flexible adapters can be rationally designed using computer programs capable of modeling DNA binding sites and the peptides themselves (Desjarlais and Berg, Proceedings of the National Academy of Sciences, 90:2256-2260, 1993; and Proceedings of the National Academy of Sciences, 91:11099-11103, 1994) or through phage display methods.

[0373] Peptide linkers can be physiologically stable or may contain releasable linkers, such as physiologically degradable or enzymatically cleavable linkers (e.g., proteolytically cleavable linkers). In some embodiments, one or more releasable linkers can result in a shorter half-life and faster clearance of the conjugate. These and related embodiments can be used, for example, to enhance the solubility and circulatory life of HRS peptides in the bloodstream while also delivering HRS peptides into the bloodstream, whereby the peptides are substantially free of one or more Fc regions after linker degradation. These aspects are particularly useful when HRS peptides exhibit reduced activity when permanently conjugated to an Fc region. By using linkers as provided herein, such HRS peptides can retain their therapeutic activity in their conjugated form. As another example, large and relatively inert HRS-Fc conjugate peptides can be administered, which are then degraded in vivo (via degradable linkers) to produce bioactive HRS peptides with or without Fc regions. In these and other ways, the properties of HRS-Fc conjugate peptides can be more effectively tailored to balance the bioactivity and cyclic half-life of HRS peptides over time.

[0374] In certain embodiments, the linker peptide includes an autocatalytic or self-cleaving peptide cleavage site. In certain embodiments, the self-cleaving peptide comprises those polypeptide sequences obtained from potato Y virus group and cardivirus 2A peptide, FMDV (foot-and-mouth disease virus), equine rhinitis virus type A, Thosea asigna virus, and swine chezinvirus. In some embodiments, the self-cleaving peptide site includes a 2A or 2A-like site, sequence, or domain (Donnelly et al., *Journal of General Virology* 82:1027-1041, 2001). Exemplary 2A sites include the following sequences: LLNFDLLKLAGDVESNPGP (SEQ ID NO:223); TLNFDLLKLAGDVESNPGP (SEQ ID NO:224); LLKLAGDVESNPGP (SEQ ID NO:225); NFDLLKLAGDVESNPGP (SEQ ID NO:226); QLLNFDLLKLAGDVESNPGP (SEQ ID NO:226); NO:227); APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:228); VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO:229); LNFDLLKLAGDVESNPGP (SEQ ID NO:230); LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:231); and EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 232). In some embodiments, the autocatalytic peptide cleavage site includes a translation 2A signal sequence, for example, the 2A region of aphthovirus foot-and-mouth disease virus (FMDV) polyprotein, which is an 18-amino acid sequence. Further examples of usable 2A-like sequences include insect virus polyproteins, the NS34 protein of type C rotavirus, and repetitive sequences from Trypanosoma spp., as described in Donnelly et al., Journal of General Virology, 82:1027-1041, 2001.

[0375] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., J. Gener. Virol. 78:699-722, 1997; and Scymczak et al., Nature Biotech. 5:589-594, 2004). Exemplary protease cleavage sites include, but are not limited to, the following cleavage sites: potato virus Y NIa protease (e.g., tobacco spot virus protease), potato virus Y HC protease, potato virus Y P1 (P35) protease, byo virus NIa protease, byo virus RNA-2 encoded protease, stomatitis virus L protease, enterovirus 2A protease, rhinovirus 2A protease, small RNA 3C protease, cowpea mosaic virus 24K protease, nematode-transmitted polyhedral virus 24K protease, RTSV (rice donggraovirus) 3C-like protease, PYVF (European parsnip yellow spot virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage strictness, some embodiments include TEV (tobacco spot virus) protease cleavage sites, such as EXXYXQ(G / S) (SEQ ID NO:233), ENLYFQG (SEQ ID NO:234), and ENLYFQS (SEQ ID NO:235), where X represents any amino acid (TEV cleavage occurs between Q and G or between Q and S).

[0376] Further examples of enzymatically degradable linkers suitable for specific embodiments include, but are not limited to, amino acid sequences cleaved by serine proteases (such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin). Illustrative examples of thrombin-cleavable amino acids include, but are not limited to: -Gly-Arg-Gly-Asp- (SEQ ID NO:236), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro- (SEQ ID NO:237), -Gly-Arg-Gly-Asp-Ser- (SEQ ID NO:238), -Gly-Arg-Gly-Asp-Ser-Pro-Lys- (SEQ ID NO:239), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-. Illustrative examples of amino acids that can be cleaved by elastase include, but are not limited to: -Ala-Ala-Ala-, -Ala-Ala-Pro-Val- (SEQ ID NO:240), -Ala-Ala-Pro-Leu- (SEQ ID NO:241), -Ala-Ala-Pro-Phe- (SEQ ID NO:242), -Ala-Ala-Pro-Ala- (SEQ ID NO:243), and -Ala-Tyr-Leu-Val- (SEQ ID NO:244).

[0377] The enzymatically degradable linker also contains an amino acid sequence that can be cleaved by matrix metalloproteinases (such as collagenase, lysosome, and gelatinase). Illustrative examples of amino acid sequences cleavable by matrix metalloproteinases include, but are not limited to: -Gly-Pro-Y-Gly-Pro-Z- (SEQ ID NO:245), -Gly-Pro-, Leu-Gly-Pro-Z- (SEQ ID NO:246), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO:247), and -Ala-Pro-Gly-Leu-Z- (SEQ ID NO:248), wherein Y and Z are amino acids. Illustrative examples of amino acids that collagenase can cleave include, but are not limited to: -Pro-Leu-Gly-Pro-D-Arg-Z- (SEQ ID NO:249), -Pro-Leu-Gly-Leu-Leu-Gly-Z- (SEQ ID NO:250), -Pro-Gln-Gly-Ile-Ala-Gly-Trp- (SEQ ID NO:251), -Pro-Leu-Gly-Cys(Me)-His- (SEQ ID NO:252), -Pro-Leu-Gly-Leu-Tyr-Ala- (SEQ ID NO:253), -Pro-Leu-Ala-Leu-Trp-Ala-Arg- (SEQ ID NO:254), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg- (SEQ ID NO:255), where Z is an amino acid. An illustrative example of an amino acid sequence that can be cleaved by lysosome is -Pro-Tyr-Ala-Tyr-Met-Arg- (SEQ ID NO:256); an example of an amino acid sequence that can be cleaved by gelatinase is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO:257).

[0378] The enzymatically degradable linker suitable for specific embodiments comprises an amino acid sequence that can be cleaved by angiotensin-converting enzyme, such as -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro- (SEQ ID NO:258), and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO:259).

[0379] The enzymatically degradable linkers suitable for specific embodiments contain amino acid sequences that can be degraded by cathepsin B, such as Val-Cit, Ala-Leu-Ala-Leu- (SEQ ID NO:260), Gly-Phe-Leu-Gly- (SEQ ID NO:261), and Phe-Lys.

[0380] In certain embodiments, the releasable linker has a half-life of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or longer, or any intermediate half-life, at pH 7.4, 25°C (e.g., physiological pH), and human body temperature (e.g., in vivo, in serum, in a given tissue). Those skilled in the art will understand that the half-life of the HRS-Fc conjugate peptide can be finely tailored by using specific releasable linkers.

[0381] However, in some embodiments, any one or more peptide linkers in the peptide linker are optional. For example, when the first and second peptides have non-essential N-terminal and / or C-terminal amino acid regions that can be used to separate functional domains and prevent steric interference, a linker sequence may not be required.

[0382] HRS peptides and polynucleotides, such as expressible polynucleotides, can be used in any of the compositions, methods, and / or kits described herein.

[0383] Neurociliin-2 polypeptide and NRP2 ligand

[0384] Embodiments of this disclosure relate to the discovery that human histyl-tRNA synthetase (HRS) peptides possess unexpected biological properties associated with the treatment of a wide range of diseases and symptoms, and that some of these properties are related to the interaction between HRS and human neuropilin 2 (NRP2). Therefore, HRS peptides can be used as a monotherapy in the treatment of diseases (e.g., NRP2-related diseases) and / or in combination with other agents to address a range of diseases and symptoms related to neuropilin-2 biology.

[0385] NRP2 is a single-transmembrane receptor whose main extracellular region contains two CUB domains (a1 / a2 combined domain), a bifactor V / VIII homologous domain (b1 / b2 combined domain), and a MAM domain (c domain) (see [link to relevant documentation]). Figure 1A-1BThe a1a2 combinatorial domain interacts with the sema region of the brachiopancreatin, and the b1 domain interacts with the PSI and Ig-like domains of the brachiopancreatin. NRP2 has a high affinity for SEMA3F and 3G; in contrast, SEMA3A, 3B, and 3E preferentially interact with NRP1. Both NRP1 and NRP2 have similar affinity for SEMA 3C. The b1b2 combinatorial domain interacts with several growth factors containing heparin-binding domains, including VEGF C and D, placental growth factor (PIGF)-2, fibroblast growth factor (FGF), galactagogue, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF)-β (see, for example, Prud'homme et al., Oncotarget 3:921-939, 2012). NRP2 also interacts with various growth factor-specific receptors, and these interactions occur independently of binding to SEMA. In this context, it has been shown that integrins and growth factor receptors (such as VEGF receptor, TGF-β receptor, c-Met, EGFR, FGFR, and PDGFR) interact with NRP and generally appear to increase the affinity of each ligand for its receptor and modulate downstream signaling. The c-domain (Mam) appears not to be essential for ligand binding but appears to be essential for signaling.

[0386] NRP2 can form homodimers and heterodimers with other cell surface receptors and co-receptors, and is highly glycosylated. NRP2 also exists in various splice variants, ranging in length from approximately 551 to 926 amino acids. The two main variants of NRP2 are classified as NRP2a and NRP2b. They differ in their intracellular C-terminal portion (see [link to NRP2]). Figure 1A-1B For NRP2a, the C-terminal domain comprises 42 amino acids and a PDZ-binding domain having a C-terminal SEA amino acid sequence. In contrast, NRP2b comprises a 46-amino acid C-terminal domain that shares approximately 11% of the intracellular and transmembrane sequence of NRP2a. Further splicing can occur between the MAM domain and the transmembrane domain, and five additional amino acids (GENFK) can be added to either the NRP2a or NRP2b form—these variants are named according to the number of additional amino acids added via alternative splicing. Thus, two additional variants of NRP2 are named NRP2a(17) and NRP2a(22), and two distinct transmembrane variants of NRP2b are named NRP2b(0) and NRP2b(5). Additionally, a soluble form called sNRP2b can also be produced (see [link to NRP2b]). Figure 2The terms “neurociliin-2” or “NRP2” or “NRP2 polypeptide” refer to all isotypes, splice variants, and naturally occurring fragments of NRP2, unless the context clearly indicates a different specific meaning. Exemplary NRP2 polypeptide sequences are provided in Table N1 below.

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395] Neurocilia-2 can respond to a variety of ligands by recruiting different co-receptors to regulate a wide range of cellular functions through its role as an essential cell surface receptor and a co-receptor for various ligands (see, for example, Guo and Vander Kooi, J. Cell. Biol. 290, 49: 29120-29126. 2015; Prud'homme et al., Tumor Targets 3: 921-939, 2012). For example, NRP2 plays a role during epithelial-mesenchymal transition (EMT) by promoting TGF-β1-mediated EMT in colorectal cancer cells and other cancer cells (see, for example, Grandclement et al., PLOS 6(7)e20444, 2011) and by mediating EMT or endo-EMT in fibroblasts, myofibroblasts and endothelial cells to promote fibrosis formation (see, for example, Pardali et al., Int. J. Mol. Sci. 18 2157 2017).

[0396] Neurociliin-2 expression also promotes lymphangiogenesis and modulates vascular permeability (see, for example, Doci et al., Cancer Research 75(14) 2937-2948, 2015; Mucka et al., American Journal of Pathology 186(11) 2803-2812 2016), and single nucleotide polymorphisms (SNPs) in NRP2 are associated with lymphedema (see, for example, Miaskowski et al., PLOS 8(4)e60164, 2013). NRP2 also regulates smooth muscle contractility (see, for example, Bielenberg et al., American Journal of Pathology 181:548-559, 2012), and is highly expressed in developing skeletal muscle (see, for example, Meye et al., PLOS DOI.10.1371 / journal.pone.0139520 2015).

[0397] Neurocilia are also multifunctional co-receptors involved in tumor initiation, growth, metastasis, lymphangiogenesis, lymphatic metastasis, and tumor immune surveillance, thereby directly contributing to tumor initiation, survival, and metastasis (see, for example, Goel et al., EMBO Mol Med 5:488-508, 2013; Cao et al., Can. Res. 73(14)4579-4590 2013; Tu et al., Oncol. Lett. 12 4224-4230, 2016; Samuel et al., PLOS 6(10)e23208, 2011).

[0398] Neurociliin-2 is expressed in various cells of the immune system, such as B cells, T cells, NK cells, neutrophils, dendritic cells, and macrophages, including alveolar macrophages, and plays an important role in the regulation of immune cell activation and migration (see, for example, Mendes-da-Cruz et al., PLOS 9(7)e103405, 2014), including the regulation of endosome maturation, autophagy, and burial (see, for example, Stanton et al., Cancer Research 73:160-171, 2013; Schellenburg et al., Molecular Immunology 90:239-244, 2017; Wang et al., Cancer Letters 418 176-184 2018).

[0399] NRP2 is also expressed in lung endothelial and epithelial cells, as well as in other tissues and cell types containing bone osteoclasts and muscle cells [see, for example, Bielenberg et al., American Journal of Pathology 181:548-559, 2012; Aung et al., PLOS ONE 11(2)e0147358, 2016; and Wild et al., International Journal of Experimental Pathology 93:81-103, 2012].

[0400] Neurocilia protein-2 also plays an active role in neuronal development, and in adults, NRP2 actively participates in peripheral nerve growth and remodeling and plays a role in pain perception in inflammatory conditions such as arthritis, osteoarthritis and rheumatoid arthritis (see, for example, Hamilton, J et al., Journal of Bone & Mineral Research, 2016 31(5)911-924; Bannerman, P et al., Journal of Neuroscience Research, 2008 86(14)3163-3169; Malykhina, A et al., BMC Physiology, 2012, 12, 15).

[0401] It is becoming increasingly clear that neurocilia-2 also plays a crucial role in endosome development and regulates late endosome maturation. These processes play important roles in phagocytosis and burial, which are key to clearing infected and apoptotic cells, respectively (see, for example, Diaz-Vera et al., J. Cell Sci. 130, 697-711 2017; Dutta et al., Cancer Research 76(2) 418-428 2016).

[0402] Neurocilia protein-2 is known to be a key factor in the pathophysiology of many diseases (“NRP2-related diseases”), and is associated with substances such as brachioside 3F, VEGF-C and D, and TGF-β, and contains a large number of cellular receptors and cofactors (“NRP2 ligands”) (see, for example, Tables N2, N3, and N2 below). Figure 1A-1B The interactions of various soluble ligands.

[0403]

[0404]

[0405]

[0406] NRP2 is also polysialylated on dendritic cells and actively interacts with the chemokine CCL21 to mediate immune cell migration via the CCR7 receptor. Single nucleotide polymorphisms in NRP2 associated with ILD and RA have been described for this purpose (see, for example, Rey-Gallardo et al., Glycobiology 20:1139-1146, 2010; Stahl et al., Nature Genetics 42:508-514, 2013; Miller et al., Arthritis Rheum 65:3239-3247). In addition, the soluble circulating form of NRP2 is known (see, for example, Parker et al., Structure 23(4):677-687, 2015), and internal studies have confirmed the presence of circulating complexes of HRS peptides and NRP2 peptides in serum and other fluids.

[0407] Given the central role of NRP2 in pathophysiology, it is evident that the interaction between NRP2 and HRS peptides offers potential for the treatment of diseases, including NRP2-related diseases. Therefore, the HRS peptides described herein, by selectively modulating the interaction of NRP2 with one or more ligands listed in Tables N2 and N3, can be used to treat a wide range of diseases and conditions as described herein.

[0408] Other therapeutic agents and compositions

[0409] Immunotherapy agents. Some embodiments employ one or more cancer immunotherapy agents. In certain cases, immunotherapy agents modulate a subject's immune response, for example, to increase or maintain a cancer-related or cancer-specific immune response, thereby increasing immune cell suppression or reducing cancer cells. Exemplary immunotherapy agents comprise peptides, such as antibodies and their antigen-binding fragments, ligands, and small peptides, and mixtures thereof. Immunotherapy agents also comprise small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapy agents, or other polynucleotide-based agents, including viral agents such as oncolytic viruses, and other agents known in the art. Thus, in some embodiments, the cancer immunotherapy agent is selected from one or more of immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies.

[0410] In some embodiments, cancer immunotherapeutic agents are immune checkpoint modulators. Specific examples include “antagonists” of one or more inhibitory immune checkpoint molecules and “agonists” of one or more stimulating immune checkpoint molecules. Typically, immune checkpoint molecules are components of enhancing (co-stimulatory) or de-stimulating signals of the immune system, and targeting these components has therapeutic potential for cancer because cancer cells may disrupt the natural function of immune checkpoint molecules (see, for example, Sharma and Allison, Science. 348:56-61, 2015; Topalian et al., Cancer Cell. 27:450-461, 2015; Pardoll, Nature Reviews Cancer. 12:252-264, 2012). In some embodiments, immune checkpoint modulators (e.g., antagonists, agonists) “bind” or “specifically bind” to one or more immune checkpoint molecules as described herein.

[0411] In certain embodiments, the immune checkpoint modulator is a polypeptide or peptide. The terms "peptide" and "polypeptide" are used interchangeably herein; however, in some cases, the term "peptide" may refer to a shorter polypeptide, such as a polypeptide consisting of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, encompassing all integers and ranges therebetween (e.g., 5-10, 8-12, 10-15). Polypeptides and peptides may consist of naturally occurring and / or non-naturally occurring amino acids as described herein.

[0412] Antibodies are also included as peptides. Therefore, in some embodiments, immune checkpoint regulatory peptide agents are antibodies or "their antigen-binding fragments" as described elsewhere herein.

[0413] In some embodiments, the agent is or includes a "ligand" of an immune checkpoint molecule, such as a naturally occurring ligand. A "ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) for biological purposes and includes a "protein ligand" that typically generates a signal by binding to a site on a target molecule or target protein. Therefore, some agents are protein ligands that naturally bind to immune checkpoint molecules and generate a signal. "Modified ligands" are also included, for example, protein ligands fused with a pharmacokinetic modifier, such as a protein ligand derived from the Fc region of an immunoglobulin.

[0414] The binding properties of peptides can be quantified using methods well known in the art (see Davies et al., *Annual Review of Biochemistry* 59:439-473, 1990). In some embodiments, the peptide binds specifically to a target molecule (e.g., an immune checkpoint molecule or its epitope) with an equilibrium dissociation constant of about ≤10⁻⁷ M to about 10⁻⁸ M. In some embodiments, the equilibrium dissociation constant is about ≤10⁻⁹ M to about ≤10⁻¹⁰ M. In some illustrative embodiments, the affinity (Kd or EC) of the peptide for the target described herein (to which the peptide specifically binds) is also considered. 50 The value is approximately, at least approximately, or less than approximately 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 40 nM, or 50 nM.

[0415] In some embodiments, the agent is a "small molecule," which refers to an organic compound having a synthetic or biological origin (biomolecule) but is not typically a polymer. Organic compounds refer to a broad class of compounds whose molecules contain carbon, typically excluding compounds containing only carbonates, simple carbon oxides, or cyanides. "Biomolecule" generally refers to organic molecules produced by living organisms, including large polymers (biopolymers) such as peptides, polysaccharides, and nucleic acids, as well as small molecules such as primary and secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerides, vitamins, and hormones. "Polymer" generally refers to a large molecule or macromolecule composed of repeating structural units, typically linked by covalent chemical bonds.

[0416] In some embodiments, the molecular weight of the small molecule is about or less than about 1,000 Daltons to 2,000 Daltons, typically between about 300 Daltons and 700 Daltons, and includes about or less than about 50 Daltons, 100 Daltons, 150 Daltons, 200 Daltons, 250 Daltons, 300 Daltons, 350 Daltons, 400 Daltons, 450 Daltons, 500 Daltons, 550 Daltons, 500 Daltons, 650 Daltons, 600 Daltons, 750 Daltons, 700 Daltons, 850 Daltons, 800 Daltons, 950 Daltons, 1,000 Daltons, or 2,000 Daltons.

[0417] Some small molecules may possess the “specific binding” properties described herein for peptides, such as antibodies. For example, in some embodiments, the small molecules have a binding affinity of about, at least about, or less than about [amount missing] (Kd or EC). 50 It specifically binds to targets, such as immune checkpoint molecules, at concentrations of 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 40 nM, or 50 nM.

[0418] In some embodiments, an immune checkpoint modulator is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include: programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), programmed death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), T-cell activation V domain Ig inhibitor (VISTA), B and T lymphocyte attenuator (BTLA), CD160, and a T-cell immune receptor having Ig and ITIM domains (TIGIT).

[0419] In some embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, which has been shown to target the restoration of immune function in the tumor setting (see, for example, Phillips et al., *International Journal of Immunology* 27:39-46, 2015). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and expressed on T cells and progenitor B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 acts as a suppressive immune checkpoint molecule, for example, by reducing or preventing T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is achieved at least in part through a dual mechanism of promoting apoptosis of antigen-specific T cells in lymph nodes while also reducing apoptosis of regulatory T cells (suppressive T cells). Some examples of PD-1 antagonists or inhibitors comprise antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1 and reduce one or more of its immunosuppressive activities, such as its downstream signaling or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab and their antigen-binding fragments (see, for example, U.S. Patents 8,008,449, 8,993,731, 9,073,994, 9,084,776, 9,102,727, 9,102,728, 9,181,342, 9,217,034, 9,387,247, 9,492,539, 9,492,540 and U.S. Applications 2012 / 0039906 and 2015 / 0203579).

[0420] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As described above, PD-L1 is one of the natural ligands of the PD-1 receptor. General examples of PD-L1 antagonists or inhibitors comprise antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and reduce one or more immunosuppressive activities, such as their binding to the PD-1 receptor. Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avelumumab (MSB0010718C), and durvalumab (MEDI4736) and their antigen-binding fragments (see, for example, U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; and 9,439,962).

[0421] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As described above, PD-L2 is one of the natural ligands of the PD-1 receptor. General examples of PD-L2 antagonists or inhibitors comprise antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L2 and reduce one or more immunosuppressive activities (e.g., their binding to the PD-1 receptor).

[0422] In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. CTLA4, or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (differentiation cluster 152), is a protein receptor that acts as an inhibitory immune checkpoint molecule, for example, by transmitting an inhibitory signal to T cells upon binding to CD80 or CD86 on the surface of antigen-presenting cells. General examples of CTLA-4 antagonists or inhibitors comprise antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Specific examples include the antibodies ipilimumab and trimemumab, and their antigen-binding fragments. It is believed that at least some of the activity of ipilimumab is mediated by antibody-dependent cell-mediated cytotoxic (ADCC) killing by cells expressing the CTLA-4 inhibitory factor Treg.

[0423] In some embodiments, the agent is an IDO antagonist or inhibitor or a TDO antagonist or inhibitor. IDOs and TDOs are tryptophan-catabolizing enzymes with immunosuppressive properties. For example, IDOs are known to inhibit T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. General examples of IDO and TDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDOs or TDOs (see, for example, Platten et al., Frontiers in Immunology 5:673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indomod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norhalman, 9H-pyrido[3,4-b]indole), rosmarinic acid, and icardolstat (see, for example, Sheridan, Nature Biotechnology 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, for example, Pilotte et al., Proceedings of the National Academy of Sciences 109:2497-2502, 2012).

[0424] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by triggering cell death upon interaction with its ligand, galactolectin-9. TIM-3 contributes to a suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (see, for example, Li et al., *Acta Oncol.* 54:1706-13, 2015). General examples of TIM-3 antagonists or inhibitors comprise antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more of its immunosuppressive activities.

[0425] In some embodiments, the agent is a LAG-3 antagonist or inhibitor. Lymphocyte activation gene-3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates T cell proliferation, activation, and homeostasis in a manner similar to that of CTLA-4 and PD-1 (see, for example, Workman and Vignali, *European Journal of Cancer* 33:970-9, 2003; and Workman et al., *Journal of Immunology* 172:5450–5, 2004), and has been reported to play a role in Treg suppression (see, for example, Huang et al., *Immunity* 21:503-13, 2004). LAG3 also keeps CD8+ T cells in a tolerogenic state and, in combination with PD-1, maintains CD8+ T cell exhaustion. General examples of LAG-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include antibody BMS-986016 and its antigen-binding fragment.

[0426] In some embodiments, the agent is a VISTA antagonist or inhibitor. T-cell activation V-domain Ig inhibitors (VISTA) are primarily expressed on hematopoietic cells and are inhibitory immune checkpoint modulators that inhibit T-cell activation, induce Foxp3 expression, and are highly expressed within the tumor microenvironment, where they suppress anti-tumor T-cell responses (see, for example, Lines et al., Cancer Research 74:1924-32, 2014). General examples of VISTA antagonists or inhibitors comprise antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA and reduce one or more of its immunosuppressive activities.

[0427] In some embodiments, the agent is a BTLA antagonist or inhibitor. B and T lymphocyte attenuator (BTLA; CD272) expression is induced during T cell activation, and it inhibits T cells by interacting with the tumor necrosis family receptor (TNF-R) and the B7 cell surface receptor family. BTLA is a member of the tumor necrosis factor (receptor) superfamily 14 (TNFRSF14), also known as a ligand for herpesvirus entry mediators (HVEM). The BTLA-HVEM complex negatively regulates T cell immune responses, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, for example, Derré et al., *Journal of Clinical Investigation (J ClinInvest)* 120:157–67, 2009). General examples of BTLA antagonists or inhibitors comprise antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

[0428] In some embodiments, the agent is an HVEM antagonist or inhibitor, such as an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD160. General examples of HVEM antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM, optionally reducing HVEM / BTLA and / or HVEM / CD160 interactions and thereby reducing one or more immunosuppressive activities of HVEM.

[0429] In some embodiments, the agent is a CD160 antagonist or inhibitor, such as an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. General examples of CD160 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CD160, optionally reduce CD160 / HVEM interaction, and thereby reduce or inhibit one or more of their immunosuppressive activities.

[0430] In some embodiments, the agent is a TIGIT antagonist or inhibitor. The T-cell Ig and ITIM domains (TIGIT) are co-inhibitory receptors found on the surface of various lymphocytes and, for example, inhibit antitumor immunity via Tregs (Kurtulus et al., *Journal of Clinical Investigation* 125:4053-4062, 2015). General examples of TIGIT antagonists or inhibitors comprise one or more antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT and reduce its immunosuppressive activity (see, for example, Johnston et al., *Cancer Cells* 26:923-37, 2014).

[0431] In some embodiments, the immune checkpoint modulator is an agonist of one or more stimulating immune checkpoint molecules. Exemplary stimulating immune checkpoint molecules include OX40, CD40, glucocorticoid-induced TNFR family-associated gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0432] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the expansion of effector T cells and memory T cells and inhibits the differentiation and activity of regulatory T cells (see, for example, Croft et al., *Immunological Reviews* 229:173-91, 2009). Its ligand is OX40L (CD252). Because OX40 signaling affects both T cell activation and survival, it plays an important role in initiating antitumor immune responses in lymph nodes and maintaining antitumor immune responses in the tumor microenvironment. General examples of OX40 agonists comprise antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and increase one or more of its immunostimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (mouse OX4 agonist), and MEDI6383 (OX40 agonist) and their antigen-binding fragments.

[0433] In some embodiments, the agent is a CD40 agonist. CD40 is expressed on antigen-presenting cells (APCs) and some malignancies. Its ligand is CD40L (CD154). On APCs, this linkage induces upregulation of co-stimulatory molecules, potentially bypassing the need for T-cell help in antitumor immune responses. CD40 agonist therapy plays an important role in APC maturation and their migration from tumors to lymph nodes, thereby enhancing antigen presentation and T-cell activation. Anti-CD40 agonist antibodies generate substantial responses and durable anticancer immunity in animal models, effects at least partially mediated by cytotoxic T cells (see, for example, Johnson et al., *Clinical Cancer Research* 21:1321-1328, 2015; and Vonderheide and Glennie, *Clinical Cancer Research* 19:1035-43, 2013). General examples of CD40 agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to CD40 and increase one or more of its immunostimulatory activities. Specific examples include CP-870, 893, dasizumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L, and their antigen-binding fragments.

[0434] In some embodiments, the agent is a GITR agonist. Glucocorticoid-induced TNFR family-associated genes (GITRs) increase T cell proliferation, inhibit the suppressive activity of Tregs, and prolong the survival of T effector daughter cells. GITR agonists have been shown to promote antitumor responses through loss of Treg lineage stability (see, for example, Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These different mechanisms suggest that GITRs play an important role in initiating immune responses in lymph nodes and maintaining immune responses in tumor tissues. Its ligand is GITRL. General examples of GITR agonists comprise antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITRs and increase one or more of their immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and their antigen-binding fragments.

[0435] In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. General examples of CD137 agonists comprise antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and increase one or more of its immunostimulatory activities. Specific examples include CD137 (or 4-1BB) ligands (see, for example, Shao and Schwarz, *J Leukoc Biol.* 89:21-9, 2011) and the antibody utomilumab, which comprises its antigen-binding fragment.

[0436] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific amplification of naïve T cells and contributes to the long-term maintenance of T cell memory and T cell immunity. Its ligand is CD70. Targeting human CD27 with an agonist antibody stimulates T cell activation and anti-tumor immunity (see, for example, Thomas et al., *Oncoimmunology*. 2014; 3:e27255. doi:10.4161 / onci.27255; and He et al., *Journal of Immunology* 191:4174-83, 2013). General examples of CD27 agonists comprise antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27 and increase one or more of its immunostimulatory activities. Specific examples include CD70 and the antibodies valilumab and CDX-1127(1F5), which contain their antigen-binding fragments.

[0437] In some embodiments, the agent is a CD28 agonist. CD28 is a constitutively expressed CD4+ T cell, some CD8+ T cells. Its ligands include CD80 and CD86, and its stimulation increases T cell proliferation. General examples of CD28 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and increase one or more of their immunostimulatory activities. Specific examples include CD80, CD86, antibody TAB08, and its antigen-binding fragment.

[0438] In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and unlike TIGIT, binding to CD226 enhances T cell activation (see, for example, Kurtulus et al., *Journal of Clinical Investigation* 125:4053-4062, 2015; Bottino et al., *Journal of Experimental Medicine* 1984:557-567, 2003; and Tahara-Hanaoka et al., *International Journal of Immunology* 16:533-538, 2004). General examples of CD226 agonists comprise antibodies or antigen-binding fragments or small molecules or ligands (e.g., CD112, CD155) that specifically bind to CD226 and increase one or more of its immunostimulatory activities.

[0439] In some embodiments, the agent is an HVEM agonist. Herpesvirus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF receptor superfamily. HVEM is found on a variety of cells including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is highly expressed on resting T cells and downregulated upon activation. HVEM signaling has been shown to play an important role in the early stages of T cell activation and during the expansion of tumor-specific lymphocyte populations in lymph nodes. General examples of HVEM agonists comprise antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM and increase one or more of its immunostimulatory activities.

[0440] In some embodiments, the cancer immunotherapy agent is a cancer vaccine. Exemplary cancer vaccines include oncophage, such as Gardex or Cervarix for human papillomavirus (HPV), hepatitis B vaccines such as Ambroxol-B, Recombivax HB, or Twinrix, and Ciproluca-T (Provinci). In some embodiments, the cancer vaccine includes or utilizes one or more cancer antigens or cancer-associated antigens. Exemplary cancer antigens include, but are not limited to: human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, VEGR-3, NRP2, CD30, C D33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tendinin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclase C, NY-ES O-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activating protein α (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PSMA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM Family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialyl ganglioside expressed on neuroectodermal tumors), phosphatidylinositol proteoglycan-3 (GPC3), and mesothelin.

[0441] In some embodiments, the cancer immunotherapy agent is an oncolytic virus. Oncolytic viruses are viruses that preferentially infect and kill cancer cells. They include naturally occurring and artificial or engineered oncolytic viruses. Most oncolytic viruses are engineered for tumor selectivity, but naturally occurring examples exist, such as reovirus and SVV-001 Seneca Valley virus. Common examples of oncolytic viruses include VSV, poliovirus, reovirus, Senecavirus, and RIGVIR, as well as their engineered versions. Non-limiting examples of oncolytic viruses include herpes simplex virus (HSV) and its engineered version, Talilach (T-VEC), and Coxsackievirus A21 (CAVATAK). TM ), Ancore (H101), Perareorepp Seneca Valley Virus (NTX-010), Seneca Virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401, etc.

[0442] In some embodiments, the cancer immunotherapy agent is a cytokine. Exemplary cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0443] In some embodiments, the cancer immunotherapy agent is a cell-based immunotherapy, such as T-cell-based adoptive immunotherapy. In some embodiments, the cell-based immunotherapy includes cancer antigen-specific T cells, optionally ex vivo T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells. In a specific embodiment, the CAR-modified T cells target CD-19 (see, for example, Maude et al., Blood 125:4017-4023, 2015).

[0444] In some cases, the cancer to be treated is associated with a cancer antigen; that is, cancer antigen-specific T cells target or are enriched against at least one antigen known to be associated with the cancer to be treated. In some embodiments, the cancer antigen is selected from one or more of the following: CD19, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD3... 3. CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tendinin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclase C, NY-ESO -1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activating protein α (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM Family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialyl ganglioside expressed on neuroectodermal tumors), phosphatidylinositol proteoglycan-3 (GPC3), and mesothelin.

[0445] Other exemplary cancer antigens include: 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, α-5-β-1-integrin, α-5-β-6-integrin, α-actin-4 / m, α-methylacyl-CoA racemicase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, β-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA 15-3 / CA 27-29, CA19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, trichomeoid protein, collagen XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAG E-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, hepsin, Her2 / neu, HERV-K-MEL, HLA-A*0201-R1 7I, HLA-A11 / m, HLA-A2 / m, HNE, homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAG E-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C 2. MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MCI R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP11, MN / CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, type I myosin / m, NA88-A, N-acetylglucosamine aminotransferase-V, neo-PAP, neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-B, NY-ESO-1, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, osteocalcin, osteopontin, pi 5. p190 small bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 kinase, Pin-1, Pml / PARα, POTE, PRAME, PRDX5 / m, prostein, protease-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD1 68, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp17. SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, Survivin, Survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF-β, TGFβRII, TGM-4, TPI / m, TRAG-3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, Tyrosinase, UPA, VEGFR1, VEGFR-2 / FLK-1, and WT1. Certain preferred antigens include p53, CA125, EGFR, Her2 / neu, hTERT, PAP, MAGE-A1, MAGE-A3, mesothelin, MUC-1, GP100, MART-1, tyrosinase, PSA, PSCA, PSMA, STEAP-1, Ras, CEA, and WT1, with PAP, MAGE-A3, WT1, and MUC-1 being more preferred.

[0446] In some embodiments, the antigen is selected from: MAGE-A1 (e.g., MAGE-A1 according to accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g., MAGE-A6 according to accession number NM_005363), MAGE-C1, MAGE-C2, melan-A (e.g., melan-A according to accession number NM_005511), GP100 (e.g., GP100 according to accession number M77348), tyrosinase (e.g., tyrosinase according to accession number NM_000372), survivin (e.g., according to accession number AF0773). 50% survivin), CEA (e.g., CEA according to accession number NM_004363), Her-2 / neu (e.g., Her-2 / neu according to accession number M11730), WT1 (e.g., WT1 according to accession number NM_000378), PRAME (e.g., PRAME according to accession number NM_006115), EGFRI (epidermal growth factor receptor 1) (e.g., EGFRI (epidermal growth factor receptor 1) according to accession number AF288738), MUC1, mucin-1 (e.g., mucin-1 according to accession number NM_002456), SEC61 G (e.g., SEC61 G according to accession number NM_014302), hTERT (e.g., hTERT accession number NM_198253), 5T4 (e.g., 5T4 according to accession number NM_006670), TRP-2 (e.g., TRP-2 according to accession number NM_001922), STEAP1 (six transmembrane prostatic epithelial antigen 1), PSCA, PSA, PSMA, etc.

[0447] In some embodiments, the cancer antigen is selected from PCA, PSA, PSMA, STEAP, and optionally MUC-1, including fragments, variants, and derivatives thereof. In some embodiments, the cancer antigen is selected from NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and optionally MUC-1, including fragments, variants, and derivatives thereof.

[0448] In some cases, cancer antigens encompass unique antigens associated with cancer or tumor diseases, particularly lymphoma or lymphoma-related diseases, where the unique antigen is an immunoglobulin unique type of lymphocytes or a T-cell receptor unique type of lymphocytes.

[0449] In some cases, cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR) modified T cells (e.g., targeting cancer antigens) and T cell receptor (TCR) modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0450] Those skilled in the art will understand that the various cancer immunotherapy agents described herein can be combined with any one or more of the various HRS peptides described herein and used according to any one or more of the methods or compositions described herein.

[0451] Chemotherapy agents. Some embodiments employ one or more chemotherapeutic agents, such as small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or II), antimicrotubule agents, etc.

[0452] Examples of alkylating agents include: nitrogen mustard (e.g., dichloromethyldiethylamine, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), formustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolamide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, and diazinon (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and atypical alkylating agents (optionally dipropylcarbazine and hexamethylmelamine).

[0453] Examples of antimetabolites include: antifolate agents (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enoxatabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0454] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, arubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, neomycin, meparone, and arubicin.

[0455] Examples of antimicrotubule agents include taxanes (e.g., paclitaxel and docetaxel) and vinblastine alkaloids (e.g., vincristine, vinorelbine, vinorelbine, vinorelbine).

[0456] Those skilled in the art will understand that the various chemotherapeutic agents described herein can be combined with any one or more of the various HRS peptides described herein and used according to any one or more of the methods or compositions described herein.

[0457] Hormone therapeutic agents. Some embodiments employ at least one hormone therapeutic agent. General examples of hormone therapeutic agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include: progestins (progesterone), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galactagogue, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, androgens, estrogens, and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors, such as aromatase inhibitors and gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), including their analogues. Also included are hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs, e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0458] It also includes inhibitors of hormonal pathways, such as antibodies targeting hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as cetuximab, dotuzumab, fentuximab, ganituzumab, isotuximab, and rotutuzumab; inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as peacecilizumab, bevacizumab, irukumab, and ramucirumab; and inhibitors of TGF-β receptors R1, R2, and R3, such as fusomumab and metilumab. Inhibitors of c-Met, such as nalcitumab; inhibitors of EGF receptors, such as cetuximab, mofotinide pertuximab, vortuximab, imatroximab, endostatin raltuximab, matezumab, mofotinide, neximetumab, nimotuzumab, panitumumab, toltuximab, zalumab; inhibitors of FGF receptors, such as ixadodine aprastatumab and bemarituzumab; and inhibitors of PDGF receptors, such as olarumab or tovertumab.

[0459] Those skilled in the art will understand that the various hormone therapeutic agents described herein can be combined with any one or more of the various HRS peptides described herein and used according to any one or more of the methods or compositions described herein.

[0460] Kinase inhibitors. Some embodiments employ at least one kinase inhibitor, including tyrosine kinase inhibitors and phosphoinositol 3 (PI3) kinase inhibitors. Examples of kinase inhibitors include, but are not limited to: adastotinib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobitinib, crizotinib, dasatinib, entrectinib, erdatinib, erlotinib, fortaminophen, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mulitinib, nilotinib, panitumumab, pazopanib, pilgatanib, panatinib, lanibimumab, regorafenib, ruxotinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib. Exemplary PI3 kinase inhibitors include: aperidilxicet, bupalixicet, cupanlixicet, CUDC-907, dattalixicet, davilixicet, GNE-477, edaraxicet, IPI-549, LY294002, ME-401, perifoxine, PI-103, picricil, PWT33597, RP6503, taselexicet, umbalixicet, votalixicet, wouma penicillin, and XL147.

[0461] Those skilled in the art will understand that the various kinase inhibitors described herein can be combined with any one or more of the various HRS peptides described herein and used according to any one or more of the methods or compositions described herein.

[0462] How to use

[0463] Some embodiments include methods for treating a disease or condition in a subject of need, improving symptoms of said disease or condition, and / or reducing the progression of said disease or condition, said methods including administering an HRS peptide to the subject. In some cases, the HRS peptide specifically binds to a human neuropilin-2 (NRP2) peptide (see Table N1). In some cases, the HRS peptide interferes with the binding of the human NRP2 peptide to an NRP2 ligand. In some cases, the HRS peptide mimics one or more signaling activities of the NRP2 ligand binding to the NRP2 peptide, for example, by acting as an agonist, partial agonist, antagonist, partial agonist, or inverse agonist of the NRP2 ligand. Exemplary NRP2 ligands are provided in Tables N2 and N3.

[0464] In some embodiments, the subject in need suffers from NRP2-related diseases or conditions. Exemplary NRP2-related diseases and conditions include, but are not limited to, cancer and cancer-related diseases or pathologies, including cancer cell growth, cancer initiation, cancer migration, cancer cell adhesion, cancer cell invasion, cancer cell chemoresistance, and cancer cell metastasis. They also include diseases related to inflammation and autoimmunity, including inflammatory lung diseases such as chronic allergic pneumonia, lung inflammation, and related inflammatory diseases. They also include diseases associated with inappropriate immune cell activation or migration, such as graft-versus-host disease (GVHD) and rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Further examples include diseases related to lymphatic development, lymphangiogenesis, and lymphatic damage, including edema, lymphedema, secondary lymphedema, inappropriate fat uptake and deposition, excessive fat deposition, and vascular permeability. This also includes diseases associated with infection (including latent infection) and diseases related to allergic conditions / diseases and allergic responses, including chronic obstructive pulmonary disease (COPD), neutrophilic asthma, systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA), systemic lupus erythematosus, rheumatoid arthritis, one or more inflammasome-related diseases, and one or more skin-related neutrophil-mediated diseases, such as pyoderma gangrenosa. Additional examples include diseases associated with granulomatous inflammatory diseases, including sarcoidosis and other pulmonary granulomatous diseases, as well as non-pulmonary granulomatous diseases. It also includes fibrotic diseases, such as endometriosis, fibrosis, endothelial-to-mesenchymal transition (EMT), and wound healing. It also includes diseases associated with inappropriate smooth muscle contractility and vascular smooth muscle cell migration and / or adhesion, as well as diseases associated with inappropriate autophagy, phagocytosis, and burial. Additional examples include neuronal diseases, including those related to peripheral nervous system remodeling and pain perception. It also includes diseases related to bone development and / or bone remodeling, as well as diseases related to inappropriate migration of cells.

[0465] In some embodiments, the subject's extracellular fluid (e.g., circulating or serum) levels of bound or free soluble NRP2 peptides (e.g., selected from Table N1) or their encoding mRNA are increased, for example, relative to healthy or matched control criteria or a reference population consisting of one or more subjects, and / or the subject is selected for treatment based on said increase. In some embodiments, extracellular fluid levels of NRP2 are detected in serum, plasma, lymph, interstitial fluid, and / or in specific tissue compartments (containing, for example, bronchoalveolar lavage (BALF) and synovial fluid) associated with NRP2-related diseases. In some embodiments, the extracellular fluid level of the soluble NRP2 peptide is about or at least about 10 pM, 20 pM, 30 pM, 50 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1000 pM, 1100 pM, 1200 pM, 1300 pM, 1400 pM, 1500 pM, 1600 pM, 1700 pM, 1800 pM, 1900 pM, 2000 pM, 3000 pM, or 4000 pM. Soluble NRP2 peptides of 5000 pM or approximately 30-50 pM, 50-100 pM, 100-2000 pM, 200-2000 pM, 300-2000 pM, 400-2000 pM, 500-2000 pM, 600-2000 pM, 700-2000 pM, 800-2000 pM, 900-2000 pM, 1000-2000 pM, 2000-3000 pM, 3000-4000 pM, or 4000-5000 pM.

[0466] In some embodiments, the level of a subject's bound or free soluble NRP2 peptide (e.g., selected from Table N1) or its encoding mRNA is increased relative to the level of non-disease control cells or tissues (e.g., non-disease control cells or tissues of the same type as NRP2-related disease cells or tissues), and / or the subject is selected for treatment based on said increase. For example, in some embodiments, the level of soluble NRP2 peptide (or its encoding mRNA) in NRP2-related disease cells or tissues is about or at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more than the level in non-disease control cells or tissues. Therefore, some embodiments include a method for selecting a subject for treatment, the method comprising: (i) determining the extracellular fluid level of the subject's soluble NRP2 peptide and / or its encoding mRNA relative to a control or reference extracellular fluid level; and (ii) administering to the subject a therapeutic composition comprising at least one HRS peptide as described herein if the subject's soluble NRP2 peptide and / or its encoding mRNA level is increased relative to a control or reference level.

[0467] In some embodiments, the extracellular fluid level of the subject's NRP2 ligand or the extracellular fluid level of the NRP2:NRP2 ligand complex (optionally selected from Tables N1 and N2) is increased relative to the level of a healthy or matched control standard or a reference population consisting of one or more subjects, optionally at a level of about or at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more of the control or reference level, and / or the subject is selected for treatment based on the increase. Therefore, some embodiments include a method for selecting subjects for treatment, the method comprising: (i) determining the extracellular fluid level of the subject's NRP2:NRP2 ligand complex relative to the extracellular fluid level of a control or reference; and (ii) if the subject's NRP2:NRP2 ligand complex level is increased relative to the control or reference level, administering to the subject a therapeutic composition comprising at least one HRS-peptide as described herein.

[0468] In some embodiments, the extracellular fluid level of the subject's HRS:NRP2 complex (optionally selected from Tables H1 and N1) is increased relative to the level of a healthy or matched control standard or a reference population consisting of one or more subjects, for example, by about or at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more of the control or reference level, and / or the subject is selected for treatment based on said increase. Therefore, some embodiments include a method for selecting subjects for treatment, the method comprising: (i) determining the subject's extracellular fluid level of the HRS:NRP2 complex relative to the extracellular fluid level of a control or reference; and (ii) if the subject's HRS:NRP2 complex level is increased relative to the control or reference level, administering to the subject a therapeutic composition comprising at least one HRS-peptide as described herein.

[0469] In some embodiments, the subject has a single nucleotide polymorphism (SNP) in an NRP2 peptide or a polynucleotide encoding NRP2, and / or the subject is selected for treatment based on having the single nucleotide polymorphism.

[0470] In some embodiments, the subject suffers from a disease associated with an increase in the level or expression of NRP2a and / or NRP2b, or an altered NRP2a:NRP2b expression ratio, relative to the levels or expression of healthy controls, matched controls, or a population of one or more subjects, and / or the subject is selected for treatment based on having said disease. In some embodiments, the level of NRP2b is increased by about or at least about 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% compared to the levels of healthy controls, matched controls, or a population of one or more subjects.

[0471] In some embodiments, the healthy controls or matched control criteria or the population of one or more subjects comprise an average range of age-matched samples of cancerous or non-cancerous cells or tissues of the same type as the cancer, the average range including specific characteristics such as drug resistance, metastatic potential, invasiveness, genetic signature (optionally one or more p53 mutations, PTEN deletion, IGFR expression) and / or expression patterns.

[0472] In some embodiments, the subject has an infection and / or the subject is selected for treatment based on having the infection. In some cases, the infection is a lymphedema-related infection, such as erysipelas, cellulitis, lymphangitis, and / or sepsis.

[0473] In some embodiments, the HRS peptide is administered in combination with a second agent, such as an antimicrobial agent, antifungal agent, and / or anti-helmintic agent. In some embodiments, the HRS peptide and the second agent are administered together as part of the same therapeutic composition. In some embodiments, the HRS peptide and the second agent are administered as separate therapeutic compositions. In some embodiments, the second agent is selected from one or more of the following: aminoglycosides, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin; carbapenems, such as ertapenem, doripenem, imipenem / cilastatin, and meropenem; cephalosporins, such as cefadroxil, cefazolin, cefadroxil, cefepime, cefotaxime, cefalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefnicillin, cefaclor, cefprozil, cefuroxime, cefixime, etc. Ferdinandibular, Ceftolun, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Cefbufen, Cefazolin, Latamoxef, Cefepime, Cefuroxime, and Cefpyrap; Glycopeptides, such as Teicoplanin, Vancomycin, Tetravancin, Dabavancin, and Orivansin; Lincosamides, such as Clindamycin and Lincomycin; Macrolides, such as Azithromycin, Clarithromycin, Erythromycin, Roxithromycin, Telithromycin, and Spiramycin; Penicillins, such as Amoxicillin, Ampicillin, Aloxicillin, Dicloxacillin, Flucloxacillin, Meropenem, Methicillin, Ethoxynaphthylpenicillin, Oxacillin, and Penicillium. Penicillin G, penicillin V, piperacillin, penicillin G, temoxicillin, and ticarcillin; polypeptides, such as bacitracin, colistin, and polymyxin B; quinolones / fluoroquinolones, such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, naflufloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grefloxacin, sparfloxacin, and temafloxacin; sulfonamides, such as sulfamidone, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadisin, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfasalazine, sulfasalazine, sulfisoxazole, and methoxyphenoxyphenidate. Benzyl-sulfamethoxazole (TMP-SMX) and sulfamethoxazole; tetracyclines, such as desmochlortetracycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline; antimycobacterial agents, such as clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, and streptomycin; chloramphenicol; metronidazole; mupirocin; tigecycline; tinidazole; and anti-helmintic agents, such as diethylcarbazine and albendazole.

[0474] HRS peptide-mediated treatment of lymphedema

[0475] Lymphedema is a chronic debilitating disease, most commonly caused by cancer surgery, obesity, congestive heart failure, hypertension, and / or peripheral vascular / venous disease in the United States and Western countries. In the cancer treatment setting, lymphedema occurs due to iatrogenic lymphatic system injury, most commonly after lymph node dissection, but also as a result of extensive skin excision and adjuvant therapy with radiation. (See, for example, Purushotham et al., J. Clin. Oncol. 23:4312-4321, 2005; Szuba et al., Cancer 95:2260-2267, 2002; Tsai et al., Ann. Surg. Oncol. 16:1959-72, 2009).

[0476] It is estimated that one in three patients who undergo lymph node dissection will go on to develop lymphedema, and conservative estimates suggest that up to 50,000 new cases are diagnosed each year. (See, for example, DiSipio et al., Lancet Oncol. 14:500-515, 2013; Petrek et al., Cancer 83:2776-2781, 1998). Because lymphedema is a lifelong condition, the number of individuals affected is increasing annually, currently estimated to be between 5 and 6 million in the United States (Rockson et al., Ann. NY Acad. Sci. 1131:147-154, 2008) and over 200 million worldwide. Since the development of lymphedema is almost linearly correlated with cancer survival, and because the prevalence of known lymphedema risk factors (such as obesity and radiation therapy) is increasing, this number is likely to continue to rise in the future (see, for example, Erickson et al., Journal of the National Cancer Institute 93:96-111, 2001).

[0477] Secondary lymphedema can also take several years to develop, suggesting that its development depends not only on lymphatic damage but may also be induced by underlying chronic inflammatory conditions. Similar to other fibroproliferative disorders, CD4+ is present in lymphedema. +Cellular responses are characterized by a mixed Th1 / Th2 cell population. (Avraham et al., FASEB J. 27:1114-1126, 2013). Primary CD4+ T cells, also known as T helper cells or Th cells, migrate through secondary lymphoid structures and differentiate upon activation along many distinct / overlapping cell types (e.g., Th1, Th2, Th17, regulatory T cells, etc.). The Th2 cell subset plays a crucial role in regulating responses to parasites and some autoimmune responses. These cells are also involved in the pathology of fibrotic diseases in many organ systems, including the heart, lungs, kidneys, and skin. Recent studies have shown an increased number of Th2 cells in tissue biopsies obtained from patients with lymphedema, and that inhibition of Th2 differentiation reduces the pathology of lymphedema in mouse models.

[0478] In preclinical mouse models, CD4 + Cells or macrophages (but not other inflammatory cell types, including CD8) + The depletion of T cells or inhibition of Th2 differentiation (but not systemic inflammation or inhibition of interleukin-6) significantly reduced the degree of fibrosis, increased lymphangiogenesis and lymph transport, and effectively treated established lymphedema. (See, for example, Avraham et al., FISB Journal 27:1114-1126, 2013; Zampell et al., PLOS 7:e49940, 2012; Ghanta et al., American Journal of Physiology: Heart and Circulatory Physiology 308:H1065-1077, 2015). These findings are supported by recent studies demonstrating that T cells effectively inhibit lymphangiogenesis by producing anti-lymphangiogenic cytokines / growth factors, including interferon-γ (IFN-γ), interleukin (IL)-4, IL-13, and TGF-β1. (See, for example, Kataru et al., *Immunology* 34:96-107, 2011; Shin et al., *Nature Communications* 6:6196, 2015; Shao et al., *Journal of Interferon-Cytokine Research* 26:568-574, 2006; Oka et al., *Hematology* 111:4571-4579, 2008; Corliss et al., *Microcirculation* 23(2):95-121, 2016). In summary, these findings suggest infiltrative CD4 in lymphedema tissues. +Cells and macrophages reduce lymphatic function through a variety of mechanisms, including inducing changes in lymphatic vessel structure secondary to tissue fibrosis and inhibiting the formation of collateral lymphatic vessels.

[0479] Lymphedema is disfiguring and debilitating; patients experience chronic swelling of the affected limbs, recurrent infections, limited mobility, and a decreased quality of life (see, for example, Hayes et al., Cancer 118:2237-2249, 2012). Furthermore, once lymphedema develops, it is usually progressive. Currently, there are no known pharmacological therapies that can halt its progression or promote its resolution (see, for example, Cormier et al., Annals of Surgical Oncology 19:642-651, 2012). Therefore, patients often need to wear tight, uncomfortable clothing for the rest of their lives to try to prevent the accumulation of lymph fluid in the affected limbs and undergo intensive and time-consuming physical therapy (see, for example, Koul et al., International Journal of Radiation Oncology, Biology and Physics, 67:841-846, 2007). Furthermore, despite receiving chronic care, some patients experience significant disease progression, with increased swelling in the lymphedema limbs and frequent infections. Therefore, the development of targeted therapies for lymphedema is an important goal and represents an unmet biomedical need.

[0480] The direct role of NRP2 in regulating adult lymphatic remodeling has been demonstrated in animal knockout systems that showed enhanced edema in NRP2 KO mice after inflammatory provocation (see, for example, Mucka et al., *American Journal of Pathology* 186(11) 2803-2812, 2016), and in subjects with SNPs in neuropilin-2 who had increased susceptibility to secondary lymphedema (see, for example, Miaskowski et al., *PLOS ONE* 8(4) e60164, 2013). Furthermore, in addition to the potential regulatory role of NRP2 in vascular biology, the expression of NRP2 on immune cells and the regulation of said activity by the HRS peptide strongly suggest that the HRS peptide represents a novel and exciting therapeutic option for the treatment of lymphedema.

[0481] In some embodiments, the subject has lymphedema selected from the following stages: stage 1, stage 2, stage 3, stage 4, stage 5, stage 6 and stage 7, and / or the subject is selected for treatment based on having lymphedema at said stage, as shown below.

[0482] Stage 1: Swelling increases during the day and usually disappears at night when the patient is lying flat in bed. The affected tissue is in the pitting stage, and when pressed with a fingertip, the affected area recedes and reverses with increasing pressure.

[0483] In stage 2, the swelling is irreversible at night and will not disappear without proper management. The affected tissue has a spongy consistency and is considered non-pitting; when pressed with a fingertip, the affected area bounces back without indentation. Fibrosis found in stage 2 lymphedema marks the onset of limb stiffening and an increase in size.

[0484] In stage 3, the swelling is irreversible and the affected tissue (e.g., limbs) typically becomes increasingly larger. The affected tissue becomes stiff (fibrotic) and unresponsive.

[0485] In stage 4, the size and periphery of the affected tissue (e.g., a limb) become significantly larger. Lumps, bulges, and / or protrusions (also known as nodules) begin to appear on the skin.

[0486] In stage 5, the affected tissues (e.g., limbs) become very large. In some cases, one or more deep skin folds are common at this stage.

[0487] In stage 6, small, elongated or round nodules aggregate together, forming a moss-like shape on the affected tissue (e.g., limbs). The subject's mobility is significantly reduced.

[0488] Phase 7 participants have physical disabilities and are unable to perform routine daily activities independently, such as walking, bathing, and cooking. They require assistance from their family and healthcare system.

[0489] In some embodiments, the subject has lymphedema selected from the following grades: grade 1 (mild edema), grade 2 (moderate edema), grade 3a (severe edema), grade 3b (very severe edema), and grade 4 (extremely severe edema), and / or the subject is selected for treatment based on having said grade of lymphedema, as shown below.

[0490] Grade 1 (mild edema) involves distal areas such as the forearm and hand or lower leg and foot. The circumference difference is less than 4 cm, and no other tissue changes have been observed.

[0491] Grade 2 (moderate edema) involves the corresponding quadrant of the entire limb or trunk. The circumference difference is 4-6 cm. Significant tissue changes such as pitting are present. Subjects may experience erysipelas (usually caused by β-hemolytic group A streptococcus bacteria, an infection of the upper dermis and superficial lymphatic vessels).

[0492] Grade 3a (severe edema): Lymphedema is present in one limb and its associated trunk quadrant. The circumference difference is greater than 6 cm. Significant skin changes are present, such as keratosis or keratoderma, cysts, and / or fistulas. Additionally, the subject may experience recurrent erysipelas.

[0493] Grade 3b (very severe edema) includes the same symptoms as Grade 3a, except that it affects two or more limbs.

[0494] Grade 4 (extremely severe edema) affected limbs are enormous due to almost complete blockage of lymphatic channels.

[0495] Therefore, some embodiments include methods for treating lymphedema in a subject in need, improving symptoms of said lymphedema, and / or reducing the progression of said lymphedema, said methods including administering the HRS peptide or therapeutic composition provided herein to the subject.

[0496] HRS peptide-mediated treatment for fibrotic diseases

[0497] Fibrotic diseases encompass a variety of clinical entities, including systemic fibrotic diseases such as systemic sclerosis, scleroderma, graft-versus-host disease, renal systemic fibrosis, lymphedema-associated fibrosis, and IgG4-associated sclerosis, as well as multi-organ-specific conditions, including radiation-induced fibrosis and fibrosis of the heart, lungs, liver, and kidneys. Although their pathogenic mechanisms differ considerably, these diseases share a common characteristic: the uncontrolled and progressive accumulation of fibrous macromolecules in the affected organs, leading to dysfunction and eventual failure. Numerous studies have identified myofibroblasts as the cells responsible for the initiation and progression of the fibrotic process. Tissue myofibroblasts in fibrotic diseases originate from various sources, including quiescent tissue fibroblasts, circulating CD34+ fibroblasts, and phenotypic transformations of various cell types (including epithelial and endothelial cells) into activated myofibroblasts.

[0498] Transforming growth factor β-1 (TGF-β1) is a key regulator of fibrosis in various organ systems. It acts through a direct mechanism to increase collagen production by fibroblasts and reduce the turnover of matrix products. (See, for example, Willis et al., *American Journal of Pathology* 166:1321-1332, 2005; Sakai et al., *American Journal of Pathology* 184:2611-2617, 2014; Qi et al., *American Journal of Pathology: Renal Physiology* 288:F800-F809, 2005; Bonniaud et al., *Journal of Immunology* 173:2099-2108, 2004). Furthermore, NRP2 plays a direct role in regulating TGF-β1-mediated EMT, directly leading to fibrosis (see, for example, Grandclement et al., PLOS Pathogens 6(7)e20444, 2011) and mediates EMT or endo-EMT in fibroblasts, myofibroblasts, and endothelial cells to promote fibrosis formation (see, for example, Pardali et al., International Journal of Molecular Sciences 18 2157 2017). Additionally, TGF-β1 is a key regulator of the inflammatory response and is thought to indirectly regulate fibrosis by modulating chronic inflammation (Pesce et al., PLOS Pathog. 5:e1000371, 2009).

[0499] Furthermore, TGF-β1 is significantly increased in lymphedema tissues, both clinically and in mouse models of lymphedema. In a mouse tail model, immunotherapy to inhibit TGF-β1 significantly accelerated lymphoid regeneration, reduced fibrosis, decreased inflammation, and improved lymphatic function. (See, for example, Avraham et al., *Plast. Reconstr. Surg.* 124:438-450, 2009; Clavin et al., *American Journal of Physiology: Heart and Circulatory Physiology* 295:H2113-H2127, 2008; Avraham et al., *American Journal of Pathology* 177:3202-3214, 2010). Inhibition of the fibrotic response preserves the lymphatic system's ability to transport interstitial fluid and inflammatory cells.

[0500] Recent studies have shown that CD4+ plays a crucial role in clinical and animal models of lymphedema. +Cells and macrophages play a crucial role in regulating fibrosis. (See, for example, Ogata et al., *Journal of Dermal Research* 136706-714, 2016; Avraham et al., *American Journal of Pathology* 177:3202-3214, 2010; Avraham et al., *Journal of the Federation for Experimental Biology* 27:1114-1126, 2013; Zampell et al., *American Journal of Pathology: Cell Physiology* 302:C392-C404, 2012; Zampell et al., *PLOS ONE* 7:e49940, 2012). For example, clinical lymphedema biopsy specimens and animal models of lymphedema have been found to be affected by CD4+. + Cellular infiltration, and the number of these cells correlates with the degree of fibrosis and the clinical severity of the disease. (See, for example, Avraham et al., FISB Journal 27:1114-1126, 2013). Typically, patients with advanced lymphedema have significantly more infiltrative T cells, especially CD4+, compared to patients with earlier-stage disease. + More cells. Following lymphovenous bypass surgery (a procedure to divert blocked lymphatic vessels into the venous circulation), clinical symptoms of lymphedema improve with reduced tissue fibrosis and CD4 counts. + Reduced cell infiltration is associated with this. (Torrisi et al., Lymphat. Res. Biol. 13:46-53, 2015).

[0501] Fibrosis is also a hallmark of many autoimmune diseases, including chronic graft-versus-host disease (GVHD). This is particularly true for cases associated with allogeneic hematopoietic stem cell transplantation (allo-HSCT), a major therapeutic treatment for many hematologic malignancies. Its antitumor activity largely depends on the immune-mediated graft-versus-host effect (GvT effect). However, donor immune cells contained in the graft can also attack healthy host tissues, leading to graft-versus-host disease (GVHD). GVHD can be divided into two syndromes: acute GVHD (historically defined as a GVHD reaction occurring within the first 100 days after allogeneic SCT) and chronic GVHD (cGVHD), which typically occurs after day 100. Although cGVHD is associated with the graft-versus-host effect, it is also a leading cause of morbidity / mortality in long-term transplant recipients.

[0502] Scleroderma-like cGVHD (scl-cGVHD) is one of the most severe forms of cGVHD and occurs in approximately 20% of cGVHD patients. Although scl-cGVHD shares common features with systemic fibrosis, the two syndromes differ in pathology (scl-cGVHD typically begins in the superficial layers of the skin and then extends to deeper layers, while systemic fibrosis usually does the opposite) and clinical symptoms (where clinical features such as Raynaud's syndrome, pulmonary hypertension, and cardiac dysfunction are frequently observed in patients with systemic fibrosis but are uncommon in patients with scl-cGVHD).

[0503] Given the role of NRP2 in regulating immune cell activation and migration, the role of TGF-β1-mediated EMT in promoting fibrosis, and the ability of HRS peptides to regulate these processes, it is clear that HRS peptides represent a novel and exciting therapeutic option for treating fibrosis in cases of lymphedema, and in other fibrotic diseases and conditions.

[0504] Therefore, some embodiments include methods for treating fibrosis in a subject in need, improving symptoms of said fibrosis, and / or reducing the progression of said fibrosis, said methods including administering the HRS peptide or therapeutic composition provided herein to the subject.

[0505] HRS peptide-mediated regulation of lymphangiogenesis

[0506] The lymphatic system, composed of an interconnected network of capillaries, collecting vessels, and lymph nodes, absorbs, collects, and transports fluids and proteins filtered from the vascular system. This system provides crucial homeostatic functions: in humans, lymphatic vessels return >4 liters of fluid and a significant amount of protein to the large veins in the neck daily.

[0507] Lymphatic dysfunction (lymphedema) leads to an excessive accumulation of fluid (edema) in the interstitium. Although lymphedema is usually not life-threatening, it has serious health consequences, including pain, immobility, fibrosis, inflammation, adipose tissue accumulation, and tissue damage. Because the lymphatic system is also a key component of the immune response, lymphedema is often associated with an increased risk of infection and other immune system problems.

[0508] Lymphangiogenesis is the formation of new lymphatic vessels from pre-existing lymphatic vessels and is associated with a variety of pathological conditions, including metastasis, graft rejection (e.g., cornea, kidney, and heart), type 2 diabetes, obesity, hypertension, and lymphedema (see, for example, Alitalo, K. et al., *Nature* 438:946-953, 2005; Karaman, S. et al., *Journal of Clinical Investigation* 124:922-928, 2014; Kim, H. et al., *Journal of Clinical Investigation* 124:936-942, 2014; Maby-El Hajjami, H. et al., *Histochemistry: Cell Biology* 130:1063-107, 2008; Machnik, A. et al., *Nature Medicine*). Med) 15:545-552; Mortimer, PS et al. 2014. Journal of Clinical Research 124:915-921; Skobe, M. et al. 2009. Nature Medicine 15:993-994).

[0509] Compared to vascular invasion, lymphatic invasion within and around the primary tumor is a prognostic marker of invasiveness in various types of cancer. Lymphatic growth is also associated with graft rejection (Dietrich, T. et al., *Journal of Immunology* 184:535-539, 2010; Hall, FT et al., *Archives of Otolaryngology-Head and Neck Surgery* 129:716-719, 2003; Maula, SM et al., *Cancer Research* 63:1920-1926, 2003; Miyata, Y. et al., *Journal of Urology* 176:348-353, 2006; Saad, RS et al., *Mod Pathol* 19:1317-1323, 2006; Schoppmann, SF et al., *Annals of Surgery*). Surg, 240:306-312, 2004; Zeng, Y. et al., Prostate, 65:222-230, 2005.

[0510] Although lymphatic vessels are recognized as playing a crucial role in the pathogenesis of many diseases, the development of anti-lymphangiogenic agents has progressed very little compared to the large number of anti-angiogenic agents already in clinical trials. Therefore, the development of additional lymphangiogenic inhibitors would be beneficial for treating a range of conditions, including lymphedema and cancer metastasis.

[0511] Anti-lymphangiogenic agents are used, for example, to treat debilitating eye diseases, where lymphangiogenesis is a major cause of corneal graft rejection and a leading contributor to neovascularization associated with age-related macular degeneration (see, for example, Dietrich et al., *Journal of Immunology* 184:535-539, 2010). Specifically, penetrating keratoplasty is the most common form of solid tissue transplantation, with approximately 40,000 corneal transplants performed annually in the United States. For uncomplicated first-time transplants in avascular, low-risk beds, penetrating keratoplasty has a success rate as high as 90%. However, the rejection rate of corneal grafts placed in high-risk vascularized host beds is extremely high (70% to 90%). Therefore, there is a need to develop safe and targeted new protocols to inhibit lymphangiogenesis to promote graft survival and reduce or inhibit neovascularization.

[0512] Anti-lymphangiogenic drugs can also be used to treat dry eye disease. Significant upregulation of pro-lymphangiogenic factors (e.g., VEGF-C, VEGF-D, and VEGFR-3) and selective lymphangiogenesis have been demonstrated in the cornea of ​​patients with dry eye disease, while angiogenesis has not occurred simultaneously (Goyal, S. et al., Archives of Ophthalmology 128:819-824, 2010). Dry eye disease is an immune-mediated condition affecting approximately 5 million Americans. It severely impacts vision-related quality of life, and symptoms can be debilitating. Current treatment options for dry eye disease are limited, mostly palliative, and expensive. Therefore, the development of lymphangiogenic inhibitors is of therapeutic value for treating dry eye disease.

[0513] It is currently accepted that metastasis is one of the causes of death in the vast majority (estimated at 90%) of solid tumors (Gupta and Massague, Cell 127, 679-695, 2006). The complex process of metastasis involves a series of different steps, including the detachment of tumor cells from the primary tumor, the infiltration of tumor cells into lymphatic vessels or blood vessels, and the extravasation and growth of tumor cells at secondary sites. Analysis of local lymph nodes in many tumor types has shown that lymphatic vessels are an important pathway for the spread of cancer in humans. Furthermore, in almost all cancers, the presence of tumor cells in lymph nodes is the most important adverse prognostic factor. While such metastasis was previously thought to exclusively involve the pathway of malignant cells along pre-existing lymphatic vessels near the tumor, recent experimental studies and clinicopathological reports (see, for example, Achen et al., *British Journal of Cancer* 94, 1355-1360, 2006 and Nathanson, *Cancer* 98, 413-423, 2003) have shown that lymphangiogenesis can be induced by solid tumors and can promote tumor spread. These and other recent studies suggest that targeting lymphatic vessels and lymphangiogenesis could be a useful therapeutic strategy for limiting the development of cancer metastasis, which would significantly benefit many patients.

[0514] Therefore, there is a need for methods and compositions that inhibit the activity of lymphangiogenic factors, as well as methods for preventing or treating graft rejection, dry eye, tumor metastasis, lymphedema, and other inflammatory conditions.

[0515] Given the role of HRS peptides in regulating the binding and activity of NRP2 ligands that interact with NRP2, such HRS peptides potentially represent powerful new tools for developing pro-lymphangiogenic and anti-lymphangiogenic therapies. This differential effect can be mediated, for example, by using different HRS peptide compositions, differential dosing, differential treatment duration, or the appropriate use of additional cofactors (e.g., VEGF-C or brachiophyll 3F and / or 3G).

[0516] Therefore, some embodiments include methods for regulating (e.g., increasing, decreasing) lymphangiogenesis in a subject in need, the methods comprising administering the HRS peptide or therapeutic composition provided herein to the subject. Some embodiments include methods for treating lymphangiogenesis and / or neovascularization in a subject in need, improving symptoms of said lymphangiogenesis and / or neovascularization, and / or reducing the progression of said lymphangiogenesis and / or neovascularization, the methods comprising administering the HRS peptide or therapeutic composition provided herein to the subject. Some embodiments include methods for treating lymphangiogenesis in a subject in need, improving symptoms of said lymphangiogenesis, and / or promoting the progression of said lymphangiogenesis or reversing said lymphangiogenesis, the methods comprising administering the HRS peptide or therapeutic composition provided herein to the subject.

[0517] HRS peptide-regulated smooth muscle contractility regulation

[0518] Decreased contractility of the smooth muscle (SM) in the bladder can result from a variety of causes, including obstruction secondary to benign prostatic hyperplasia (BPH), posterior urethral valves, diabetes, multiple sclerosis, spinal cord injury, or idiopathic causes. (See, for example, Drake et al., *Nature Reviews Urology* 11(8):454–464, 2014). In cases such as BPH or posterior urethral valves, the bladder contracts due to outlet obstruction. The initial response is adaptive, involving a compensatory phase of SM hypertrophy that enables increased force generation to overcome increased outlet resistance. When demand exceeds the bladder's adaptive capacity, contractile efficienc...

Claims

1. A method of preparing a medicament for inhibiting autophagy, phagocytosis or efferocytosis in a subject in need thereof, wherein the subject suffers from a neuropilin-2 (NRP2)-related disease, wherein the disease is prostate cancer, pancreatic cancer or colorectal cancer that overexpresses NRP2, and wherein the HRS polypeptide consists of the amino acid sequence shown in SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 159 or SEQ ID NO:

171.

2. The use according to claim 1, wherein the prostate cancer, pancreatic cancer or colorectal cancer exhibits NRP2-dependent growth, NRP2-dependent adhesion, NRP2-dependent migration, NRP2-dependent chemoresistance and / or NRP2-dependent invasion.

3. Use according to claim 1 or 2, wherein the prostate cancer, pancreatic cancer or colorectal cancer is a primary cancer.

4. Use according to claim 1 or 2, wherein the prostate cancer, pancreatic cancer or colorectal cancer is a metastatic cancer.

5. The use according to claim 4, wherein the metastatic cancer expresses NRP2a and / or NRP2b.

6. The use according to any one of claims 1, 2 and 5, wherein the prostate cancer, pancreatic cancer or colorectal cancer is chemoresistant to at least one cancer therapy selected from an immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent and a kinase inhibitor.

7. The use according to claim 3, wherein the prostate cancer, pancreatic cancer or colorectal cancer is chemoresistant to at least one cancer therapy selected from the group consisting of an immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent and a kinase inhibitor.

8. The use according to claim 4, wherein the prostate cancer, pancreatic cancer or colorectal cancer is chemoresistant to at least one cancer therapy selected from the group consisting of an immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent and a kinase inhibitor.

9. The use according to any one of claims 1, 2, 5 and 7-8, wherein the subject is administered at least one additional agent selected from one or more of the following: a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent and a kinase inhibitor.

10. The use according to claim 3, wherein the subject is administered at least one additional agent selected from one or more of the following: a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent, and a kinase inhibitor.

11. The use according to claim 4, wherein the subject is administered at least one additional agent selected from one or more of the following: a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent, and a kinase inhibitor.

12. The use according to claim 6, wherein the subject is administered at least one additional agent selected from one or more of the following: a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent, and a kinase inhibitor.

13. purposes according to claim 9, wherein said at least one HRS polypeptide and said at least one medicament are separately administered as separate compositions.

14. The method of claim 10, wherein the HRS polypeptide and the at least one medicament are administered separately as separate compositions.

15. The method of claim 9, wherein the HRS polypeptide and the at least one agent are administered together as part of the same therapeutic composition.

16. The method of claim 10, wherein the HRS polypeptide and the at least one agent are administered together as part of the same therapeutic composition.

17. The use according to any one of claims 10-13 and 15, wherein the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint regulator, a cancer vaccine, an oncolytic virus and a cytokine.

18. The use according to claim 9, wherein the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint regulator, a cancer vaccine, an oncolytic virus and a cytokine.

19. The use according to claim 14, wherein the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint regulator, a cancer vaccine, an oncolytic virus and a cytokine.

20. The use according to claim 16, wherein the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint regulator, a cancer vaccine, an oncolytic virus and a cytokine.

21. The use according to any one of claims 10-13 and 15, wherein the cancer immunotherapeutic agent is a cell-based immunotherapy.

22. The use according to claim 9, wherein the cancer immunotherapeutic agent is a cell-based immunotherapy.

23. The use according to claim 14, wherein the cancer immunotherapeutic agent is a cell-based immunotherapy.

24. The use according to claim 16, wherein the cancer immunotherapeutic agent is a cell-based immunotherapy.

25. The use according to any one of claims 10-13 and 15, wherein the chemotherapeutic agent is selected from one or more of the following: an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a type 1 or type II topoisomerase inhibitor, and an anti-microtubule agent.

26. The use according to claim 9, wherein the chemotherapeutic agent is selected from one or more of the following: alkylating agents, antimetabolites, cytotoxic antibiotics, type 1 or type II topoisomerase inhibitors, and anti-microtubule agents.

27. The use according to claim 14, wherein the chemotherapeutic agent is selected from one or more of the following: alkylating agents, antimetabolites, cytotoxic antibiotics, type 1 or type II topoisomerase inhibitors, and anti-microtubule agents.

28. The use according to claim 16, wherein the chemotherapeutic agent is selected from one or more of the following: alkylating agents, antimetabolites, cytotoxic antibiotics, type 1 or type II topoisomerase inhibitors, and anti-microtubule agents.

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