Compositions and methods for treating NRP2-associated diseases

Abstract

To provide compositions and methods for treating NRP2-associated diseases.SOLUTION: Provided are therapies, including monotherapies and combination therapies, for treating neuropilin-2 (NRP2)-associated diseases and conditions, which include the use of at least one histidyl-tRNA synthetase (HRS) polypeptide. Embodiments of the present disclosure include methods for treating a neuropilin-2 (NRP2) associated disease or condition in a subject in need thereof, the methods comprising administering to the subject a therapeutic composition comprising a histidyl-tRNA synthetase (HRS) polypeptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 703,757, filed July 26, 2018, U.S. Provisional Patent Application No. 62 / 776,208, filed December 6, 2018, U.S. Provisional Patent Application No. 62 / 800,035, filed February 1, 2019, and U.S. Provisional Patent Application No. 62 / 849,440, filed May 17, 2019, each of which is incorporated by reference in its entirety.

[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference. The text file containing the sequence listing is named ATYR_135_04WO_ST25.txt. This text file is approximately 392 KB, was created on July 26, 2019, and has been submitted electronically via EFS-Web.

[0003] Embodiments of the present disclosure relate to therapies, including monotherapy and combination therapy, for treating diseases and conditions associated with neuropilin-2 (NRP2), comprising the use of at least one histidyl-tRNA synthetase (HRS) polypeptide. [Background technology]

[0004] Recent advances in research suggest that tRNA synthetases play important roles in cellular responses beyond their well-characterized role in protein synthesis. Specifically, there is growing recognition that tRNA synthetases may participate in a variety of previously unrecognized roles in responses to cellular stress and in tissue homeostasis in both the intracellular and extracellular environments.

[0005] The resokine protein family (HRS polypeptides) are derived from the histidyl-tRNA synthetase gene (HARS) via proteolysis or alternative splicing and are important extracellular modulators of cellular activity. Extracellular HARS can be readily detected in the circulation of healthy volunteers, and autoantibodies against HARS (Jo-1 antibodies) have been characterized in some inflammatory myopathies (IM) and in subjects with inflammatory lung diseases (ILD). While the role of Jo-1 antibodies in disease progression remains poorly understood, subjects with Jo-1 antibodies tend to be less susceptible to cancer than subjects with inflammatory myopathies who lack Jo-1 antibodies (see, e.g., 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)doi 10.3899 / jrheum.161480).

[0006] Recently, important progress has been made in elucidating the role of extracellular HARS-derived proteins, including the identification of a putative cellular receptor, neuropilin-2 (NRP2 ​​or NRP-2). It has been revealed that the interaction of HARS with NRP2 is mediated by the N-terminal region of HARS and can lead to important changes in the cellular function of NRP2. Thus, the current discovery of the resokine / neuropilin-2 axis represents a previously unknown mechanism that acts as a central regulator of cellular processes directly relevant to muscle, vascular, neural, bone, and immune homeostasis. Deregulation of any of these processes can lead to a variety of diseases, potentially addressable by novel HRS polypeptide-based therapeutics. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Lu et al.,PLOS ONE 9(4)e94128,2014 [Non-patent document 2] Modan et al.,Clin.Exp.Dermatol.34(5)561-565,2009 [Non-patent document 3] Shi et al., J. Rheum 44(7)doi 10.3899 / jrheum.161480 Summary of the Invention

[0008] Embodiments of the present disclosure include a method for treating a disease or condition associated with neuropilin-2 (NRP2) in a subject in need thereof, the method comprising administering to the subject a therapeutic composition comprising a histidyl-tRNA synthetase (HRS) polypeptide.

[0009] In some embodiments, the disease or condition associated with NRP2 is cancer and cancer-related diseases or conditions, such as cancer cell growth, cancer initiation, cancer migration, cancer cell adhesion, cancer cell invasion, cancer cell chemotherapy resistance, and cancer cell metastasis; diseases associated with inflammation and autoimmunity, optionally inflammatory lung diseases, such as hypersensitivity pneumonitis and pulmonary inflammation, and diseases associated with inappropriate immune cell activation or migration, optionally graft-versus-host disease (GVHD) and rheumatoid arthritis-associated interstitial lung disease (RA-ILD); diseases associated with lymphatic vessel development, lymphangiogenesis, and lymphatic vessel damage, optionally edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excess fat deposition, and vascular permeability; diseases associated with infection, such as latent infection; allergic disorders and diseases associated with allergic responses, optionally and diseases associated with inappropriate smooth muscle contractility, vascular smooth muscle cell migration and adhesion; diseases associated with inappropriate autophagy, phagocytosis, and efferocytosis; diseases associated with neuronal cell disorders, optionally associated with peripheral nervous system remodeling and pain sensation; diseases associated with bone development and / or bone remodeling; and diseases associated with inappropriate migratory cell migration.

[0010] In some embodiments, the subject has, and / or is selected for treatment based on having, an increased extracellular fluid level of soluble NRP2 polypeptide, either bound or free (optionally selected from Table N1), compared to levels in a population of healthy or matched controls or subjects, optionally levels of about or at least about 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 79 The soluble NRP2 polypeptide is 0, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, or 5000 pM, or about 30 to 50, 50 to 100, 100 to 2000, 200 to 2000, 300 to 2000, 400 to 2000, 500 to 2000, 600 to 2000, 700 to 2000, 800 to 2000, 900 to 2000, 1000 to 2000, 2000 to 3000, 3000 to 4000, or 4000 to 5000 pM.

[0011] In some embodiments, the subject has, and / or is selected for treatment based on having, an increased extracellular fluid level of an NRP2:NRP2-ligand complex (optionally selected from Table N1 and Table N2) compared to a population of healthy or matched controls or subjects, optionally a level that is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more of the control or reference level.

[0012] In some embodiments, the subject has, and / or is selected for treatment based on having, an increased extracellular fluid level of the HRS:NRP2 complex (optionally selected from Table H1 and Table N1) compared to a population of healthy or matched controls or subjects, optionally a level that is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more of the control or reference level.

[0013] In some embodiments, a subject has, and / or is selected for treatment based on having, a single nucleotide polymorphism (SNP) in an NRP2 polypeptide or polynucleotide encoding NRP2 from the subject.

[0014] In some embodiments, the subject has, and / or is selected for treatment based on having, a disease associated with increased levels or expression of NRP2a and / or NRP2b, or an altered ratio of NRP2a:NRP2b expression, compared to a population of healthy or matched control standards or subjects. 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%, 1000% compared to a population of healthy or matched control standards or subjects.

[0015] In some embodiments, a population of healthy or matched controls or subjects comprises an average range of age-matched samples of cancerous or non-cancerous cells or tissues of the same type as the cancer, which samples contain specific characteristics, such as drug resistance, metastatic potential, invasiveness, genetic characteristics (optionally p53 mutation, PTEN deletion, IGFR expression), and / or expression patterns.

[0016] In some embodiments, the subject in need thereof has an infection and / or a treatment is selected based on having an infection, and optionally the method further comprises administering at least one antibacterial, antifungal, and / or antihelminthic agent to the subject. In some embodiments, (a) the HRS polypeptide and (b) the antibacterial, antifungal, and / or antihelminthic agent are administered together as part of the same therapeutic composition. In some embodiments, (a) the HRS polypeptide and (b) the antibacterial, antifungal, and / or antihelminthic agent are administered as separate therapeutic compositions. In some embodiments, the antibacterial, antifungal, and / or antihelminthic agent is an aminoglycoside, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin; a carbapenem, such as ertapenem, doripenem, imipenem / cilastatin, and meropenem; a cephalosporin, such as cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cephalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime , moxalactam, cefepime, ceftaroline fosamil, and ceftobiprole; glycopeptides such as teicoplanin, vancomycin, telavancin, dalbavancin, and oritavancin; lincosamines such as clindamycin and lincomycin; macrolides such as azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, and spiramycin; penicillins such as amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, and ticarcillin; polypeptides such as bacitracin, colistin, and polymyxin B;Quinolones / fluoroquinolones, such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin; sulfonamides, such as mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (cotrimoxazole) (TMP-SMX) ), and sulfonamide chrysoidine; tetracyclines such as demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline; antimycobacterial agents such as clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin (rifampin), rifabutin, rifapentine, and streptomycin; chloramphenicol; metronidazole; mupirocin; tigecycline; tinidazole; and antihelminthic agents such as diethylcarbamazine and albendazole.

[0017] Also included are methods selected from one or more of the following: - A method for improving or restoring lymphatic function in a subject in need thereof; - a method for modulating lymphangiogenesis in a subject in need thereof; - a method for treating a disease or disorder associated with semaphorin signaling in a subject in need thereof; - A method for modulating vascular endothelial growth factor C (VEGF-C) signaling in a subject in need thereof; - A method for modulating integrin signaling in a subject in need thereof; - a method for modulating TGF-β signaling in a subject in need thereof; - A method for modulating autophagy, phagocytosis, or efferocytosis in a subject in need thereof; - A method for modulating neurogenesis in a subject in need thereof; - a method for reducing lymphatic endothelial cell migration or adhesion in a subject in need thereof; - a method for modulating endothelial-mesenchymal transition (EMT) in a subject in need thereof; - A method for modulating bone development in a subject in need thereof; - a method for modulating vascular permeability in a subject in need thereof; - a method for modulating the binding or functional interaction between an NRP2 polypeptide and an NRP2 ligand in a subject in need thereof; - a method for inhibiting immune cell activity, migration, or adhesion in a subject in need thereof; and - A method for reducing tumor cell migration or adhesion in a subject in need thereof, said method comprising administering to a subject in need thereof a therapeutic composition comprising a histidyl-tRNA synthetase (HRS) polypeptide.

[0018] In some embodiments, the lymphangiogenesis is secondary to cancer, corneal trauma, dry eye disease, inflammation, lymphedema, graft rejection, or a combination thereof. In some embodiments, the neurogenesis is peripheral nerve remodeling associated with inflammatory or autoimmune conditions. In some embodiments, the NRP2 ligand is selected from VEGF-C, VEGF-D, VEGF-A145, VEGFA165, PIGF-2, semaphorin 3B, 3C, 3D, and 3F, heparin, integrin, and TGF-beta. 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 semaphorin 3B, 3C, 3D, 3F, and 3G. In some embodiments, the immune cells are selected from bone marrow-derived cells, macrophages, neutrophils, eosinophils, granulocytes, dendritic cells, T cells, B cells, and natural killer (NK) cells. REG cell, T H1 cells, or T H2 In some embodiments, the macrophage is an M1 or M2 macrophage. In some embodiments, the method includes reducing tumor cell migration within the lymphatic system.

[0019] In some embodiments, the subject has a disease or condition associated with neuropilin-2 (NRP2), and optionally the subject has, and / or is selected for treatment based on having, increased extracellular levels of soluble NRP2 polypeptide, increased extracellular levels of NRP2:NRP2 ligand complex, increased extracellular levels of HRS:NRP2 complex, and / or a single nucleotide polymorphism (SNP) in an NRP2 polypeptide or polynucleotide encoding NRP2 from the subject.

[0020] In some embodiments, the disease is cancer, for example, 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, and optionally, the metastatic cancer expresses NRP2a and / or NRP2b.

[0021] In some embodiments, the cancer is resistant to at least one cancer therapy, such as a cancer immunotherapeutic agent, a chemotherapy agent, a hormonal therapy agent, and / or a kinase inhibitor. In some cases, the method includes selecting a subject with a cancer that is resistant to at least one cancer therapy before administering an HRS polypeptide.

[0022] In some embodiments, HRS polypeptides regulate autophagy, efferocytosis, or phagocyte maturation in cancer cells or cancer-associated macrophages. In certain embodiments, HRS polypeptides regulate autophagy in cancer cells.

[0023] In some embodiments, the cancer is selected from one or more of 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, hepatocellular carcinoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), 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.

[0024] In some embodiments, the metastatic cancer is selected from one or more of the following: (a) bladder cancer that has metastasized to bone, liver, and / or lung; (b) breast cancer that has metastasized to the bone, brain, liver, and / or lungs; (c) colorectal cancer metastasizing to the liver, lung, and / or peritoneum; (d) kidney cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or lungs; (e) lung cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or other lung sites; (f) melanoma that has metastasized to bone, brain, liver, lung, and / or skin / muscle; (g) ovarian cancer metastasizing to the liver, lung, and / or peritoneum; (h) pancreatic cancer metastasizing to the liver, lung, and / or peritoneum; (i) prostate cancer that has metastasized to the adrenal glands, bone, liver, and / or lungs; (j) gastric cancer metastasizing to the liver, lung, and / or peritoneum; (l) thyroid cancer that has metastasized to the bone, liver, and / or lungs; and (m) Uterine cancer that has metastasized to the bone, liver, lung, peritoneum, and / or vagina.

[0025] Some embodiments include administering to the subject at least one additional agent selected from one or more of a cancer immunotherapy agent, a chemotherapeutic agent, a hormonal therapy agent, and a kinase inhibitor. In some embodiments, the at least one HRS polypeptide and the at least one agent are administered separately as separate compositions. In some embodiments, the at least one HRS polypeptide and the at least one agent are administered together as part of the same therapeutic composition.

[0026] In some embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy. In some embodiments, the immune checkpoint modulator is a polypeptide, optionally an antibody or antigen-binding fragment thereof, or a ligand, or a small molecule. In some embodiments, the immune checkpoint modulator comprises: (a) an antagonist of an inhibitory immune checkpoint molecule; or (b) An agonist of a stimulatory immune checkpoint molecule, e.g., the immune checkpoint modulator specifically binds to an immune checkpoint molecule.

[0027] In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of 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 and mucin domain 3 (TIM-3), lymphocyte-activation gene-3 (LAG-3), V-domain Ig suppressor of T-cell activation (VISTA), B- and T-lymphocyte attenuator (BTLA), CD160, herpesvirus entry mediator (HVEM), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0028] In some embodiments, the antagonist is an antagonist of PD-L1 and / or PD-L2, optionally chosen from one or more of: an antibody or antigen-binding fragment or small molecule that specifically binds thereto, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and optionally, the cancer is selected from one or more of: colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma; the antagonist is a PD-1 antagonist optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514 PDR001, and pidilizumab, optionally wherein the PD-1 antagonist is nivolumab, and the cancer is optionally chosen from one or more of Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer; the PD-1 antagonist is pembrolizumab, and the cancer is optionally selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma; the antagonist is a CTLA-4 antagonist, optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, ipilimumab, tremelibumab, and optionally the cancer is chosen from one or more of melanoma, prostate cancer, lung cancer, and bladder cancer; the antagonist is an IDO antagonist, optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, and the cancer is optionally chosen from one or more of metastatic breast cancer and brain cancer, optionally glioblastoma multiforme, glioma, gliosarcoma, or malignant brain tumor; the antagonist is a TDO antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91, and LM10; the antagonist is a TIM-3 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds thereto; the antagonist is a LAG-3 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, and BMS-986016; the antagonist is a VISTA antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds thereto; the antagonist is an antagonist of BTLA, CD160, and / or HVEM, optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds thereto; The antagonist is a TIGIT antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds thereto.

[0029] In some embodiments, the stimulatory immune checkpoint molecule is selected from one or more of OX40, CD40, glucocorticoid-inducible TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0030] In some embodiments, the agonist is an OX40 agonist, optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, OX86, Fc-OX40L, and GSK3174998; the agonist is a CD40 agonist, optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, CP-870, 893, dacetuzumab, Chi Lob7 / 4, ADC-1013, and rhCD40L, and the cancer is optionally chosen from one or more of melanoma, pancreatic cancer, mesothelioma, and hematological cancers, optionally lymphomas such as non-Hodgkin's lymphoma; the agonist is a GITR agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, INCAGN01876, DTA-1, and MEDI1873; the agonist is a CD137 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, utomilumab, and a 4-1BB ligand; the agonist is a CD27 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, varlilumab, and CDX-1127 (1F5); the agonist is a CD28 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, and TAB08; and / or The agonist is an HVEM agonist, optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule, or a ligand that specifically binds thereto.

[0031] In some embodiments, the cancer vaccine is selected from one or more of Oncophage, a human papillomavirus HPV vaccine, optionally Gardasil or Cervarix, a hepatitis B vaccine, optionally Engerix-B, Recombivax HB, or Twinrix, and Sipuleucel-T (Provenge), or a cancer vaccine selected from 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, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, 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 activation protein alpha (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), EGP40pan 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 (a disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin; and optionally, the subject isHaving or being at risk of having a cancer that contains the corresponding cancer antigen.

[0032] In some embodiments, the oncolytic virus is selected from one or more of talimogene laherparepvec (T-VEC), Coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0033] In some embodiments, the cytokine is selected from one or more of interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0034] In some embodiments, the cell-based immunotherapeutic comprises cancer antigen-specific T cells, optionally ex vivo derived T cells, hi some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0035] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and a microtubule inhibitor.

[0036] In some embodiments, the alkylating agent is selected from one or more of nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and derivatives thereof (optionally carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine); the antimetabolite is selected from one or more of antifolates (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 mercaptopurine); the cytotoxic antibiotic is selected from one or more of anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin; the topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin; and / or The microtubule inhibitor is selected from one or more of taxanes (optionally paclitaxel and docetaxel), and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0037] In some embodiments, the at least one hormonal 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: progestogen (progestin), corticosteroid (optionally prednisolone, methylprednisolone, or dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factor (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen, and somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, optionally an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or analogues thereof; a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an antiandrogen; or an antibody against a hormone receptor, optionally including cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, robatumumab, alacizumab pegol and / or one or more of: pegol, bevacizumab, icrucumab, ramucirumab, frezolimumab, metelimumab, naxitamab, cetuximab, depatuximab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, zalutumumab, aprituximab ixadotin, bemarituzumab, olaratumab, or tobetumab.

[0038] In some embodiments, the kinase inhibitor is selected from one or more of adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozanib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib. In some embodiments, the kinase inhibitor is a PI3 kinase inhibitor selected from one or more of 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.

[0039] Also included is a method for treating an inflammatory lung disease in a subject in need thereof, comprising administering to the subject a therapeutic composition comprising a histidyl-tRNA synthetase (HRS) polypeptide. In some embodiments, the inflammatory lung disease is a disease or condition associated with neuropilin-2 (NRP2), e.g., RA-ILD, chronic hypersensitivity pneumonitis, pulmonary inflammation, granulomatous lung disease, or sarcoidosis. In some embodiments, the subject has, and / or is selected for treatment based on, having increased extracellular levels of a soluble NRP2 polypeptide, increased extracellular levels of an NRP2:NRP2 ligand complex, increased extracellular levels of an HRS:NRP2 complex, and / or a single nucleotide polymorphism (SNP) in an NRP2 polypeptide or polynucleotide encoding NRP2 from the subject, as described herein.

[0040] Some embodiments include a method for treating lymphedema in a subject in need thereof, the method comprising administering to the subject a therapeutic composition comprising a histidyl-tRNA synthetase (HRS) polypeptide. In some embodiments, the subject has, and / or is selected for treatment based on having, a stage of lymphedema selected from Stage 1, Stage 2, Stage 3, Stage 4, Stage 5, Stage 6, and Stage 7. In some embodiments, the subject has, and / or is selected for treatment based on having, a grade of lymphedema selected from Grade 1 (mild edema), Grade 2 (moderate edema), Grade 3a (severe edema), Grade 3b (widespread edema), and Grade 4 (massive edema). In some embodiments, the subject has, and / or is selected for treatment based on having, lymphedema-associated fibrosis. In some embodiments, the subject has, and / or is selected for treatment based on having, lymphedema secondary to cancer (optionally breast cancer), surgery (optionally cancer surgery, optionally breast cancer surgery), radiation therapy, obesity, congestive heart failure, hypertension, peripheral vascular / venous disease, or any combination thereof.

[0041] 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, thickening of the skin, hardening of the skin, fullness, pain, discomfort, limited range of motion, and any combination thereof.

[0042] In some embodiments, lymphedema is a neuropilin-2 (NRP2)-associated disease or condition. In some embodiments, the subject has, and / or is selected for treatment based on having, increased extracellular levels of soluble NRP2 polypeptide, increased extracellular levels of NRP2:NRP2 ligand complex, increased extracellular levels of HRS:NRP2 complex, and / or a single nucleotide polymorphism (SNP) in an NRP2 polypeptide or polynucleotide encoding NRP2 from the subject.

[0043] In some embodiments, the subject in need thereof has and / or is selected for treatment based on having a lymphedema-associated infection, optionally erysipelas, cellulitis, lymphangitis, and / or sepsis, and optionally the method further comprises administering to the subject at least one antibacterial, antifungal, and / or antihelminthic agent. In some embodiments, (a) the HRS polypeptide and (b) the antibacterial, antifungal, and / or antihelminthic agent are administered together as part of the same therapeutic composition. In some embodiments, (a) the HRS polypeptide and (b) the antibacterial, antifungal, and / or antihelminthic agent are administered as separate therapeutic compositions. In some embodiments, the antibacterial, antifungal, and / or antihelminthic agent is an aminoglycoside, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin; a carbapenem, such as ertapenem, doripenem, imipenem / cilastatin, and meropenem; a cephalosporin, such as cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cephalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, cef glycopeptides such as teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin; lincosamines such as clindamycin and lincomycin; macrolides such as azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, and spiramycin; penicillins such as amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, and ticarcillin;Polypeptides, such as bacitracin, colistin, and polymyxin B; quinolones / fluoroquinolones, such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin; sulfonamides, such as mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole ( tetracyclines, such as demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline; antimycobacterial agents, such as clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin (rifampin), rifabutin, rifapentine, and streptomycin; chloramphenicol; metronidazole; mupirocin; tigecycline; tinidazole; and antihelminthic agents, such as diethylcarbamazine and albendazole.

[0044] In some embodiments, the HRS polypeptide is SEQ ID NO: 156 (Fc-HRS(2-60) or HRS FC1) or 100% identical to a sequence selected from Table H1, Table H2, and Table H4. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H1, Table H2, and Table 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)), and lacks residues 507 to 509 of SEQ ID NO:1.

[0045] In some embodiments, the HRS polypeptide is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide includes an Fc region to form an HRS-Fc fusion polypeptide, optionally comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H8. In some embodiments, the heterologous polypeptide includes a cartilage oligomeric protein (COMP) polypeptide, optionally a COMP pentamer domain polypeptide, to form an HRS-COMP fusion polypeptide, optionally comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H9.

[0046] Also included are therapeutic compositions comprising: (a) a histidyl-tRNA synthetase (HRS) polypeptide; and (b) at least one additional agent selected from an antibacterial agent, an antifungal agent, an antihelminthic agent, a cancer immunotherapy agent, a chemotherapeutic agent, a hormonal therapy agent, and a kinase inhibitor.

[0047] In some embodiments, the HRS polypeptide is SEQ ID NO: 156 (Fc-HRS(2-60) or HRS FC1 ) or 100% identical to a sequence selected from Table H1, Table H2, and Table H4. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H1, Table H2, and Table 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)), and lacks residues 507 to 509 of SEQ ID NO:1.

[0048] In some embodiments, the HRS polypeptide is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide includes an Fc region to form an HRS-Fc fusion polypeptide, optionally comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H8. In some embodiments, the heterologous polypeptide includes a cartilage oligomeric protein (COMP) polypeptide, optionally a COMP pentamer domain polypeptide, to form an HRS-COMP fusion polypeptide, optionally comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H9.

[0049] In some embodiments, the antibacterial, antifungal, and / or antihelminthic agent is an aminoglycoside, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin; a carbapenem, such as ertapenem, doripenem, imipenem / cilastatin, and meropenem; a cephalosporin, such as cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cephalosporin ... Falexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, cefepime, ceftaroline fosamil, and ceftobiprole; glycopeptides, such as teicoplanin, vancomycin, telavancin, dalba Vancin, oritavancin, etc.; lincosamines, such as clindamycin and lincomycin; macrolides, such as azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, and spiramycin; penicillins, such as amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin such as cillin G, temocillin, and ticarcillin; polypeptides such as bacitracin, colistin, and polymyxin B; quinolones / fluoroquinolones such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin;Sulfonamides, such as mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (cotrimoxazole) (TMP-SMX), and sulfonamide chrysoidine; tetracyclines, such as demeclocycline, doxycycline, methacycline, minocycline, and oxytetracycline. , and tetracycline; antimycobacterial agents such as clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin (rifampin), rifabutin, rifapentine, and streptomycin; chloramphenicol; metronidazole; mupirocin; tigecycline; tinidazole; and antihelminthic agents such as diethylcarbamazine and albendazole.

[0050] In some embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy.

[0051] In some embodiments, the immune checkpoint modulator is a polypeptide, and optionally an antibody or antigen-binding fragment thereof, or a ligand, or a small molecule. In some embodiments, the immune checkpoint modulator comprises: (a) an antagonist of an inhibitory immune checkpoint molecule; or (b) An agonist of a stimulatory immune checkpoint molecule, e.g., the immune checkpoint modulator specifically binds to an immune checkpoint molecule.

[0052] In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of 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 and mucin domain 3 (TIM-3), lymphocyte-activation gene-3 (LAG-3), V-domain Ig suppressor of T-cell activation (VISTA), B- and T-lymphocyte attenuator (BTLA), CD160, herpesvirus entry mediator (HVEM), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0053] In some embodiments, the antagonist is an antagonist of PD-L1 and / or PD-L2, optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736); the antagonist is a PD-1 antagonist, optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514, PDR001, and pidilizumab; the antagonist is a CTLA-4 antagonist, optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, ipilimumab, and tremelibumab; the antagonist is an IDO antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat; the antagonist is a TDO antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91, and LM10; the antagonist is a TIM-3 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds thereto; the antagonist is a LAG-3 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, and BMS-986016; the antagonist is a VISTA antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds thereto; the antagonist is an antagonist of BTLA, CD160, and / or HVEM, optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds thereto; and / or The antagonist is a TIGIT antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds thereto.

[0054] In some embodiments, the stimulatory immune checkpoint molecule is selected from one or more of OX40, CD40, glucocorticoid-inducible TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0055] In some embodiments, the agonist is an OX40 agonist, optionally chosen from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, OX86, Fc-OX40L, and GSK3174998; the agonist is a CD40 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, CP-870, 893, dacetuzumab, Chi Lob7 / 4, ADC-1013, rhCD40L; the agonist is a GITR agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, INCAGN01876, DTA-1, and MEDI1873; the agonist is a CD137 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, utomilumab, and a 4-1BB ligand; the agonist is a CD27 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, varlilumab, and CDX-1127 (1F5); the agonist is a CD28 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, and TAB08; and / or The agonist is an HVEM agonist, optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule, or a ligand that specifically binds thereto.

[0056] In some embodiments, the cancer vaccine is selected from one or more of Oncophage, a human papillomavirus HPV vaccine, optionally Gardasil or Cervarix, a hepatitis B vaccine, optionally Engerix-B, Recombivax HB, or Twinrix, and sipuleucel-T (Provenge), or a vaccine selected from human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD 22, 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, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), charcoal Acid 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 activation protein alpha (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), EGP40pan 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 (a disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0057] In some embodiments, the oncolytic virus is selected from one or more of talimogene laherparepvec (T-VEC), Coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0058] In some embodiments, the cytokine is selected from one or more of interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0059] In some embodiments, the cell-based immunotherapeutic comprises cancer antigen-specific T cells, optionally ex vivo derived T cells, hi some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0060] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and a microtubule inhibitor.

[0061] In some embodiments, the alkylating agent is selected from one or more of nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and derivatives thereof (optionally carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine); the antimetabolite is selected from one or more of antifolates (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 mercaptopurine); the cytotoxic antibiotic is selected from one or more of anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin; the topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin; and / or The microtubule inhibitor is selected from one or more of taxanes (optionally paclitaxel and docetaxel), and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0062] In some embodiments, the at least one hormonal 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: progestogen (progestin), corticosteroid (optionally prednisolone, methylprednisolone, or dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factor (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen, and somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, optionally an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or analogues thereof; a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an antiandrogen; or an antibody against a hormone receptor, optionally including cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, robatumumab, alacizumab pegol and / or one or more of: pegol, bevacizumab, icrucumab, ramucirumab, frezolimumab, metelimumab, naxitamab, cetuximab, depatuximab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, zalutumumab, aprituximab ixadotin, bemarituzumab, olaratumab, or tobetumab.

[0063] In some embodiments, the kinase inhibitor is selected from one or more of adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozanib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib. In some embodiments, the kinase inhibitor is a PI3 kinase inhibitor selected from one or more of 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.

[0064] In some embodiments, the therapeutic composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis with respect to the HRS polypeptide and is substantially free of aggregates. In some embodiments, the therapeutic composition is substantially free of endotoxins. In some embodiments, the therapeutic composition is a sterile injectable solution, optionally suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration.

[0065] Additionally, a patient care kit containing: (a) a histidyl-tRNA synthetase (HRS) polypeptide described herein; and (b) at least one additional agent selected from an antibacterial agent, an antifungal agent, an antihelminthic agent, a cancer immunotherapy agent, a chemotherapeutic agent, a hormonal therapy agent, and a kinase inhibitor, as described herein.

[0066] In some embodiments, (a) and (b) are in separate therapeutic compositions. In some embodiments, (a) and (b) are in the same therapeutic composition.

[0067] Certain embodiments include isolated fusion proteins comprising a histidyl-tRNA synthetase polypeptide fused to a cartilage oligomeric protein (COMP) polypeptide, optionally a COMP pentamer domain polypeptide, to form an HRS-COMP fusion polypeptide, wherein the HRS-COMP fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H9.

[0068] Some embodiments include a therapeutic composition, the therapeutic composition comprising an 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% on a protein basis with respect to the HRS-COMP fusion polypeptide, and is substantially free of aggregates. In certain embodiments, the therapeutic composition is substantially free of endotoxins. In some embodiments, the therapeutic composition is a sterile injectable solution, optionally suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration. [Brief explanation of the drawings]

[0069] [Figure 1] 1A-1B show the overall domain structure of neuropilins (1A) and exemplary neuropilin coreceptor functions (1B). [Figure 2] Figure 2 shows the domain structure of specific NRP2 isoforms and the NRP2 ligand-binding domain. [Figure 3] Figure 3 shows the binding of human NRP2 to Fc-HRS(2-60) on an SPR chip. 50 nM NRP2 (black line), NRP1 (gray line), and mouse Plexin A1 (dotted line) were flowed as analytes over an SPR chip coated with immobilized Fc-HRS(2-60). [Figure 4] Figures 4A-4B show that human, mouse, and rat NRP2 bound to Fc-HRS(2-60) but not to the truncated form, Fc-HRS(2-11). 50 nM human NRP2 (black line), mouse NRP2 (dashed line), rat NRP2 (gray line), or NRP1 (dotted line) was flowed as analyte over an SPR chip coated with immobilized full-length Fc-HRS(2-60) (4A) or the truncated form, Fc-HRS(2-11), lacking the C-terminal 49 amino acids. [Figure 5]Figures 5A-5D show the binding of human NRP2 to Fc-HRS(2-60) and t-RNA synthetases containing domains that share homology with the WHEP domain of Fc-HRS(2-60). 20 nM NRP2 was flowed as an analyte over an SPR chip surface coated with immobilized Fc-HRS(2-60) (5A), GARS Fc-WHEP (5B), MARS Fc-WHEP (5C), or WARS WHEP (5D). [Figure 6] Figures 6A-6B show the binding of human NRP2 to Fc-HRS(2-60) on an SPR chip coated with immobilized Fc-HRS(2-60) in the presence and absence of divalent cations. The running buffer for this experiment was 50 mM HEPES, 300 mM NaCl, 0.005% Tween® 20, pH 7.4. For each analyte, 20 nM NRP2 was prepared in running buffer supplemented with either 5 mM CaCl, EDTA (6A) or MgCl, MgCl + CaCl, or ZnCl (6B). [Figure 7] Figures 7A-7B show that preformed complexes of Fc-HRS(2-60) and NRP2 bind to the 4D4 monoclonal antibody 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) (black line), a mixture of 100 nM Fc-HRS(2-60) and 200 nM NRP2 (gray line), or a mixture of 100 nM Fc-HRS(2-60) and 200 nM 1C8 mAb (dashed line). [Figure 8]Figures 8A–8D show that the binding of Fc-HRS(2-60) to NRP2 was captured by several monoclonal antibodies against Fc-HRS(2-60), but not by others. 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. Simultaneous loading was then performed, with one analyte loaded immediately followed by the second. The timing of the two loadings is indicated by arrowheads. In each of the above panels, 2000 nM Fc-HRS(2-60) was loaded as the first analyte to saturate the antibody surface, followed by either additional Fc-HRS(2-60) (gray line) or 200 nM NRP2 (black line). To exclude nonspecific binding of NRP2 to the antibody surface, a simultaneous injection of buffer followed by 200 nM NRP2 was also performed (dotted line). [Figure 9] Figures 9A-9B show the dose-dependent binding of Fc-HRS(2-60) to cells expressing NRP2a-GFP fusion protein. Quantification of staining intensity (9A) and staining intensity CV (9B) of the Fc-HRS(2-60) / anti-Fc-PE complex on HEK293T cells overexpressing NRP2v2-GFP. Intensity values ​​are from cells gated on high NRP2 expression (GFP Bright). Fc-HRS(2-60) was titrated in 2-fold steps and then mixed with 87.5 nM anti-Fc-PE. 175 nM Fc-HRS(2-11) / anti-Fc-PE was included as a specificity control. [Figure 10]Figure 10 shows the inhibition of Fc-HRS(2-60) binding to cells expressing NRP2a-GFP fusion protein in the presence of anti-HRS antibody clone 1C8. Quantification of staining intensity of Fc-HRS(2-60) / anti-Fc-PE complexes pre-incubated with either an isotype antibody control or anti-HRS (WHEP) clone 1C8 on HEK293T cells overexpressing NRP2v2-GFP. Intensity is the value from cells gated on high NRP2 expression (GFP Bright). 175 nM Fc-HRS(2-60) / anti-Fc-PE was used. 175 nM Fc-HRS(2-11) / anti-Fc-PE was included as a specificity control. [Figure 11] Figures 11A-11B show that the anti-HRS antibody KL31 series inhibited the binding of Fc-HRS(2-60) to NRP2 in a concentration-dependent manner, whereas other antibodies in the AB04 and AB13 series did not exhibit significant inhibitory properties in this assay. Quantification of staining of cells stably expressing Expi293-NRP2 with biotinylated Fc-HRS-streptavidin-PE 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 (filled circles), KL31-467 (filled triangles), KL31-356 (partially filled triangles), and mouse clone 13C8 (crosses), and Figure 11B shows control human IgG1 (filled circles), AB04-425 (open triangles), AB13-288 (partially filled squares), and KL31-478 (filled triangles), shown as the percentage of streptavidin-PE+ / NRP2+ cells in the live single gate. [Figure 12]Figures 12A-12B show the inhibition of Fc-HRS(2-60) binding to cells expressing NRP2v2-GFP fusion protein in the presence of VEGF-C. Quantification of staining intensity of the Fc-HRS(2-60) / anti-Fc-PE complex pre-incubated with different doses of VEGF-C on HEK293T cells overexpressing NRP2v2-GFP. Intensity is from cells gated on high NRP2 expression (GFP Bright). 175 nM Fc-HRS(2-60) / anti-Fc-PE was used. 175 nM Fc-HRS(2-11) / anti-Fc-PE was included as a specificity control. [Figure 13] Figure 13 shows the quantification of circulating NRP2 levels in serum and plasma from normal healthy donors. Serum and plasma from normal healthy volunteers (n=72) were isolated and quantified for circulating levels of NRP-2. Serum samples (filled circles) and plasma samples (open squares) were validated with an ELISA specific for human NRP-2. The mean values ​​for serum (16.3 pM) and plasma (15.6 pM) for all 72 samples are shown. The limit of quantification for the NRP2 ELISA was 1.5 pM. [Figure 14] Figure 14 shows a comparison of circulating HRS and NRP2 levels. Serum HRS (filled circles) levels show a wide range in circulation among 72 normal healthy volunteers examined. Serum NRP2 levels from the same donors were matched and overlaid on the same axis. Donors with low HRS levels exhibited low or undetectable levels of soluble NRP2 (limit of quantification 1.5 pM). Donors with high HRS levels generally had correspondingly high levels of circulating NRP2. [Figure 15]Figure 15 shows HRS N-terminal interference in human serum from healthy donors. Normal serum from healthy donors was analyzed in two separate HARS ELISAs. Samples were analyzed in an ELISA detecting full-length HARS (HARS_FL; filled circles) and an ELISA specifically directed against the N-terminus (HARS_NT; open squares). Increased full-length HARS levels and a lack of correlation between these two ELISAs are considered N-terminal interference and may indicate the presence of a cofactor, binding partner, or soluble receptor for HRS. [Figure 16] Figure 16 shows the correlation between HARS N-terminal analytical interference and soluble NRP2 levels. Serum from normal healthy individuals was analyzed for differences in detection with the two HARS ELISAs and compared to circulating NRP2 levels. The difference in detection levels between the full-length HARS ELISA and the N-terminal HARS ELISA was termed HARS N-terminal interference units. These interference units were graphed against soluble NRP2 levels. The results demonstrate a correlation between increased N-terminal interference and soluble NRP2 in normal serum. [Figure 17] Figure 17 shows the detection of soluble complexes of endogenous HRS and NRP2. Serum samples from normal healthy donors were analyzed in a multiplex HRS & NRP-2 complex ELISA. These assay formats utilized capture of circulating HRS (HARS_NT or HARS_CT) and detection with an NRP2 monoclonal antibody. Similarly, a reverse format was used, whereby circulating NRP2 was captured and detected with an anti-HRS antibody. In both formats, elevated signal was observed in high-interference samples compared to low-interference serum samples. [Figure 18]Figure 18 shows that complexed HRS and NRP2 in high-interference samples interfered with detection using site-specific HRS antibodies. Serum from low- and high-HRS N-terminal interference samples was analyzed in an HRS and NRP2 complex ELISA. Serum samples were supplemented with an NRP2 monoclonal antibody and detected with one of two specific HRS N-terminal monoclonal antibodies. When detected with HRS_NT (black bars), the high-interference sample showed complex formation, but this signal was completely blocked with the N-terminal anti-HRS antibody (HRS blocking antibody, gray bars). [Figure 19] Figures 19A-19C show the activity of Fc-HRS(2-60) on skin markers in a mouse model of scleroderma chronic graft-versus-host disease. Figure 19A shows the effect of treatment with vehicle, Fc-HRS(2-60), or nintedanib, starting 7 days (7D) or 21 days (21D) after allografting, on skin thickness. Figure 19B shows the effect of treatment with vehicle, Fc-HRS(2-60), or nintedanib, starting 7 days (7D) or 21 days (21D) after allografting, on the number of myofibroblasts counted in skin sections. Figure 19C shows the effect of treatment with vehicle, Fc-HRS(2-60), or nintedanib, starting 7 days (7D) or 21 days (21D) after allografting, on hydroxyproline content in the skin (an indicator of collagen content). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 Kruskal-Wallis nonparametric ANOVA followed by Dunn's multiple comparison test relative to vehicle animals (third group from the left) euthanized 8 weeks after allotransplantation. [Figure 20]Figures 20A-20D show the activity of Fc-HRS(2-60) on lung markers in a mouse model of scleroderma chronic graft-versus-host disease. Figure 20A shows the effect of treatment with vehicle, Fc-HRS(2-60), or nintedanib, initiated 7 days (7D) or 21 days (21D) after allograft transplantation, on Ashcroft scores. Figure 20B shows the effect of treatment with vehicle, Fc-HRS(2-60), or nintedanib, initiated 7 days (7D) or 21 days (21D) after allograft transplantation, on the percentage of each section occupied by tissue stained with picrosirius red, a stain that specifically stains collagen fibers. Figure 20C shows the effect of treatment with vehicle, Fc-HRS(2-60), or nintedanib starting 7 days (7D) or 21 days (21D) after allografting on the number of myofibroblasts counted in lung sections. Figure 20D shows the effect of treatment with vehicle, Fc-HRS(2-60), or nintedanib starting 7 days (7D) or 21 days (21D) after allografting on hydroxyproline content in the skin (an indicator of collagen content). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Kruskal-Wallis nonparametric ANOVA followed by Dunn's multiple comparison test for vehicle animals (third group from the left) euthanized 8 weeks after allografting. [Figure 21] Figures 21A-21C show the effect of HRS(2-60)-containing proteins on LPS-induced leukocyte infiltration into mouse lungs. Mice were intravenously treated with Fc-HRS(2-60) or HRS(2-60)-COMP at the indicated doses. The following day, saline or LPS (10 μg / mouse) was instilled into the airways via oropharyngeal administration. 24 hours later, infiltrating cells were collected from bronchoalveolar lavage fluid and analyzed by flow cytometry. LPS-treated groups are separated by horizontal bars. Individual animal data (circles) are shown along with the mean and SEM. Asterisks indicate significant differences (p<0.05) from the LPS / vehicle group by ANOVA followed by Dunnett's post-hoc test. [Figure 22]Figures 22A-22B show that incubation of bone marrow-derived macrophages with either 100 nM or 200 nM Fc-HRS(2-60) (Imod) during 5 days of monocytic differentiation significantly inhibited phagocyte maturation, as revealed by a dramatically reduced spectral shift, as indicated by the pH-sensitive fluorescent dye pH rhodo™. [Figure 23] Figures 23A-23B show that incubation of bone marrow-derived macrophages with 100 nM HRS(2-60)-COMP, but not the control compound COMP, during 5 days of monocytic differentiation significantly inhibited efferocytosis, as revealed by a dramatically reduced spectral shift, indicated by the pH-sensitive fluorescent dye pH rhodo™. [Figure 24] Figures 24A-24B show histological confirmation of model induction by the presence of increased inflammation (H&E) and fibrosis (Masson's trichrome) in mice treated with P. acnes (groups 3 and 4) compared to mice not treated with P. acnes (group 2). [Figure 25] Figures 25A-25B show measurements of lung inflammation (25A) and fibrosis (25B) at the end of the study. [Figure 26] Figures 26A-26H show that several pro-fibrotic cytokines in the lung were reduced in response to 3 mg / kg Fc-HRS(2-60) treatment, as indicated in the figures. [Figure 27] Figure 27A shows that in the control groups (Groups 3 and 4), mice exposed to thermophilic actinomycete S. rectivirgula had robust and consistent multifocal chronic pneumonia compared with mice exposed to PBS in Group 2. Figure 27B shows histological scores determined by a veterinary pathologist. Figure 27C shows the reduction in individual BALT areas in the Fc-HRS(2-60) 3 mg / kg group and detailed analysis of H&E-stained lung tissue sections using the HALO platform. [Figure 28]Figures 28A-28G show that several pro-inflammatory cytokines and chemokines were significantly reduced in the presence of Fc-HRS(2-60) at both 0.4 mg / kg and 3 mg / kg. [Figure 29] Figures 29A-29E show that matrix metalloproteinases (MMPs) were significantly reduced in the presence of Fc-HRS(2-60) at both 0.4 mg / kg and 3 mg / kg. [Figure 30] Figures 30A-30H show that administration of 5 mg of zymosan successfully induced inflammatory arthritis in SKG mice, and that Fc-HRS(2-60) was able to reduce the numbers of specific immune cells, especially B cells and T cells, in the lungs of SKG mice. [Figure 31] 31A-31D show the overall clinical arthritis scores on days 35, 42, 49, and 56. DETAILED DESCRIPTION OF THE INVENTION

[0070] The practice of the present invention will employ, unless specifically indicated to the contrary, standard methods of molecular biology and recombinant DNA technology within the skill of the art, many of which are described below for illustrative purposes. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); DNA Cloning: A Practical Approach, vol. & II(D.Glover,ed.);Oligonucleotide Synthesis(N.Gait,ed.,1984);Oligonucleotide Synthesis:Methods and Applications(P.Herdewijn,ed.,2004);Nucleic Acid Hybridization(B.Hames & S.Higgins,eds.,1985);Nucleic Acid Hybridization:Modern Applications(Buzdin and Lukyanov,eds.,2009);Transcription and Translation(B.Hames & S.Higgins,eds.,1984);Animal Cell Culture(R.Freshney,ed.,1986);Freshney,R.I.(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 Edition 2010);Farrell,R.,RNA Methodologies:A Laboratory Guide for Isolation and Characterization(3rd Edition 2005)、Poly(ethylene glycol),Chemistry and Biological Applications,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(Ethylene Glycols)for modification of polypeptides”in Polyethylene Glycol See Chemistry:Biotechnical and Biomedical Applications. definition

[0071] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods, materials, compositions, reagents, cells similar to, or similar to, those described herein, equivalent to, or equivalent to those described herein, can be used to implement or test the subject matter of this disclosure, preferred methods and materials are described. All publications and references cited herein, including but not limited to patents and patent applications, are incorporated herein by reference as if each individual publication or reference were specifically and individually indicated to be incorporated herein by reference in its entirety. All patent applications to which this application claims priority are also incorporated herein by reference in their entirety in the manner described above for publications and references.

[0072] For purposes of this disclosure, the following terms are defined below.

[0073] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0074] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0075] "Antagonist" or "inhibitor" refers to a biological structure or chemical agent that interferes with or otherwise reduces the physiological action of another agent or molecule. In some instances, antagonists specifically bind to other agents or molecules. Full antagonists and partial antagonists are included.

[0076] "Agonist" refers to a biological structure or chemical agent that increases or potentiates the physiological action of another agent or molecule. In some instances, agonists specifically bind to other agents or molecules. Full agonists and partial agonists are included.

[0077] The term "anergy" refers to the functional inactivation of T or B cell responses to restimulation with an antigen.

[0078] As used herein, the term "amino acid" is intended to refer to all natural and unnatural amino acids, as well as amino acid analogs and mimetics. Natural amino acids include the 20 (L)-amino acids utilized in protein biosynthesis, as well as other amino acids such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Unnatural amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to those skilled in the art. Amino acid analogs include modified forms of natural and unnatural amino acids. Such modifications include, for example, substitutions or replacements of chemical groups and chemical moieties on amino acids, or derivatization of amino acids. Amino acid mimetics include organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristics of reference amino acids. For example, organic structures that mimic arginine (Arg or R) have a similar positive charge moiety in the molecular space and have the same degree of flexibility as the e-amino group of the side chain of the natural Arg amino acid. Mimetics also include constrained structures that maintain optimal spacing and charge interactions of amino acids or amino acid functional groups. Those skilled in the art will know or can determine which structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0079] As used herein, a subject "at risk" of developing a disease or an adverse reaction may or may not have detectable disease or disease symptoms before undergoing the treatment methods described herein, and may or may not exhibit detectable disease or disease symptoms. "At risk" refers to a subject having one or more risk factors, which are measurable parameters that correlate with the development of a disease, as described herein and known in the art. A subject who has one or more of these risk factors has a higher probability of developing a disease or an adverse reaction than a subject who does not have one or more of these risk factors.

[0080] By "coding sequence" is meant any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene. In contrast, a "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of the polypeptide product of a gene.

[0081] The term "binding" refers to a direct association between two molecules, including interactions such as, for example, salt bridges and water bridges, such as by covalent interactions, electrostatic interactions, hydrophobic interactions, and ionic and / or hydrogen bonding interactions.

[0082] The term "clonal deletion" refers to the elimination (elimination or death) of autoreactive T cells. Clonal deletion can be achieved centrally in the thymus, or peripherally, or both.

[0083] The term "chemotherapeutic drug resistance" refers to the gradual change in the therapeutic sensitivity of cancer cells after exposure to cancer treatment, including resistance to at least one of cancer immunotherapeutics, chemotherapy, hormonal therapy, and / or kinase inhibitors. Chemotherapy drug resistance ultimately leads to cancer recurrence and / or metastasis, posing a challenge to improving clinical outcomes for cancer patients. Chemotherapy drug resistance remains a major challenge to the success of long-term cancer treatment. For example, approximately 30% of women diagnosed with early-stage breast cancer develop chemotherapy drug resistance and ultimately progress to metastatic breast cancer. Molecular mechanisms of chemotherapy drug resistance include the induction of transport pumps, oncogenes, tumor suppressor genes, mitochondrial alterations, DNA repair, autophagy, epithelial-mesenchymal transition (EMT), cancer stemness, and exosome production. Although these mechanisms can be manipulated separately, they ultimately work together to prevent cell death in response to chemotherapy. For example, the proteins encoded by the oncogene (EGFR-Akt-NF-κB) regulate the expression of apoptosis-related genes and may contribute to EMT, stem cell properties, and autophagy. Autophagic cells are characterized by anti-apoptotic properties during chemotherapy resistance. Therefore, agents that reduce chemotherapy resistance, including those that modulate autophagy, endosomal maturation, phagocytosis, and / or efferocytosis, may find utility in treating or reducing chemotherapy-resistant cancers.

[0084] Throughout this disclosure, unless necessarily otherwise stated, the words "comprise" and "comprising" will be understood to imply the inclusion of the stated step or element, or group of steps or elements, but not the exclusion of any other step or element, or group of steps or elements.

[0085] "Consisting of" means including, but limited to, what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements that are limited to the elements listed after the phrase, and other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present, depending on whether they do or do not materially affect the activity or function of the listed elements.

[0086] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or containers that contain at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects in a subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, e.g., bacteria, typically gram-negative bacteria, although gram-positive bacteria, such as Listeria monocytogenes, may also contain endotoxins. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), which are present in the outer membrane of various gram-negative bacteria and represent the pathogenic properties central to the ability of these bacteria to cause disease. In humans, small amounts of endotoxin can cause fever, a drop in blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0087] In pharmaceutical manufacturing, it is often desirable to remove most or all traces of endotoxin from drug products and / or drug containers, since even small amounts can cause harmful effects in humans. Temperatures above 300°C are typically required to destroy nearly all endotoxin, so a depyrogenation oven may be used for this purpose. Depending on the primary packaging material, e.g., syringe or vial, a glass temperature of 250°C combined with a 30-minute hold time is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods of endotoxin removal are also contemplated, including, for example, chromatographic and filtration methods described herein and known in the art.

[0088] Endotoxin can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes horseshoe crab blood, is a highly sensitive assay for detecting the presence of endotoxin. In this test, even very low levels of LPS can produce detectable clotting of Limulus lysate due to a powerful enzyme cascade that amplifies the clotting reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, endotoxin levels may be less than approximately 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 approximately 1-10 EU.

[0089] As used herein, the terms "contacting a cell," "introducing," or "delivering" include delivering an agent described herein (e.g., a polypeptide agent, a polynucleotide agent) into a cell by methods common in the art, such as transfection (e.g., liposomes, calcium phosphate, polyethyleneimine), electroporation (e.g., nucleofection), microinjection, or administration to a subject.

[0090] The terms "cell-penetrating peptide" (CPP) or "peptide moiety that enhances cellular uptake" are used interchangeably and refer to cationic cell-penetrating peptides, also referred to as "transport peptides," "carrier peptides," or "peptide transduction domains." In some embodiments, the peptides have the ability to induce cell (e.g., muscle cell) infiltration into about, or at least about, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in a given cell culture population, and translocate macromolecules within multiple tissues (e.g., muscle tissue) in vivo upon systemic or other administration. In some embodiments, the CPP has the formula -[(C(O)CHR'NH) m wherein R' is the side chain of a natural amino acid or its one-carbon or two-carbon homolog; R" is selected from hydrogen or acyl; and m is an integer up to 50. Additional CPPs are known in the art and are disclosed, for example, in U.S. Patent Application 2010 / 0016215, which is incorporated herein by reference in its entirety. In some embodiments, m is an integer selected from 1-50, and when m is 1, the moiety is a single amino acid or derivative thereof. Any of the polynucleotide agents (e.g., antisense, RNAi agents) described herein may be conjugated to a CPP to improve uptake into target cells, such as muscle cells.

[0091] The term "half maximal effective concentration" or "EC50" refers to the concentration of an agent described herein (e.g., an HRS polypeptide or other agent) that induces a response halfway between baseline and maximum after some specified exposure time. Thus, the EC50 of a graded dose-response curve represents the compound concentration at which 50% of its maximum effect is observed. EC50 is also the plasma concentration required to achieve 50% of its maximum effect in vivo. Similarly, "EC90" refers to the concentration of an agent or composition at which 90% of its maximum effect is observed. "EC90" can be calculated from "EC50" and the Hill slope, or can be determined directly from data using knowledge common in the art. In some embodiments, the EC50 of the agent is less than about 0.01, 0.05, 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, 100, 200, or 500 nM. In some embodiments, the biotherapeutic composition has an EC50 of about 1 nM or less.

[0092] "Homology" refers to the percentage of amino acids that are identical or that make conservative substitutions. Homology can be determined, for example, using a sequence comparison program such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this method, sequences of similar length or substantially different length to the sequences cited herein can be compared by inserting gaps in the alignment. Such gaps are determined, for example, by the comparison algorithm used in GAP.

[0093] The term "innate immune response" refers to the responses of immune cells (e.g., including macrophages, neutrophils, eosinophils, granulocytes, and bone marrow-derived cells such as natural killer (NK) cells) and mechanisms of regulating cytokine expression and release (e.g., interferon and interferon signaling), inducing cell death, and inhibiting protein synthesis that protect the host from infection by pathogens.

[0094] "Isolated" refers to a material that is substantially or essentially free from components that normally accompany it in its natural state. For example, as used herein, "isolated polynucleotide," "isolated oligonucleotide," or "isolated oligonucleotide" refers to a polynucleotide that has been purified or removed from the sequences adjacent to it in nature, and may refer to a DNA fragment that has been removed from the sequences adjacent to the fragment in a genome, for example. When referring to cells, the term "isolating" refers to the purification of cells (e.g., fibroblasts, lymphoblasts, etc.) from a source of interest (e.g., a subject with a polynucleotide repeat sequence). In the context of mRNA or protein, "isolating" refers to the recovery of mRNA or protein from a source, such as a cell.

[0095] The term "migratory cell" refers to a cell that has the ability to move from one location to another in response to a stimulus. Exemplary migratory cells include, for example, monocytes, natural killer (NK) cells, dendritic cells (immature or mature), myeloid dendritic cells, plasmacytoid dendritic cells (also called lymphoid dendritic cells), and dendritic cell subsets including Langerhans cells, macrophages, e.g., Kupffer cells, microglia of the CNS, tissue-resident macrophages such as alveolar macrophages and peripheral macrophages, e.g., M0, M1, Mox, M2a, M2b, and M Immune cells include macrophages, such as macrophage subtypes such as 2c macrophages, neutrophils, eosinophils, mast cells, basophils, plasma B cells, memory B cells, B cells including B-1 cells and B-2 cells, CD45RO (naive T) cells, CD45RA (memory T) cells, CD4 helper T cells, including Th1, Th2, and Tr1 / Th3 cells, CD8 cytotoxic T cells, regulatory T cells, gamma delta T cells, and thymocytes. Additional examples of migratory cells include fibroblasts, fibrosis-related cells, tumor cells, and stem cells. Thus, the term "cell migration" refers to the movement of migratory cells, and the term "modulation of cell migration" refers to the modulation of the movement of any such migratory cells.

[0096] The term "modulate" includes "increasing" or "decreasing" one or more quantifiable parameters, optionally by a predetermined and / or statistically significant amount. "Increase" or "increasing," "enhance" or "enhancing," or "stimulate" or "stimulating" generally refer to the ability of one or more agents or compounds to produce a greater physiological response (i.e., downstream effect) in a cell or subject compared to the response produced by either the absence of the agent / compound or a control compound. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to one of skill in the art and can include an increase in skeletal muscle mass in a tissue or subject in need thereof. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more (e.g., 500-fold, 1000-fold), including all integers and decimals therebetween and greater than 1 (e.g., 1.5, 1.6, 1.7, 1.8, etc.), over the amount produced by the absence of the agent / compound or a control compound. The terms "reducing" or "inhibiting" generally refer to the ability of one or more agents or compositions to "reduce" expression of a target gene or an associated physiological or cellular response, such as a symptom of a disease or condition, as described herein, as measured by routine techniques in the diagnostic field. The associated physiological or cellular response (in vivo or in vitro) will be apparent to one of skill in the art and can include a reduction or improvement in the symptoms or pathology of pulmonary inflammation or ILDs described herein. A "reduction" in response may be "statistically significant" compared to the absence of the agent or composition or the response produced by a control agent or composition and may include a 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% reduction, including all integers in between.

[0097] In certain embodiments, the "purity" of any given agent in a composition may be specifically defined. For example, a particular composition may contain an agent that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all fractions therebetween, as measured by, for example, but by no means limited to, high performance liquid chromatography (HPLC), a well-known form of column chromatography frequently used in the biochemistry and analytical chemistry fields to separate, identify, and quantify compounds.

[0098] "Lipid nanoparticles" or "solid lipid nanoparticles" refer to one or more spherical nanoparticles, having an average diameter of about 10 to about 1000 nanometers, comprising a solid lipid core matrix capable of solubilizing lipid-soluble molecules. The lipid core is stabilized by a surfactant (e.g., an emulsifier) ​​and may comprise one or more of a triglyceride (e.g., tristearin), a diglyceride (e.g., glycerol bahenate), a monoglyceride (e.g., glycerol monostearate), a fatty acid (e.g., stearic acid), a steroid (e.g., cholesterol), and a wax (e.g., cetyl palmitate), or a combination thereof. Lipid nanoparticles can be prepared by, for example, Petrilli et al., Curr Pharm. Biotechnol. 15:847-55, 2014, and U.S. Patent Nos. 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 by reference in their entireties.

[0099] The term "neuropilin 2-related disease" or "NRP2-related disease" refers to diseases and conditions in which the activity, expression, and / or spatial distribution of NRP2 plays a role in the pathophysiology of the disease or condition. In some cases, NRP2-related diseases are regulated by the HRS polypeptide of the present disclosure, for example, by altering the interaction of NRP2 with at least one NRP2 ligand, thereby affecting the activity, signal transduction, expression, and / or spatial distribution of NRP2. Exemplary NRP2-related diseases and conditions include, but are not limited to, 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. Also included are diseases related to inflammation and autoimmunity, such as inflammatory lung diseases, such as hypersensitivity pneumonitis and pulmonary inflammation, and related inflammatory diseases. Also included are 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). Additional examples include diseases associated with lymphatic vessel development, lymphangiogenesis, and lymphatic vessel damage, such as edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability. Also included are diseases associated with infection, including latent infection, and allergic disorders / diseases and diseases associated with allergic responses, such as chronic obstructive pulmonary disorder (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic lupus erythematosus, rheumatoid arthritis, inflammasome-related diseases, and skin-related neutrophil-mediated diseases, such as pyoderma gangrenosum. Additional examples include diseases associated with granulomatous inflammatory disorders, such as sarcoidosis, other granulomatous lung diseases, and non-pulmonary granulomas, as well as fibrotic disorders, such as endometriosis, fibrosis, endothelial-mesenchymal transition (EMT), and wound healing, among others.Further examples include the disease associated with inappropriate smooth muscle contractility and vascular smooth muscle cell migration and / or adhesion, and the disease associated with inappropriate autophagy, phagocytosis and efferocytosis.Additional examples include nerve cell diseases, including the disease associated with peripheral nervous system remodeling and pain sensation.Further examples include the disease associated with bone development and / or bone remodeling, and the disease associated with inappropriate migratory cell migration.

[0100] As used herein, "nucleobase (Nu)," "base pair moiety," or "base" are used interchangeably and refer to purine or pyrimidine residues present in natural DNA or natural RNA (uracil, thymine, adenine, cytosine, and guanine), as well as analogs of natural purines and natural pyrimidines that improve performance, such as binding affinity to oligonucleotides. Exemplary analogs include hypoxanthine (the inosine base component of nucleosides), 2,6-diaminopurine, 5-methylcytosine, C5-propynyl-modified pyrimidines, 9-(aminoethoxy)phenoxazine (G-clamp), and the like.

[0101] Further examples of base pairing moiety include but are not limited to uracil, thymine, adenine, cytosine, guanine and hypoxanthine, with amino group protected by acyl protecting group, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogues such as pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purine, xanthine or hypoxanthine (the latter two are natural degradation products).Also anticipated are the modified nucleobases disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol.7, 313.

[0102] Further examples of base pairing portion include but are not limited to the size-expanded nucleobase that one or more benzene rings are added to.The nucleobase substitutions described in Glen Research catalog (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 are also expected to be useful for oligonucleotide synthesis as described herein. Examples of size-expanded nucleobases are shown below: [ka]

[0103] Nucleobases covalently linked to ribose, sugar analogs, or morpholinos comprise nucleosides. A "nucleotide" consists of a nucleoside and one phosphate group. The phosphate groups are covalently linked to adjacent nucleotides to form oligonucleotides.

[0104] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-natural amino acids, e.g., chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers.

[0105] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. The terms typically refer to polymeric forms of nucleotides at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The terms include single- and double-stranded forms of DNA. The terms "isolated DNA," "isolated polynucleotide," and "isolated nucleic acid" refer to molecules isolated from total genomic DNA of a particular species. Thus, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences but has been substantially isolated and freed, or purified, from total genomic DNA of the species from which the DNA segment was obtained. Also included are non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode a polypeptide. Also included are recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, and the like.

[0106] Additional coding or non-coding sequences may, but need not, be present within the polynucleotides described herein, and polynucleotides may, but need not, be linked to other molecules and / or support materials. Thus, regardless of the length of the coding sequence itself, a polynucleotide, or an expressible polynucleotide, may be combined with other sequences, such as, for example, expression control sequences.

[0107] "Expression control sequences" include nucleic acid or corresponding amino acid regulatory sequences, such as promoters, leaders, enhancers, introns, RNA recognition motifs or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, and subcellular localization signals, which are capable of influencing the transcription or translation of a coding sequence in a host cell, or its intracellular or cellular location. Examples of expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0108] A "promoter" is a DNA regulatory region capable of initiating transcription of a downstream (3') coding sequence in a cell by binding RNA polymerase. As used herein, a promoter sequence is attached at its 3' end to a transcription initiation site, extends upstream (5'), and contains the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. A transcription initiation site (conveniently defined by mapping with nuclease S1) can be present within the promoter sequence, as well as within protein binding domains (consensus sequences) responsible for RNA polymerase binding. Eukaryotic promoters often, but not always, contain "TATA" and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

[0109] Many promoters, including constitutive promoters, inducible promoters, and repressible promoters, from various different sources are known in the art. Representative sources include, for example, viruses, mammalian, insect, plant, yeast, and bacterial cells. Suitable promoters from these sources are readily available or can be synthetically produced based on publicly available sequences online, or from depositories such as the ATCC, as well as other commercial or personal sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either 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 (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93(8):3346-3351); the T-REx™ system (Invitrogen, Carlsbad, CA), LacSwitch® (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., al. Nuc. Acid. Res. (1999) 27(22):4324-4327; Nuc. Acid. Res. (2000) 28(23):e99; U.S. Patent No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308:123-144), or any promoter known in the art that is suitable for expression in the desired cell.

[0110] An "expressible polynucleotide" includes cDNA, RNA, mRNA, or other polynucleotide that contains at least one coding sequence and, optionally, at least one expression control sequence, e.g., a transcriptional regulator and / or a translational regulator, which, when introduced into a cell, e.g., a cell of a subject, is capable of expressing an encoded polypeptide (e.g., an HRS polypeptide).

[0111] In some embodiments, the expressible polynucleotide is a modified RNA or mRNA polynucleotide, e.g., a non-naturally occurring RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide contains one or more modified or non-natural bases, such as a nucleotide base other than adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). In some embodiments, the modified mRNA contains one or more modified or non-natural internucleotide linkages. Expressible RNA polynucleotides for delivering encoded therapeutic polypeptides are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011; and U.S. Patent Applications 2015 / 0111248; 2014 / 0243399; 2014 / 0147454; and 2013 / 0245104, which are incorporated by reference in their entireties.

[0112] In some embodiments, various viral vectors that can be used to deliver expressible polynucleotides include adenoviral vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retroviral vectors. In some examples, the retroviral vector is a derivative of a rodent or avian retrovirus, or a lentiviral vector. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV, and Lewis sarcoma virus (RSV). Many additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate genes for selectable markers, allowing transduced cells to be identified and generated. Inserting a polypeptide sequence of interest into the viral vector along with another gene encoding a ligand for a receptor on a specific target cell can make the vector target-specific. Retroviral vectors can be made target-specific, for example, by inserting a polynucleotide encoding a protein. An exemplary targeting can be achieved by using an antibody to target the retroviral vector. Those skilled in the art will know, or can readily ascertain without undue experimentation, specific polynucleotide sequences that can be inserted into the retroviral genome to enable target-specific delivery of the retroviral vector.

[0113] In particular examples, the expressible polynucleotides described herein are engineered to localize within the cell, potentially within a particular cellular compartment such as the nucleus, or are engineered to be secreted from the cell, or are engineered to translocate to the plasma membrane of the cell. In exemplary embodiments, the expressible polynucleotides are engineered for nuclear localization.

[0114] Also included are biologically active "variants" and "fragments" of the polypeptides described herein, as well as the polynucleotides encoding them. "Variants" contain one or more substitutions, additions, deletions, and / or insertions compared to a reference polypeptide or polynucleotide (see, e.g., Tables and Sequence Listings). Variant polypeptides or polynucleotides contain amino acid or polynucleotide sequences that have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, similarity, or homology to the reference sequences described herein, and substantially retain the activity of the reference sequences. Also included are sequences that consist of, or differ from, a reference sequence by 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 acid or nucleotide additions, deletions, insertions, or substitutions, which sequences substantially retain the activity of the reference sequence. In certain embodiments, the additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0115] The term "sequence identity," or, for example, "a sequence 50% identical to," as used herein, refers to the degree to which sequences are identical nucleotide by nucleotide or amino acid by amino acid over a comparison window.Therefore, "percentage of sequence identity" can be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exists in both sequences; calculating the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size); and multiplying the result by 100 to calculate the percentage of sequence identity. Optimal alignment of sequences for alignment of a comparison window may be performed using computer-implemented algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA), or by inspection and optimal alignment (i.e., the alignment that produces the highest percentage of homology over the comparison window) performed by any of a variety of methods selected. Reference may also be made to the BLAST family of programs, as disclosed, for example, by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0116] "Statistically significant" means that the result is unlikely to occur by chance. Statistical significance can be determined by any method known in the art. A commonly used measure of significance is the p-value. The p-value is the frequency or probability that the observed event would occur if the null hypothesis were true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In a simple case, the significance level is defined as a p-value of 0.05 or less.

[0117] The term "solubility" refers to the ability of an agent provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is usually expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg / ml, etc.), molarity, molar concentration, mole fraction, or other similar concentration descriptions. The maximum equilibrium amount of solute that can be dissolved per volume of solvent is the solubility of the solute in that solvent under specific conditions, including temperature, pressure, pH, and solvent properties. In certain embodiments, solubility is measured at physiological pH or other pHs, such as pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In certain embodiments, solubility is measured in water or a physiological buffer solution, such as PBS or NaCl (with or without NaP). In particular embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a relatively high salt concentration (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent), such as blood or serum. In certain embodiments, the temperature may be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain 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.

[0118] A "subject" or "subject in need thereof" includes a mammalian subject, such as, for example, a human subject.

[0119] "Substantially" or "essentially" means entirely or completely, e.g., approaching 95% or more of some given amount.

[0120] "Therapeutic response" refers to an improvement in symptoms (regardless of duration) based on a therapeutic response to administration.

[0121] As used herein, the term "target" refers to an RNA region, specifically to the RNA region of a target gene described herein. Targets can include coding and non-coding sequences, 5' upstream sequences, 3' downstream sequences, and other RNA sequences described herein.

[0122] The term "target sequence" refers to the portion of the target RNA to which an antisense or RNAi agent is directed. For example, it refers to the sequence to which an antisense oligonucleotide hybridizes by Watson-Crick base pairing of a complementary sequence, or the sequence corresponding to the sense strand of an RNAi agent.

[0123] As used herein, the terms "quantifying," "quantitation," or other related phrases refer to determining the amount, mass, or concentration of a nucleic acid, polynucleotide, oligonucleotide, peptide, polypeptide, or protein per unit volume.

[0124] As used herein, the terms "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount" refer to the amount of agent required to elicit a desired biological response following administration. Similarly, the terms "antisense therapy" or "RNAi therapy" include therapies that maintain an average steady-state concentration of an antisense or RNAi agent in a patient's plasma or other tissue compartment (e.g., muscle tissue) that exceeds the minimum effective therapeutic level.

[0125] As used herein, "treatment" of a subject (e.g., a mammal such as a human) or cell is any type of intervention used in an attempt to alter the natural course of the subject or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be performed prophylactically, or may be performed after the onset of a pathological event, or after contact with a pathogenic agent. Also included is "prophylactic" treatment. Prophylactic treatment may aim to reduce the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete elimination, cure, or prevention of the disease or condition, or its associated symptoms.

[0126] The term "wild-type" refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and is thus optionally intended as the "normal" or "wild type" of the gene.

[0127] Specific embodiments of histidyl-tRNA synthetase (HRS) polypeptides and polynucleotides include histidyl-tRNA synthetase polypeptides ("HRS" or "HisRS" polypeptides), including conjugates (e.g., fusion proteins, Fc conjugates, Fc fusion proteins, COMP conjugates, COMP fusion proteins), variants, and fragments thereof, and expressible polynucleotides encoding HRS polypeptides. Histidyl-tRNA synthetases belong to the class II tRNA synthetase family and share three highly conserved sequence motifs. Class I and class II tRNA synthetases are widely recognized as responsible for the specific attachment of amino acids to their associated tRNAs in a two-step reaction: an amino acid (AA) is first activated by ATP to form AA-AMP, which is then transferred to the acceptor end of the tRNA. Full-length histidyl-tRNA synthetases typically exist as either cytoplasmic homodimers or spliced ​​mitochondrial forms.

[0128] Certain biological fragments or splice isoforms of eukaryotic histidyl-tRNA synthetase, or in some circumstances, the entire full-length synthetase, regulate specific therapeutically relevant cell signaling pathways, bind to one or more neuropilin polypeptides (see, for example, Table N1), and / or have anti-inflammatory properties. These activities are different from the role of classical tRNA synthetases in protein synthesis and are herein referred to as "non-canonical activities." For example, as presented herein, HRS polypeptides, such as the N-terminal region of histidyl-tRNA synthetase (e.g., HRS1-48, HRS1-60), have the ability to bind to neuropilin polypeptides, thereby regulating the migration, activation, and / or differentiation of inflammatory or migratory cells, thereby treating neuropilin-related diseases. Furthermore, compared to the full-length HRS polypeptide sequence, certain fragments, splice variants, mutations, and / or deletions (e.g., HRS1-60) may contribute to increased activity and / or improved pharmacological properties. The sequences of certain exemplary HRS polypeptides are provided in Table H1 below. [Table H1-1] [Table H1-2] [Table H1-3] [Table H1-4] [Table H1-5] [Table H1-6] [Table H1-7] [Table H1-8] [Table H1-9] [Table H1-10] [Table H1-11] [Table H1-12]

[0129] Thus, in certain embodiments, an HRS polypeptide comprises, consists of, or consists essentially of a mammalian HRS amino acid sequence of Table H1 (e.g., SEQ ID NOS: 1-116 and 172) or an active variant or fragment thereof. In some embodiments, an HRS polypeptide comprises, consists of, or consists essentially of a human HRS amino acid sequence of Table H1 (e.g., SEQ ID NOS: 1-108 and 172) or an active variant or fragment thereof. In some embodiments, an expressible polynucleotide encodes an HRS polypeptide that comprises, consists of, or consists essentially of an amino acid sequence of Table H1 (e.g., SEQ ID NOS: 1-116 and 172), e.g., a human HRS sequence of Table H1 (SEQ ID NOS: 1-108 and 172) or an active variant or fragment thereof.

[0130] As described herein, HRS polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation, addition, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the HRS reference polypeptide can be generated by mutations in the DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al. (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, J.D. et al. ("Molecular Biology of the Gene", Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and the references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0131] Biologically active truncated and / or variant HRS peptides may contain conservative amino acid substitutions at various positions along their sequence compared to the reference HRS amino acid residue. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and can generally be subclassified as follows:

[0132] Acidic: This residue has a negative charge due to the loss of an H ion at physiological pH. This residue is attracted to aqueous solutions and therefore seeks a position on the surface of the peptide's conformation when the peptide is in aqueous media at physiological pH. Amino acids with acidic side chains include glutamic acid and aspartic acid.

[0133] Basic: The residue has a positive charge due to its association with H ions at physiological pH or within 1 or 2 pH units thereof (e.g., histidine). The residue is attracted to aqueous solutions and therefore seeks a position on the surface of the peptide's conformation when the peptide is in aqueous medium at physiological pH. Amino acids with basic side chains include arginine, lysine, and histidine.

[0134] Charged: The residue is charged at physiological pH and thus includes amino acids with acidic or basic side chains (ie, glutamic acid, aspartic acid, arginine, lysine, and histidine).

[0135] Hydrophobic: This residue is uncharged at physiological pH. Because this residue is repelled by aqueous solutions, it seeks a position on the interior side of the peptide's conformation when the peptide is in aqueous media. Amino acids with hydrophobic side chains include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine, and tryptophan.

[0136] Neutral / polar: This residue is uncharged at physiological pH. However, because it is not significantly repelled by aqueous solution, it will likely seek a position on the interior side of the peptide's conformation when the peptide is in aqueous media. Amino acids with neutral / polar side chains include asparagine, glutamine, cysteine, histidine, serine, and threonine.

[0137] This disclosure further characterizes certain amino acids as "small" because their side chains are not large enough to confer hydrophobicity, even if a polar group is absent. With the exception of proline, "small" amino acids are those with four or fewer carbons when at least one polar group is present on the side chain, or three or fewer carbons when no polar group is present 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 effect on the secondary structure of peptide chains. The structure of proline differs from all other natural amino acids in that its side chain is attached to the nitrogen and α-carbon of the α-amino group. Several amino acid similarity matrices are known in the art (see, for example, the PAM120 and PAM250 matrices disclosed in Dayhoff et al., 1978, which are models of evolutionary change in proteins). However, matrices for determining distance relatedness in M. O. Mayhoff, (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) place proline in the same group as glycine, serine, alanine, and threonine, and therefore classify proline as a "small" amino acid.

[0138] The degree of attraction or repulsion required to classify as polar or nonpolar is arbitrary, and therefore, amino acids specifically contemplated by the present invention have been classified as either. Most amino acids not specifically named can be classified based on known properties.

[0139] Amino acid residues can be further subclassified as cyclic or acyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the residue's side chain substituents, and as small or large. Residues are considered small if they contain a total of four or fewer carbon atoms, including the carboxyl carbon if an additional polar substituent is present, or three or fewer carbon atoms if none. Small residues are, of course, always nonaromatic. Depending on their structural properties, amino acid residues can be divided into more than one classification. For amino acids in natural proteins, subclassification according to this scheme is shown in Table A. [Table A]

[0140] Conservative amino acid substitutions also include groupings based on side chains. For example, amino acids with aliphatic side chains include glycine, alanine, valine, leucine, and isoleucine. Amino acids with aliphatic-hydroxyl side chains include serine and threonine. Amino acids with amide-containing side chains include asparagine and glutamine. Amino acids with aromatic side chains include phenylalanine, tyrosine, and tryptophan. Amino acids with basic side chains include lysine, arginine, and histidine. And amino acids with sulfur-containing side chains include cysteine ​​and methionine. For example, substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, serine with threonine, or similar substitutions of structurally related amino acids do not significantly affect the properties of the resulting variant polypeptide, and therefore, it is reasonable to exclude them. Whether an amino acid change results in a functional truncated and / or variant HRS polypeptide can be readily determined by assaying its non-canonical activity, as described herein. Conservative substitutions are shown in Table B under the heading of exemplary substitutions. Amino acid substitutions within the scope of the invention are generally achieved by selecting substituents that do not significantly alter (a) the structure of the peptide backbone in the region of the substitution, (b) the charge or hydrophobicity of the target site molecule, (c) the bulk of the side chain, or (d) maintain biological function. After the substitutions are introduced, the variants are screened for biological activity. [Table B-1] [Table B-2]

[0141] Alternatively, similar amino acids for conservative substitution can be grouped into three categories based on the identity of their side chains. As described in Zubay, G., Biochemistry, Third Edition, Wm. C. Brown Publishers (1993), 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. The third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, and methionine.

[0142] In some embodiments, the HRS polypeptide has one or more cysteine ​​insertions or substitutions, e.g., one or more non-cysteine ​​residues are substituted with cysteine ​​residues (e.g., to alter stability, facilitate thiol-based conjugation of an Fc fragment, facilitate thiol-based addition of PEG or other molecules). In some embodiments, the one or more cysteine ​​substitutions are near the N-terminus and / or C-terminus of the HRS polypeptide, or in other surface-exposed regions of the HRS polypeptide. Specific embodiments include those in which 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 of the N-terminus and / or C-terminus of the HRS polypeptide are substituted with cysteine ​​residues. In some embodiments, cysteine ​​residues may be added to the HRS polypeptide via the generation of an N- or C-terminal fusion protein. Such fusion proteins can be of any length, but are typically about 1-5, or about 5-10, about 10-20, or about 20-30 amino acids in length.

[0143] Specific examples of cysteine ​​engineered proteins are shown in Table H2. The table is based on the HRS polypeptide HRS(1-60). This method can also be applied to the HRS polypeptides of Table H1 and other HRS polypeptides described herein. [Table H2-1] [Table H2-2]

[0144] Thus, in certain embodiments, an HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence of Table H2 (e.g., SEQ ID NOs:117-119), or an active variant or fragment thereof. In some embodiments, an expressible polynucleotide encodes an HRS polypeptide comprising, consists of, or consists essentially of an amino acid sequence of Table H2 (e.g., SEQ ID NOs:117-119), or an active variant or fragment thereof.

[0145] In some embodiments, the HRS polypeptide has a mutation in which an endogenous or natural cysteine ​​residue is mutated to an alternative amino acid or deleted. In some embodiments, the insertion or substitution of a cysteine ​​residue into the HRS polypeptide is combined with the elimination of other surface-exposed reactive cysteine ​​residues. Thus, in some embodiments, the HRS polypeptide comprises 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 by SEQ ID NO: 1), thereby removing the natural cysteine ​​residues, including, for example, a combination thereof.

[0146] Certain embodiments include HRS polypeptides of Table H1 having a mutation or deletion of any one or more of Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, Cys455, or a deletion of Cys507 and Cys509 by deleting the C-terminal three amino acids (Δ507-Δ509). Examples of mutations at these positions include, for example, a cysteine ​​to serine, alanine, leucine, valine, or glycine mutation. In certain embodiments, the amino acid residue for a particular cysteine ​​substitution can be selected from naturally occurring substitutions present in HRS orthologs from other species or organisms. Examples of this type of substitution are provided in Table H3. [Table H3-1] [Table H3-2]

[0147] In some embodiments, the native cysteines selected for mutagenesis are selected 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, resulting in a deletion of residues 507-509. In some embodiments, cysteines are selected for mutation or deletion to eliminate an intramolecular cysteine ​​pair, such as Cys174 and Cys191.

[0148] Specific examples of cysteine ​​mutations / substitutions (shown in bold and underlined) to reduce surface-exposed cysteine ​​residues include those listed in Table H4 below. [Table H4-1] [Table H4-2]

[0149] Thus, in certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence of Table H4 (SEQ ID NOS:120-126), or an active variant or fragment thereof. In some embodiments, the expressible polynucleotide encodes an HRS polypeptide comprising, consists of, or consists essentially of an amino acid sequence of Table H4 (e.g., SEQ ID NOS:120-126), or an active variant or fragment thereof.

[0150] In some embodiments, such cysteine ​​substitution mutations are modified to incorporate, insert, or otherwise introduce new surface-exposed cysteine ​​residues at predetermined surface-exposed positions, such that the introduced residues do not significantly interfere with the non-canonical activities of the HRS polypeptide. Specific examples include, for example, inserting (or reinserting) additional cysteine ​​residues at the N- or C-terminus of any of the above-described cysteine-reduced HRS polypeptides. In some embodiments, such insertion of N- or C-terminal surface-exposed cysteines includes reinserting the last one, last two, or last three naturally occurring C-terminal amino acids of full-length human HRS into a cysteine-reduced variant of the HRS polypeptide, e.g., reinserting all or part of the sequence CIC (Cys Ile Cys). Examples of cysteine-reducing mutations include, for example, any combination of mutations (or deletions) at residues Cys174, Cys191, Cys224, and Cys235 of any of the HRS polypeptides of Table H1, or deletions or substitutions of Cys507 and Cys509 (based on the numbering of full-length human cytoplasmic HRS (SEQ ID NO: 1)).

[0151] For some types of site-specific conjugation or attachment to a heterologous molecule, such as an Fc region or PEG or other heterologous molecule, the HRS polypeptide may have one or more glutamine substitutions, in which one or more naturally occurring (non-glutamine) residues are substituted with glutamine to facilitate transglutaminase-catalyzed addition of the molecule to, for example, the amide group of glutamine. In some embodiments, glutamine substitutions are introduced near the N-terminus and / or C-terminus of the HRS polypeptide. Specific embodiments include those in which 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 of the N-terminus and / or C-terminus of the HRS polypeptide are substituted with glutamine residues. These and related HRS polypeptides may also contain substitutions (e.g., conservative substitutions) to remove any native glutamine residues, if desired, and to control the degree of site-specific conjugation or attachment.

[0152] For certain types of site-specific conjugation or attachment to heterologous molecules, such as Fc regions or PEG or other heterologous molecules, the HRS polypeptide may have one or more lysine substitutions, in which one or more naturally occurring (non-lysine) residues are substituted with lysine to facilitate, for example, alkylation-based attachment of molecules to the amino groups of lysine. Typically, these methods result in attachment of molecules to the N-terminal residue. In some embodiments, the lysine substitutions are near the N-terminus and / or C-terminus of the HRS polypeptide. Specific embodiments include those in which 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 of the N-terminus and / or C-terminus of the HRS polypeptide are substituted with lysine residues. These and related HRS polypeptides may also contain substitutions (e.g., conservative substitutions) to remove any naturally occurring lysine residues, if desired, and to control the degree of site-specific conjugation or attachment.

[0153] Site-specific conjugation to an HRS polypeptide may be achieved by substituting one or more adjacent soluble surface amino acids of the HRS polypeptide. For example, suitable adjacent soluble amino acids may be determined based on solvent accessibility predictions using the SPIDDER server (http: / / sppider.cchmc.org / ) using published crystal structures of exemplary HRS polypeptides (see Xu et al., Structure. 20:1470-7, 2012; and U.S. Patent 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. Surface accessibility scores for amino acids based on the crystal structure can also be calculated, with higher scores indicating better accessibility. In certain embodiments, high scores (e.g., >40) are preferred. According to some embodiments, amino acid positions with a surface accessibility score greater than 40 may be used to introduce cysteine, lysine, glutamine, or other unnatural amino acids.

[0154] In certain embodiments, the solvent-accessible surface amino acids are selected from the group consisting of alanine, glycine, and serine, and may be substituted with natural amino acids, including but not limited to, cysteine, glutamine, or lysine, or non-natural amino acids that are optimized for site-specific conjugation or attachment.

[0155] Certain embodiments include site-specific conjugation or addition to an HRS polypeptide at any amino acid position by substituting a non-natural amino acid containing a functional group that forms a covalent bond with a functional group attached to an Fc region or a heterologous molecule, such as PEG or another heterologous molecule. The non-natural amino acid 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 of the N-terminus and / or C-terminus of an HRS polypeptide described herein, at the N-terminus and / or C-terminus, or at a solvent-accessible surface amino acid residue.

[0156] In certain embodiments, unnatural amino acids include, but are not limited to, selenocysteine ​​and any amino acid, modified amino acid, or amino acid analog other than the 20 genetically encoded alpha amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine. The generic structure of the alpha amino acids is shown in the following formula: [ka]

[0157] Unnatural amino acids are often any structure having the aforementioned formula, where the R group is any substituent other than those used in the 20 natural amino acids. For example, for the structures of the 20 natural amino acids, see biochemistry texts such as *Biochemistry* by L. Stryer, 3rd ed. 1988, Freeman and Company, New York. Note that the unnatural amino acids disclosed herein can be natural compounds other than the 20 alpha-amino acids described above. The unnatural amino acids disclosed herein often differ from natural amino acids only in their side chains, such that the unnatural amino acids form amide bonds, e.g., with other amino acids, natural or unnatural, in the same manner as they are formed in natural proteins. However, the unnatural amino acids have side chain groups that distinguish them from the natural amino acids. For example, R in the preceding formula optionally includes alkyl, aryl, aryl halide, vinyl halide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl, keto, azide, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boronic acid, boronate ester, borane, phenylboronic acid, thiol, seleno, sulfonyl, borate, boronate, phospho, phosphono, phosphine, heterocyclic, pyridyl, naphthyl, benzophenone, constrained ring such as cyclooctyne, thioester, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid, alpha ketocarboxylic acid, alpha or beta unsaturated acids and amides, glyoxylamide, or organosilane group, or any combination thereof.

[0158] Specific examples of unnatural 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, L-Dopa, fluorine p-amino-L-phenylalanine, isopropyl-L-phenylalanine, ...

[0159] Thus, unnatural amino acids may be selected that contain a functional group that will form a covalent bond with any desired functional group on a desired molecule (e.g., an Fc region, PEG). Once selected, the unnatural amino acids can be purchased from a vendor or chemically synthesized. Any number of unnatural amino acids may be incorporated into a target molecule, and the number of unnatural amino acids may vary depending on the number of desired molecules to be added. Molecules may be added to all or only a portion of the unnatural amino acids. Additionally, the same or different unnatural amino acids may be incorporated into an HRS polypeptide depending on the desired result. In certain embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unnatural amino acids may be incorporated into an HRS polypeptide, all or any of which may be attached to a molecule containing a desired functional group.

[0160] In certain embodiments, the use of unnatural amino acids can be utilized to modify (e.g., increase) selected non-canonical activities of HRS polypeptides or to alter the in vivo or in vitro half-life of the protein. Furthermore, unnatural amino acids can be used to facilitate (selective) chemical modification (e.g., pegylation) of HRS polypeptides, as described herein. For example, certain unnatural amino acids allow for the selective addition of an Fc region or a polymer such as PEG to a given protein, thereby improving its pharmacokinetic properties.

[0161] Specific examples of amino acid analogs or mimetics can be found, for example, in Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, NY (1983), which is incorporated herein by reference in its entirety. Other examples include peralkylated amino acids, particularly permethylated amino acids. See, for example, Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, John Wiley & Sons Inc., New York, NY (1997), which is incorporated herein by reference in its entirety. Still other examples include amino acids whose amide moiety (and therefore the amide backbone of the resulting peptide) is substituted with a sugar ring, a steroid, a benzodiazepine, or a hydrocarbon ring. See, for example, Burger's Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, NY (1995). The entirety of this bibliography is incorporated herein by reference. Methods for the synthesis of peptides, polypeptides, peptidomimetics and proteins are also known in the art (see, for example, U.S. Patent No. 5,420,109; Chapter 7 of M. Bodanzsky, Principles of Peptide Synthesis (1st ed. & 2nd rev. ed.), Springer-Verlag, New York, NY (1984 & 1993); Stewart and Young, Solid Phase Peptide Synthesis, (2nd ed.), Pierce Chemical Co., Rockford, Ill. (1984). Each of these documents is incorporated herein by reference). Thus, HRS polypeptides can be composed of natural and unnatural amino acids, as well as amino acid analogs and mimetics.

[0162] In certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of the smallest active fragment of a full-length HRS polypeptide that has the ability to modulate anti-inflammatory activity in vivo or has neuropilin polypeptide binding activity. In some embodiments, such a smallest active fragment comprises, consists of, or consists essentially of the WHEP domain (e.g., about amino acids 1-43 of SEQ ID NO: 1) or an active variant or active fragment thereof.

[0163] In certain embodiments, the HRS polypeptide has a nucleotide sequence of about, at least about, and / or up to 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, 54, 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, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, , 48, 49, 50, 51, 52, 53, 54, 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, 4 506, 507, 508 or 509 amino acids in length and comprising, consisting of or consisting essentially of an amino acid sequence of Table H1, Table H2 or Table H4.

[0164] In certain embodiments, the HRS polypeptide possesses at least one non-canonical activity, such as, for example, anti-inflammatory activity or binding to a neuropilin polypeptide, examples of which are described herein. Assays for determining anti-inflammatory activity or neuropilin polypeptide binding or receptor binding, including routine in vitro cell-based cytokine release assays and animal studies, are well established in the art (see, e.g., Wittmann et al., J Vis Exp.(65):e4203.doi:10.3791 / 4203, 2012; Feldman et al., Mol Cell.47:585-95, 2012; Clutterbuck et al., J Proteomics.74:704-15, 2011; Giddings and Maitra, J Biomol Screen.15:1204-10, 2010; Wijnhoven et al., Glycoconj J.25:177-85, 2008; and Frow et al., Med Res Rev.24:276-98, 2004), and can be readily used to analyze and optimize anti-inflammatory activity. Exemplary in vivo experimental systems are also described in the accompanying Examples.

[0165] It will be appreciated that in any of the HRS polypeptides, the N-terminal amino acid of the HRS polypeptide (eg, the N-terminal Met) can be deleted and replaced with a different amino acid.

[0166] In some embodiments, fusion proteins of HRS polypeptides with other (non-HARS) proteins (e.g., heterologous proteins or polypeptides) are also included, and these fusion proteins may modulate the biological activity, secretion, antigenicity, targeting, biological useful life, ability to penetrate cell membranes or the blood-brain barrier, or pharmacokinetic properties of the HRS polypeptides. Fusion proteins that improve pharmacokinetic properties (PK modifiers) include, but are not limited to, human albumin (Osborn et al.: Eur. J. Pharmacol. 456(1-3):149-158, (2002)), the Fc domain of an antibody, a polyGlu or polyAsp sequence, and transferrin. Furthermore, fusion with a conformationally disordered polypeptide sequence composed of the amino acids Pro, Ala, and Ser (PASylation) or hydroxyethyl starch (sold under the trademark HESYLATION®) provides a simple method for increasing the hydrodynamic volume of HRS polypeptides. This additional elongation introduces large random structures, resulting in a significant increase in the size of the resulting fusion protein. By this means, the typically rapid clearance of small HRS polypeptides via renal filtration is delayed by several orders of magnitude. Furthermore, using IgG fusion proteins, it has been shown that some fusion proteins can cross the blood-brain barrier (Fu et al., (2010) Brain Filtration). Res.1352:208-13).

[0167] Examples of fusion proteins that modulate antigenicity or other properties of HRS polypeptides include fusions to T cell binding ligands, including MHC class I and II proteins, b-2 microglobulin, LFA-3 portions, Fc region portions of heavy chains, and conjugates and derivatives thereof. Examples of such fusion proteins are described in EP 1 964 854; U.S. Patent Nos. 5,468,481; 5,130,297; 5,635,363; and 6,451,314; and U.S. Patent Application 2009 / 0280135.

[0168] In some embodiments, the HRS polypeptide can contain a synthetic or natural secretory signal sequence derived from another well-characterized secreted protein. In some embodiments, such proteins are processed by proteolytic cleavage to form the HRS polypeptide in situ. In some embodiments, the HRS polypeptide contains a heterologous proteolytic cleavage site, allowing for in situ expression and production of the HRS polypeptide in either an intracellular or extracellular location. Other fusion proteins include, for example, fusing the HRS polypeptide to ubiquitin to generate a new N-terminal amino acid, or using a secretion signal to mediate high-level secretion of the HRS polypeptide into the extracellular medium, or using N- or C-terminal epitope tags to improve purification or detection, and fusion to a cell-penetrating peptide.

[0169] In certain embodiments, the use of unnatural amino acids can be utilized to modify (e.g., increase) selected non-canonical activities of HRS polypeptides or to alter the in vivo or in vitro half-life of the protein. Additionally, unnatural amino acids can be used to facilitate (selective) chemical modification (e.g., pegylation) of HRS proteins, as described elsewhere herein. For example, certain unnatural amino acids allow for the selective addition of polymers such as PEG to a given protein, thereby improving its pharmacokinetic properties.

[0170] Certain embodiments include HRS-Fc conjugates, which comprise at least one Fc region covalently linked to one or more HRS polypeptides. Examples of HRS-Fc conjugates include various forms of fusion proteins and chemically crosslinked proteins. Various Fc region sequences may be employed in HRS-Fc conjugates, including wild-type sequences from any number of species, as well as variants, fragments, hybrids, and chemically modified versions thereof. The HRS-Fc polypeptide may further (optionally) comprise one or more linkers, which typically separate the Fc region from the HRS polypeptide and include peptide and chemical linkers described herein and known in the art. It should be recognized that in any of these HRS-Fc conjugates, the native N- or C-terminal amino acids of the HRS polypeptide, or the native N- or C-amino acids in the Fc domain, may be deleted and / or substituted with non-natural amino acids to facilitate expression and / or cloning or to serve as a linker sequence between the two proteins.

[0171] HRS-Fc conjugate polypeptides may offer various advantages over unconjugated or unmodified HRS polypeptides, such as corresponding HRS polypeptides of the same or similar sequence without the added Fc region. For illustrative purposes, the co-incorporation of one or more Fc regions can alter (e.g., increase or decrease) the solubility, half-life (e.g., in serum, in selected tissues, or in a test tube under storage conditions, e.g., room temperature or refrigerated), dimerization or multimerization properties, or biological activity of the HRS polypeptide compared to an unmodified HRS polypeptide having the same or similar sequence, by, for example, providing Fc region-associated effector functions (e.g., activation of the classical complement cascade, interaction with immune effector cells via Fc receptors (FcRs), immunoglobulin compartmentalization), cellular uptake, intracellular trafficking, tissue distribution, and / or bioavailability. In certain embodiments, the Fc region can contribute to 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 an important role in the elimination of specific target cells, such as tumor cells or infected cells.

[0172] Certain embodiments employ HRS-Fc fusion proteins. "Fusion proteins" are defined separately herein and are known in the art, as are methods for producing fusion proteins (for example, for reviews and methods related to Fc fusion proteins, see 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 the 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 fused internally to the HRS sequence, for example, by placing the Fc region between a first HRS sequence (e.g., domain) and a second HRS sequence (e.g., domain), where 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 certain embodiments, the first and second HRS sequences are identical. In some embodiments, the first and second HRS sequences are different (e.g., comprise different functional domains of the HRS polypeptide). Certain HRS-Fc fusion proteins may further comprise additional heterologous protein sequences, i.e., non-Fc region sequences and non-HRS polypeptide sequences.

[0173] The term "HRS-Fc" can, but does not necessarily, indicate that an Fc region has been added to an HRS polypeptide at the N-terminus or C-terminus. For example, in a specific example, the term "Fc-HRS" indicates that an Fc region has been fused to the N-terminus of an HRS polypeptide, and the term "HRS-Fc" indicates that an Fc region has been fused to the C-terminus of an HRS polypeptide. However, either term can be used more generally to refer to any fusion protein or conjugate of an Fc region and an HRS polypeptide.

[0174] In some embodiments, an HRS-Fc fusion protein may comprise tandem repeat copies of an HRS polypeptide linked to a single Fc domain, optionally separated by a linker peptide. Exemplary tandem repeat HRS-Fc fusion proteins are provided in Table H5. The preparation and sequences of specific tandem repeat HRS-Fc conjugates are described in the Examples. [Table H5]

[0175] Certain embodiments relate to HRS-Fc conjugates, in which, for example, one or more Fc regions are chemically conjugated or crosslinked to an HRS polypeptide. In these and related embodiments, the Fc region may be conjugated to the HRS polypeptide at the N-terminal region (e.g., the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or so amino acids), at an internal region (between the N-terminal and C-terminal regions), and / or at the C-terminal region (the last 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or so amino acids). Polypeptides may be conjugated or crosslinked to other polypeptides using a variety of techniques commonly known in the art. For example, certain techniques employ the carboxyl-reactive carbodiimide crosslinker EDC (or EDAC), which covalently couples via the D-, E-, and C-terminal carboxyl groups. Other techniques employ activated EDC, which covalently couples via the K- and N-terminal amino groups. Yet other techniques employ m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or sulfo-MBS, which covalently couple via thiol groups or cysteine ​​residues (see also U.S. Patent Application Publication No. 2007 / 0092940 for cysteine-engineered Ig domains that can be used for thiol coupling). Such cross-linked proteins may further comprise linkers, including cleavable or otherwise releasable linkers (e.g., enzyme-cleavable linkers, hydrolyzable linkers), and non-cleavable linkers (i.e., physiologically stable linkers). Certain embodiments may employ non-peptide polymers as cross-linkers between the Fc domain and the HRS polypeptide (e.g., PEG polymers; HRS-N-PEG-N-Fc conjugates), for example, as described in U.S. Patent Application Publication No. 2006 / 0269553. See also U.S. Patent Application Publication No. 2007 / 0269369 for a discussion of Fc domain binding sites.

[0176] In certain embodiments, as discussed in more detail below, variant or otherwise modified Fc regions may be employed, including Fc regions that have altered properties or altered biological activity compared to the wild-type Fc region. Examples of modified Fc regions include Fc regions that have altered sequence compared to the wild-type sequence, e.g., by one or more amino acid substitutions, insertions, deletions, or truncations, hybrid Fc polypeptides composed of domains from different immunoglobulin classes / subclasses, Fc polypeptides with altered glycosylation / sialylation patterns, and Fc polypeptides that have been modified or derivatized, e.g., by biotinylation (see, e.g., U.S. Patent Application 2010 / 0209424), phosphorylation, sulfation, or any combination thereof. Such modifications can be employed to improve, among other properties described herein, the binding ability of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn), its pharmacokinetic performance (e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, C max , t max , C min , variation), immunogenicity, complement fixation or activation, and / or CDC / ADCC / ADCP-related activity of the Fc region may be altered (e.g., increased, decreased) compared to the corresponding wild-type Fc sequence.

[0177] The "Fc region" of the HRS-Fc conjugates presented herein is usually derived from the heavy chain of an immunoglobulin (Ig) molecule. A typical Ig molecule is composed of two heavy chains and two light chains. The heavy chain is divided into at least three functional regions: the Fd region, the Fc region (fragment crystallizable region), and the hinge region. The latter is present only in IgG, IgA, and IgD immunoglobulins. The Fd region is the variable (V) region of the heavy chain. H ) domain and constant (CH1) domain, and the light chain variable (V L ) domain and stationary (C L ) domains together form the antigen-binding fragment of the Fab region.

[0178] The Fc region of IgG, IgA, and IgD immunoglobulins comprises heavy chain constant domains 2 and 3, designated CH2 and CH3, respectively. The Fc region of IgE and IgM immunoglobulins comprises heavy chain constant domains 2, 3, and 4, designated CH2, CH3, and CH4, respectively. The Fc region is primarily responsible for the effector functions of the immunoglobulin, including, for example, complement fixation and binding to cognate Fc receptors on effector cells.

[0179] The hinge region (present in IgG, IgA, and IgD) serves as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region. In contrast to the constant region, the hinge region is structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. The hinge region may also contain one or more glycosylation sites, which contain many structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of the hinge region, thereby rendering the hinge region polypeptide exceptionally resistant to gastrointestinal proteases. Residues in the hinge-proximal region of the CH2 domain can also influence the specificity of the interaction between immunoglobulins and their respective Fc receptors (see, e.g., Shin et al., Intern. Rev. Immunol. 10:177-186, 1993).

[0180] Thus, as used herein, the term "Fc region" or "Fc fragment" or "Fc" refers to a protein comprising one or more CH2, CH3, and / or CH4 regions from one or more selected immunoglobulins, including fragments and variants and combinations thereof. An "Fc region" may further comprise one or more hinge regions of the heavy chain constant region of an immunoglobulin. In certain embodiments, an Fc region comprises the CH1, CH2, CH3, and CH4 regions of an immunoglobulin. L , V L , and / or VH Does not include one or more of the regions.

[0181] The Fc region may be derived from the CH2 region, CH3 region, CH4 region, and / or hinge region of any one or more immunoglobulin classes, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, including subclasses and combinations thereof. In some embodiments, the Fc region is derived from an IgA immunoglobulin, including the IgA1 and / or IgA2 subclasses. In certain embodiments, the Fc region is derived from an IgD immunoglobulin. In certain embodiments, the Fc region is derived from an IgE immunoglobulin. In some embodiments, the Fc region is derived from an IgG immunoglobulin, including the IgG1, IgG2, IgG2, IgG3, and / or IgG4 subclasses. In certain embodiments, the Fc region is derived from an IgM immunoglobulin.

[0182] Certain Fc regions exhibit specific binding to one or more Fc receptors (FcR). Exemplary classes of Fc receptors include Fcγ receptors (FcγR), Fcα receptors (FcαR), Fcε receptors (FcεR), and neonatal Fc receptors (FcRn). For example, certain Fc regions have increased binding (or affinity) to one or more FcγRs relative to FcαR, FcεR, and / or FcRn. In some embodiments, an Fc region has increased binding to FcαR relative to one or more FcγRs, FcεR, and / or FcRn. In some embodiments, an Fc region has increased binding to FcεR (e.g., FcαRI) relative to one or more FcγRs, FcαR, and / or FcRn. In some embodiments, an Fc region has increased binding to FcRn relative to one or more FcγRs, FcαR, and / or FcεR. In certain embodiments, the binding (or affinity) of the Fc region to one or more selected FcRs is increased, typically by about 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x or more (including all integers therebetween) compared to the binding (or affinity) to one or more different FcRs.

[0183] 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 endocytic trafficking of dendritic cells. FcγRI expression is upregulated by both GM-CSF and gamma interferon (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 gamma-IFN and downregulated by IL-4. FcγIIb is expressed on B cells, PMNs, macrophages, and mast cells. FcγIIb inhibits immunoreceptor tyrosine-based activation motif (ITAM)-mediated responses and is therefore an inhibitory receptor. FcγRIIc expression is upregulated by intravenous immunoglobulin (IVIG) and IL-4 and decreased 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, 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.

[0184] Some Fc regions have increased binding to FcγRI compared to FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. In some embodiments, increased binding to FcγRIIa compared to FcγRI, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Certain Fc regions have increased binding to FcγRIIb compared to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Some Fc regions have increased binding to FcγRIIc compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and / or FcγRIIIb. Some Fc regions have increased binding to FcγRIIIa relative to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and / or FcγRIIIb. Certain Fc regions have increased binding to FcγRIIIb relative to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and / or FcγRIIIa.

[0185] FcαR includes FcαRI (CD89), which is present on the surface of neutrophils, eosinophils, monocytes, certain macrophages (e.g., Kupffer cells), and certain dendritic cells. FcαRI is composed of two extracellular Ig-like domains and is associated with the immunoglobulin superfamily and multi-chain immune recognition receptors (MIRRs). It is a member of both FcRγ signaling chains (FcRγ signaling chains) and signals by associating with the two FcRγ signaling chains.

[0186] FcεR includes FcεRI and FcεRII. The high-affinity receptor, FcεRI, is a member of the immunoglobulin superfamily and is expressed on epidermal Langerhans cells, eosinophils, mast cells, and basophils, where it plays a major role in regulating 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 proliferation and differentiation and blocks IgE binding on eosinophils, monocytes, and basophils. Some Fc regions exhibit enhanced binding to FcεRII compared to FcεRII. Other Fc regions exhibit enhanced binding to FcεRII compared to FcεRI. Table H6 below summarizes the characteristics of certain FcRs. [Table H6-1] [Table H6-2] [Table H6-3]

[0187] The Fc region may be derived from an immunoglobulin molecule of any animal, including vertebrates such as mammals, for example, cows, goats, pigs, dogs, mice, rabbits, hamsters, rats, guinea pigs, non-human primates, and humans. The amino acid sequences of the CH2, CH3, CH4, and hinge regions from exemplary wild-type human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM immunoglobulins are shown in Table H7. [Table H7-1] [Table H7-2]

[0188] The Fc region of the HRS-Fc conjugate may comprise, consist of, or consist essentially of one or more of the human Fc region amino acid sequences of Table H7, including variants, fragments, homologs, orthologs, paralogs, and combinations thereof. Specific exemplary embodiments comprise an Fc region 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 acids in length, and optionally comprising, consisting of, or consisting essentially of any one or more of the sequences in Table H7. In certain embodiments, the Fc region comprises 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, optionally comprising, consisting of, or consisting essentially of any one or more of the amino acid sequences in Table H7.

[0189] Particular Fc regions comprise, consist of, or consist essentially of the human IgA1 sequences of Table H7, read in any order from N-terminus to C-terminus, including combinations thereof, and variants and fragments thereof. Particular Fc regions comprise, consist of, or consist essentially of the human IgA1 sequences of Table H7. Particular Fc regions comprise, consist of, or consist essentially of the human IgA1 sequences of Table H7. Particular Fc regions comprise, consist of, or consist essentially of the human IgA1 sequences of Table H7.

[0190] Some Fc regions comprise, consist of, or consist essentially of the human IgA2 sequences of Table H7, taken in any order from N-terminus to C-terminus, including combinations thereof, and variants and fragments thereof. Particular Fc regions comprise, consist of, or consist essentially of the human IgA2 sequences of Table H7. Particular Fc regions comprise, consist of, or consist essentially of the human IgA2 sequences of Table H7. Particular Fc regions comprise, consist of, or consist essentially of the human IgA2 sequences of Table H7.

[0191] Particular Fc regions comprise, consist of, or consist essentially of the human IgD sequences of Table H7, read in any order from N-terminus to C-terminus, including combinations thereof, and variants and fragments of these sequences and combinations. Particular Fc regions comprise, consist of, or consist essentially of the human IgE sequences of Table H7, read in any order from N-terminus to C-terminus, including combinations thereof, and variants and fragments of these sequences and combinations. Particular Fc regions comprise, consist of, or consist essentially of the human IgG1 sequences of Table H7, read in any order from N-terminus to C-terminus, including combinations thereof, and variants and fragments of these sequences and combinations. Particular Fc regions comprise, consist of, or consist essentially of the human IgG2 sequences of Table H7, read in any order from N-terminus to C-terminus, including combinations thereof. Particular Fc regions comprise, consist of, or consist essentially of the human IgG3 sequences of Table H7, read in any order from N-terminus to C-terminus, including combinations thereof. Particular Fc regions comprise, consist of, or consist essentially of the human IgG4 sequences of Table H7, read in any order from N-terminus to C-terminus, including combinations thereof. Particular Fc regions comprise, consist of, or consist essentially of the human IgM sequences of Table H7, read in any order from N-terminus to C-terminus, including combinations thereof, and variants and fragments of these sequences and combinations. Exemplary HRS-Fc fusion proteins are shown in Table H8 below. [Table H8-1] [Table H8-2] [Table H8-3]

[0192] Thus, in certain embodiments, the HRS polypeptide is fused or otherwise linked to an Fc region and comprises, consists of, or consists essentially of an amino acid sequence of Table H8 (SEQ ID NOS:156-171), or an active variant or fragment thereof. In some embodiments, an expressible polynucleotide encodes an HRS polypeptide that comprises, consists of, or consists essentially of an amino acid sequence of Table H8 (e.g., SEQ ID NOS:156-171), or an active variant or fragment thereof.

[0193] As noted above, certain embodiments employ variants, fragments, hybrids, and / or otherwise modified forms of Fc regions described herein and known in the art. Also included are variants having one or more amino acid substitutions, insertions, deletions, and / or truncations compared to a reference sequence, such as any one or more of the reference sequences in Table H7 or Table H8. Polypeptide and polynucleotide variants are described elsewhere herein.

[0194] Also included are hybrid Fc regions, such as Fc regions that contain a combination of Fc domains (eg, hinge, CH2, CH3, CH4) derived from immunoglobulins of different species, different Ig classes, and / or different Ig subclasses.Common examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following CH2 / CH3 domain combinations: 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 / IgG 4, IgA2 / IgM, IgD / 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, I gG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG 3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / I 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 a fragment or variant thereof), and optionally a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2 domain, CH3 domain, and CH4 domain are derived from human Ig.

[0195] Additional examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following CH2 / CH4 domain combinations: 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 IgIg / IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or a fragment or variant thereof), and optionally a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In certain embodiments, the hinge, CH2 domain, CH3 domain, and CH4 domain are derived from human Ig.

[0196] Particular examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following CH3 / CH4 domain combinations: 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 IgIg / IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or a fragment or variant thereof), and optionally a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In certain embodiments, the hinge, CH2 domain, CH3 domain, and CH4 domain are derived from human Ig.

[0197] Particular examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following hinge / CH2 domain combinations: 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 ...4, IgA1 / IgG5, IgA1 / IgG6, IgA1 / IgG7, IgA1 / IgG8, IgA1 / IgG9, IgA1 / IgG10, IgA1 / IgG11, IgA1 / IgG12, IgA1 / IgG13, IgA1 / IgG14, IgA1 / IgG15, IgA1 / IgG16, IgA1 / IgG17, IgA1 / IgG18, IgA1 / IgG19, IgA1 / IgG20, IgA1 / IgG21, IgA1 / IgG22, IgA1 / IgG23, IgA1 / IgG24, IgA1 / IgG25, IgA1 / IgG26, IgA1 / IgG27, IgA1 / IgG28, IgA1 / IgG29, IgA1 / IgG31, IgA1 / IgG32, IgA1 / IgG33, IgA1 / IgG34, IgA1 / IgG35, IgA1 / IgG36, IgA1 / IgG37, IgA1 / gA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / I gG4, 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 / IgM, IgG4 / IgA1, IgG IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM (or a fragment or variant thereof), and optionally a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2 domain, CH3 domain, and CH4 domain are derived from a human Ig.

[0198] Particular examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following hinge / CH3 domain combinations: 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 ...4, IgA1 / IgG5, IgA1 / IgG6, IgA1 / IgG7, IgA1 / IgG8, IgA1 / IgG9, IgA1 / IgG10, IgA1 / IgG11, IgA1 / IgG12, IgA1 / IgG13, IgA1 / IgG14, IgA1 / IgG15, IgA1 / IgG16, IgA1 / IgG17, IgA1 / IgG18, IgA1 / IgG19, IgA1 / IgG20, IgA1 / IgG21, IgA1 / IgG22, IgA1 / IgG23, IgA1 / IgG24, IgA1 / IgG25, IgA1 / IgG26, IgA1 / IgG27, IgA1 / IgG28, IgA1 / IgG29, IgA1 / IgG31, IgA1 / IgG32, IgA1 / IgG33, IgA1 / IgG44, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA gA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / I gG4, 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 / IgM, IgG4 / IgA1, IgG IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM (or a fragment or variant thereof), and optionally a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2 domain, CH3 domain, and CH4 domain are derived from a human Ig.

[0199] Some examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following hinge / CH4 domain combinations: IgAl / IgE, IgAl / IgM, IgA2 / IgE, IgA2 / IgM, IgD / IgE, IgD / IgM, IgGl / IgE, IgGl / IgM, IgG2 / IgE, IgG2 / IgM, IgG3 / IgE, IgG3 / IgM, IgG4 / IgE, IgG4 / IgM (or fragments or variants thereof), and optionally a CH2 domain derived from one or more of IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM, and / or a CH3 domain derived from one or more of IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM.

[0200] Particular examples of hybrid Fc regions can be found, for example, in WO2008 / 147143, such examples being derived from combinations of IgG subclasses or combinations of human IgD and IgG.

[0201] Also included are derivatized or otherwise modified Fc regions. In certain aspects, the Fc region may be modified compared to a wild-type or native Fc region by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc. In certain embodiments, the Fc region may comprise a wild-type or native glycosylation pattern, or may have increased glycosylation compared to a native form, decreased glycosylation compared to a native form, or may be completely aglycosylated. As one example of an altered Fc glycoform, decreased glycosylation of the Fc region reduces binding of the first complement component C1 to the C1q region, reduced ADCC-related activity, and / or reduced CDC-related activity. Thus, certain embodiments employ a deglycosylated or aglycosylated Fc region. For example, see 2005 / 047337 for the production of exemplary aglycosylated Fc regions. Another example of an Fc region glycoform can be generated by substituting position Q295 with a cysteine ​​residue according to the numbering system of Kabat et al. (See, e.g., U.S. Patent Application Publication No. 2010 / 0080794). An embodiment can include an Fc region in which approximately 80-100% of the glycoprotein of the Fc region comprises a mature core carbohydrate structure lacking fructose (See, e.g., U.S. Patent Application Publication No. 2010 / 0255013). Some embodiments can include an Fc region optimized by reducing fucosylation levels through substitution or deletion to increase affinity for FcγRI, FcγRIa, or FcγRIIIa and / or improve phagocytosis by FcγRIIa-expressing cells (See, U.S. Patent Applications Nos. 2010 / 0249382 and 2007 / 0148170).

[0202] As another example of an altered Fc glycotype, the Fc region may comprise oligomannose-type N-glycans and, optionally, have one or more of the following: increased ADCC activity, increased binding affinity for FcγRIIIA (and certain other FcRs), equivalent or higher binding specificity for the target of the HRS polypeptide, equivalent or higher binding affinity for the target of the HRS polypeptide, and / or equivalent or lower binding affinity for the mannose receptor, compared to a corresponding Fc region or HRS-Fc conjugate containing complex-type N-glycans (see, e.g., U.S. Patent Application Publication No. 2007 / 0092521 and U.S. Patent No. 7,700,321). As another example, enhanced affinity of the Fc region for FcγRs has been achieved using engineered glycoforms generated by expressing antibodies in engineered or variant cell lines (see, e.g., Umana et al., Nat Biotechnol. 17:176-180, 1999; Davies et al., Biotechnol Bioeng. 74:288-294, 2001; Shields et al., J Biol Chem. 277:26733-26740, 2002; Shinkawa et al., 2003, J Biol Chem. 278:3466-3473, 2003; and U.S. Patent Application 2007 / 0111281). Certain Fc region glycoforms have an increased proportion of complex N-glycosidically linked carbohydrate chains. The carbohydrate chain does not have a fucose at position 1 attached to position 6 of the N-acetylglucosamine at the reducing end of the carbohydrate chain (see, e.g., U.S. Patent Application Publication No. 2010 / 0092997). Certain embodiments include IgG Fc regions that are glycosylated with at least one galactose moiety attached to each terminal sialic acid moiety by an α-2,6-linkage, optionally in which case the Fc region has enhanced anti-inflammatory activity compared to the corresponding wild-type Fc region (see, e.g., U.S. Patent Application Publication No. 2008 / 0206246). Certain of these and related methods of engineered glycosylation, as described herein, significantly enhance the ability of the Fc region to selectively bind to FcRs, e.g., FcγRIII, mediate ADCC, and alter other properties of the Fc region.

[0203] Particular variant, fragment, hybrid, or otherwise modified Fc regions may have altered binding to one or more FcRs compared to the corresponding wild-type Fc sequence (e.g., allogeneic, same Ig class, same Ig subclass). For example, such Fc regions may have increased binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal Fc receptors compared to the corresponding wild-type Fc sequence. In some embodiments, variant, fragment, hybrid, or modified Fc regions may have decreased binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal Fc receptors compared to the corresponding wild-type Fc sequence. Specific FcRs are described elsewhere herein.

[0204] Specific examples of Fc variants with altered (e.g., increased or decreased) FcR binding can be found, for example, in U.S. Patent Nos. 5,624,821 and 7,425,619; U.S. Patent Applications 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046; and WO2000 / 42072 and WO2004 / 016750. Specific examples include human Fc regions with one or more substitutions at positions 298, 333, and / or 334, such as S298A, E333A, and / or K334A (based on the EU index numbering of Kabat et al.). They have been shown to have increased binding to the activating receptor FcγRIIIa and decreased binding to the inhibitory receptor FcγRIIb. These mutations can be combined to obtain double and triple mutant variants with further improved binding to FcRs. A particular example is the S298A / E333A / K334A triple mutant, which has increased binding to FcγRIIIa, decreased binding to FcγRIIb, and increased ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604, 2001; and Presta (See Umana et al., Biochem Soc Trans. 30:487-490, 2002). See also Umana et al., supra; and the engineered Fc glycoforms with increased binding to FcRs disclosed in U.S. Pat. No. 7,662,925. Some embodiments comprise 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. Patent Applications 2009 / 0163699 and 20060173170).

[0205] Certain variant, fragment, hybrid, or modified Fc regions may have altered effector functions compared to the corresponding wild-type Fc sequence. For example, such Fc regions may have increased complement fixation or activation, increased CIq binding affinity, increased CDC-related activity, increased ADCC-related activity, and / or increased ADCP-related activity compared to the corresponding wild-type Fc sequence. In some embodiments, such Fc regions may have decreased complement fixation or activation, decreased CIq binding affinity, decreased CDC-related activity, decreased ADCC-related activity, and / or decreased ADCP-related activity compared to the corresponding wild-type Fc sequence. As an illustrative example, the Fc region may include deletions or substitutions in a complement binding site, such as a C1q binding site, and / or deletions or substitutions in an ADCC site. 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 assayed according to techniques common in the art (see, e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Effector cells useful for such assays include, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMCs). Alternatively, or in addition, specific Fc effector functions can be assayed using Clynes The present invention can be analyzed in vivo by employing the animal model described in the publication by Toshiba et al. PNAS. 95:652-656, 1998.

[0206] Certain variant, hybrid, or modified Fc regions may have altered stability or half-life compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have an extended half-life compared to the corresponding wild-type Fc sequence. In some embodiments, variant, hybrid, or modified Fc regions may have a shortened half-life compared to the corresponding wild-type Fc sequence. Half-life can be measured in vitro (e.g., under physiological conditions) or in vivo according to techniques well known in the art, such as radiolabeling, ELISA, or other methods. In vivo measurements of stability or half-life can be measured in one or more body fluids, including blood, serum, plasma, urine, or cerebrospinal fluid, or a given tissue, such as liver, kidney, muscle, central nervous system tissue, or bone. As one example, modifications to an Fc region that alter its ability to bind to FcRn can alter its in vivo half-life. Non-limiting examples of analytical methods for measuring in vivo pharmacokinetic properties (e.g., in vivo mean elimination half-life) and Fc modifications that alter binding to FcRn are described, for example, in U.S. Pat. Nos. 7,217,797 and 7,732,570; and U.S. patent applications US2010 / 0143254 and 2010 / 0143254.

[0207] Another non-limiting example of a stability- or half-life-altering modification includes substitution / deletion of one or more amino acid residues selected from 251-256, 285-290, and 308-314 of the CH2 domain, and 385-389 and 428-436 of the CH3 domain, according to the numbering system of Kabat et al. See U.S. Patent Application 2003 / 0190311. Specific examples include substitution with leucine at position 251, substitution with tyrosine, tryptophan, or phenylalanine at position 252, substitution with threonine or serine at position 254, substitution with arginine at position 255, substitution with glutamine, arginine, serine, threonine, or glutamic acid at position 256, substitution with threonine at position 308, substitution with proline at position 309, substitution with serine at position 311, substitution with aspartic acid at position 312, substitution with leucine at position 314, substitution with arginine, asparagine, or glutamic acid at position 385. substitution with threonine or proline at position 386, with arginine or proline at position 387, with proline, asparagine, or serine at position 389, with methionine or threonine at position 428, with tyrosine or phenylalanine at position 434, with histidine, arginine, lysine, or serine at position 433, and / or with histidine, tyrosine, arginine, or threonine at position 436, including combinations thereof. Such modifications optionally increase the affinity of the Fc region for FcRn, thereby resulting in an increased half-life compared to the corresponding wild-type Fc region.

[0208] Certain variant, hybrid, or modified Fc regions may have altered solubility compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have increased solubility compared to the corresponding wild-type Fc sequence. In some embodiments, variant, hybrid, or modified Fc regions may have decreased solubility compared to the corresponding wild-type Fc sequence. Solubility can be measured in vitro (e.g., under physiological conditions) according to techniques commonly known in the art. Exemplary solubility measurements are described elsewhere herein.

[0209] Additional exemplary variants include IgG Fc regions with conservative or non-conservative substitutions at one or more of heavy chain positions 250, 314, or 428, 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, e.g., U.S. Patent Application Publication 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 of amino acid residues at positions 214-238, 297-299, 318-322, and / or 327-331 may be used as suitable targets for modification (e.g., conservative or non-conservative 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 Ser228Pro and Leu235Ala mutations) to attenuate the effector function of the Fc region (see, e.g., U.S. Pat. No. 7,030,226). As used herein, the numbering of residues in the heavy chain is that of the EU index (Kabat et al., "Sequences of Proteins of Immunological Interest," 5 th (See, Ed., National Institutes of Health, Bethesda, Md. (1991)). Certain of these and related embodiments have altered (e.g., increased, decreased) FcRn binding and / or serum half-life, optionally without reducing effector function, e.g., activity associated with ADCC or CDC.

[0210] Additional examples include variant Fc regions containing one or more amino acid substitutions at positions 279, 341, 343, or 373 of a wild-type Fc region, or any combination thereof (see, e.g., U.S. Patent Application 2007 / 0224188). For human IgG, the wild-type amino acid residues at these positions are valine (279), glycine (341), proline (343), and tyrosine (373). The substitutions may be conservative or non-conservative, or may include non-natural amino acids or mimetics as described herein. Alone or in combination with these substitutions, certain embodiments may also employ variant Fc regions 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, 254 V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V, 256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 26 2L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 279G, 2 79H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 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, 341 Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343E, 343F, 343G, 34 3H, 343I, 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 343V, 343W, 34 3Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373Q, 3 73R, 373S, 373T, 373V, 373W, 375R, 376E, 376F, 376G, 376H, 376I, 376L, 3 76M, 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, 382L, 382M, 382N, 382P, 382Q, 382R, 382S, 382T, 382V , 382W, 382Y, 385E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, 427N , 429A, 429F, 429M, 430A, 430D, 430F, 430G, 430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431K, 431P, 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 noted above, the numbering of residues in the heavy chain is:EU index numbering (see Kabat et al., supra). Such variant Fc regions often confer altered effector function or altered serum half-life to the HRS polypeptide to which the variant Fc region is operably attached. Preferably, the altered effector function is increased ADCC, decreased ADCC, increased CDC, decreased CDC, increased CIq binding affinity, decreased CIq binding affinity, increased FcR (preferably FcRn) binding affinity, or decreased FcR (preferably FcRn) binding affinity, compared to a corresponding Fc region lacking the amino acid substitution.

[0211] Additional examples include 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, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 35 and variant Fc regions comprising amino acid substitutions at one or more of positions 88, 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, e.g., U.S. Patent No. 7,662,925). In a specific embodiment, the variant Fc region comprises 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, K274 T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L32 8M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R and T335Y. In other specific embodiments, the variant Fc region comprises 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 / I332 D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332E, V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S23 9T, V240M, V264Y, A330I, N325T, L328D / I332E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I332E, S239Q / V264I / I3 32E, S239E / V264I / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E, V264I / S 298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, S239D / V264I / A330L / I332E, S239D / I332E / A330I, P230A, P230A / E233D / I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I , E318R, S324D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L235D, 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 comprises a series of substitutions selected from the group consisting of: N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E, S239D / N297D / I332E, S239E / N297D / I332E,In a specific embodiment, the variant Fc region comprises an amino acid substitution at position 332 (using EU index numbering, Kabat et al., supra). Exemplary substitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, and 332Y. The numbering of residues in the Fc region is that of the EU index of Kabat et al. Among the properties described herein, such variant Fc regions may have increased affinity for FcγRs, increased stability, and / or increased solubility compared to the corresponding wild-type Fc region.

[0212] Further 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, 267K, 268K, 269K, 270K, 271K, 272K, 273K, 274K, 275K, 276K, 277K, 278K, 279K, 280K, 281K, 282K, 283K, 284K, 285K, 286K, 67G / N, 268N, 269K / G, 273A, 276D, 278H, 279M, 280N, 283G, 285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A, 306I / F, 311R, 312N, 315D / K / S, 320R, 322E, 323 A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 35 1S, 352A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 365Q / P, 370R, 372L, 377V, 378T, 38 3N, 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, in this case, the variant has altered Fc ligand recognition and / or altered effector function compared to the parent Fc polypeptide, and the numbering of residues is that of Kabat et al.Specific examples of these and related embodiments include variant Fc regions comprising or consisting of the following sets of substitutions: (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, S267G, G341E, and E356G; (7) M252T, P291L, P352A, R355W, N390D, S408G, S426F, and A431S; (8) P228L, T289A, L365Q, N389S, and 5440G; (9) F241L, V273A, K340Q, and L441F; (10) F241L, T299A, I332T, and M428T; (11) E269K, Y300H, Q342R, V422I, and G446A; (12) T225A, R301c, S3 04G, D312N, N315D, L351S, 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, N361 D and S426P; (19) C261Y, K290E, L306F, Q311R, E333G and Q438L; (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.The residue numbering here is that of the EU index as in Kabat et al. (see, eg, US Patent Application 2010 / 0184959).

[0213] Another specific example of an Fc variant includes the Fc sequences of Table H7, in which Xaa at position 1 is Ala or absent, 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 absent (see, e.g., U.S. Patent Application 2007 / 0253966). Certain of these Fc regions, and related HRS-Fc conjugates, have extended half-lives, reduced effector activity, and / or are significantly less immunogenic than wild-type Fc sequences.

[0214] The variant Fc region may have one or more mutant hinge regions, such as those described in U.S. Patent Application Publication No. 2003 / 0118592. For example, one or more cysteines in the hinge region may be deleted or replaced with different amino acids. The mutant hinge region may contain no cysteine ​​residues, or may contain one, two, or three fewer cysteine ​​residues than the corresponding wild-type hinge region. In some embodiments, an Fc region having this type of mutant hinge region exhibits a reduced ability to dimerize compared to a wild-type Ig hinge region.

[0215] As discussed above, HRS-Fc conjugates, such as HRS-Fc fusion proteins, often have altered (e.g., improved, increased, or decreased) pharmacokinetic properties compared to the corresponding HRS polypeptide. Examples of pharmacokinetic properties include stability or half-life, bioavailability (fraction of drug absorbed), tissue distribution, volume of distribution (apparent volume into which a drug is distributed and equilibrated between plasma and surrounding tissues immediately after intravenous injection), concentration (initial or steady-state concentration of drug in plasma), elimination rate constant (rate at which a drug is eliminated from the body), elimination rate (infusion rate required to reverse elimination), area under the curve (AUC or exposure; integral of the concentration-time curve after a single dose or during steady state), clearance (amount of drug cleared from plasma per unit time), C max (peak plasma concentration of drug after oral administration), t max (C max time to reach C min These include peak-to-trough fluctuation (the minimum concentration reached by the drug before the next dose is administered) and variability (peak-to-trough fluctuation within one administration interval at steady state). In some embodiments, these improved properties are achieved without significantly altering the secondary structure of the HRS polypeptide and / or reducing non-canonical biological activities. Indeed, some HRS-Fc conjugates have increased non-canonical biological activities.

[0216] Thus, in some embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has a plasma or serum pharmacokinetic AUC profile that is 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 higher than the corresponding unmodified or differently modified HRS polypeptide when administered to a mammal under the same or equivalent conditions. In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater stability (e.g., as measured by half-life) than a corresponding unmodified or differently modified HRS polypeptide when compared under similar conditions at room temperature, e.g., in PBS at pH 7.4, for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 1, 2, 3, or 4 weeks.

[0217] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has a biological half-life of 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 more, or any intermediate half-life, at, for example, physiological pH, such as human body temperature, pH 7.4, 25°C (e.g., in vivo, in serum, in a given tissue, in a given species, e.g., rat, mouse, monkey, or human).

[0218] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has increased bioavailability after subcutaneous (SC) administration compared to the corresponding unmodified HRS polypeptide, ie, 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 polypeptide.

[0219] In certain embodiments, the HRS-Fc fusion polypeptide has substantially the same secondary structure as the corresponding unmodified HRS polypeptide or a differently modified HRS polypeptide, as determined by UV circular dichroism analysis. In certain embodiments, the HRS-Fc fusion polypeptide has substantially the same activity as the corresponding unmodified HRS polypeptide or a differently modified HRS polypeptide in an anti-inflammatory activity assay. In some embodiments, the HRS-Fc fusion polypeptide has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times greater activity in an anti-inflammatory activity assay than the corresponding unmodified HRS polypeptide or a differently modified HRS polypeptide.

[0220] An example of an HRS fusion protein that modulates oligomerization of an HRS polypeptide includes a fusion of HRS with cartilage oligomeric matrix protein (COMP). Accordingly, certain embodiments include fusion proteins comprising an HRS polypeptide described herein fused to COMP, e.g., the pentamerization domain derived from COMP, which consists of approximately residues 28-73 of COMP (see, e.g., Prodeus et al., JCI Insight. 2017;2(18):e94308; and Kim et al. (See, e.g., et al., Biochim Biophys Acta. 2009 May;1793(5):772-80). Exemplary HRS-COMP fusion proteins are provided below in Table H9. [Table H9]

[0221] Thus, in certain embodiments, an HRS polypeptide is fused or otherwise linked to COMP or a COMP pentamerization domain and comprises, consists of, or consists essentially of an amino acid sequence of Table H9, or an active variant or fragment thereof. In some embodiments, an expressible polynucleotide encodes an HRS polypeptide that comprises, consists of, or consists essentially of an amino acid sequence of Table H9 (e.g., SEQ ID NOs:173-175), or an active variant or fragment thereof.

[0222] In certain embodiments, a peptide linker sequence may be employed to separate the HRS polypeptide and the Fc region or PEG or other fusion partner by a distance sufficient to ensure folding of each polypeptide into the desired secondary and tertiary structures, and such a peptide linker sequence can be incorporated into a conjugate or fusion polypeptide using techniques standard in the art.

[0223] A particular peptide linker sequence may be selected based on the following exemplary factors: (1) the ability to adopt a flexible, extended conformation, (2) the ability to avoid secondary structures that may interact with functional epitopes on the first and second polypeptides, (3) physiological stability, and (4) the absence of hydrophobic or charged residues or other properties that may react with functional epitopes of the polypeptides. See, e.g., George and Heringa, J. See Protein Eng. 15:871-879, 2002.

[0224] Linker sequences generally can be from 1 to about 200 amino acids in length. Particular linkers can be from about 1 to 200 amino acids, 1 to 150 amino acids, 1 to 100 amino acids, 1 to 90 amino acids, 1 to 80 amino acids, 1 to 70 amino acids, 1 to 60 amino acids, 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, 1 to 10 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids. , 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 in total amino acid length.

[0225] Peptide linkers may employ any one or more natural amino acids, unnatural amino acids, amino acid analogs, and / or amino acid mimetics, as otherwise described herein and known in the art. Specific amino acid sequences that may be usefully employed as linkers are described in Maratea et al., Gene 40:39-46, 1985; Murphy et al., J. Am. Chem. Soc. 1999, 11:111-112, 1999; al., PNAS USA. 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Certain peptide linker sequences contain Gly, Ser, and / or Asn residues. Other closely related neutral amino acids, such as Thr and Ala, can also be employed in peptide linker sequences if desired.

[0226] Certain exemplary linkers include linkers 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), wherein: x is 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 of skill in the art.

[0227] Additional examples of linker peptides include, but are not limited to, the following amino acid sequences: Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 209); Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 210); Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly Asp-Ala-Ala-Ala-Lys-Glu-Ala-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).

[0228] Further non-limiting examples of linker peptides include DGGGS (SEQ ID NO: 214); TGEKP (SEQ ID NO: 215) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997); GGRR (SEQ ID NO: 216) (Pomerantz et al. 1995); (GGGGS) n (SEQ ID NO: 204) (Kim et al., PNAS. 93: 1156-1160, 1996); EGKSSGSGSESKVD (SEQ ID NO: 217) (Chaudhary et al., PNAS. 87: 1066-1070, 1990); KESGSVSSEQLAQFRSLD (SEQ ID NO: 218) (Bird et al., Science. 242: 423-426, 1988), GGRRGGGS (SEQ ID NO: 219); LQRDGERP (SEQ ID NO: 220); LRQKDGGGSERP (SEQ ID NO: 221); LRQKd(GGGS)2ERP (SEQ ID NO: 222). In a specific embodiment, the linker sequence comprises a Gly3 linker sequence containing three glycine residues. In certain embodiments, flexible linkers can be rationally designed using computer programs capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994) or by phage display methods.

[0229] The peptide linker may be physiologically stable or may include a releasable linker, such as a physiologically degradable linker or an enzymatically cleavable linker (e.g., a proteolytic cleavage linker). In certain embodiments, one or more releasable linkers may provide a short half-life and more rapid clearance of the conjugate. These and related embodiments can be used, for example, to deliver an HRS polypeptide into the bloodstream while enhancing solubility and circulating half-life of the HRS polypeptide in the bloodstream, followed by degradation of the linker to substantially release the Fc region. These embodiments are particularly useful in cases where the activity of the HRS polypeptide would be reduced if the Fc region were permanently attached. By using the linkers provided herein, such HRS polypeptides can maintain therapeutic activity even in their conjugated form. As another example, a large, relatively inactive HRS-Fc conjugate polypeptide may be administered, which is then degraded in vivo (via the degradable linker) to generate a biologically active HRS polypeptide that retains a portion of the Fc region or lacks the entire Fc region. In these and other ways, the properties of the HRS-Fc conjugate polypeptide can be more effectively tailored to balance the biological activity and circulating half-life of the HRS polypeptide over time.

[0230] In certain embodiments, the linker peptide comprises an autocatalytic or self-cleaving peptide cleavage site. In certain embodiments, the self-cleaving peptide comprises a polypeptide sequence obtained from potyvirus and cardiovirus 2A peptides, FMDV (foot and mouth disease virus), equine rhinitis A virus, Thosea asigna virus, and porcine teschovirus. In some embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence, or domain (Donnelly et al., J. Gen. Virol. 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: 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 cleavage site of the autocatalytic peptide comprises a translational 2A signal sequence, e.g., the 2A region of the aphthovirus foot and mouth disease virus (FMDV) polyprotein, which is an 18-amino acid sequence. Additional examples of 2A-like sequences that can be used include those found in insect virus polyproteins, the NS34 protein of type C rotavirus, and those described, for example, by Donnelly et al., Journal of General Examples include the repetitive sequences of Trypanosoma species described in Virology. 82:1027-1041, 2001.

[0231] Suitable protease cleavage sites and self-cleaving peptides are known to those of skill 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). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to their high cleavage stringency, some embodiments include TEV (tobacco etch virus) protease cleavage sites, such as EXXYXQ(G / S) (SEQ ID NO: 233), including 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).

[0232] Further examples of enzyme-degradable linkers suitable for use in certain embodiments include, but are not limited to, amino acid sequences that are cleaved by a serine protease, such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin. Examples of thrombin-cleavable amino acid sequences 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-. Examples of elastase-cleavable amino acid sequences 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).

[0233] Enzymatically degradable linkers further include amino acid sequences that can be cleaved by matrix metalloproteases, such as collagenase, stromelysin, and gelatinase. Examples of matrix metalloprotease-cleavable amino acid sequences 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), where Y and Z are amino acids. Examples of collagenase-cleavable amino acid sequences 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 example of a stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg- (SEQ ID NO: 256), and an example of a gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO: 257).

[0234] Enzymatically degradable linkers suitable for use in certain embodiments include amino acid sequences that can be cleaved by angiotensin converting enzyme, such as, for example, -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro- (SEQ ID NO: 258), and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO: 259).

[0235] Enzymatically degradable linkers suitable for use in certain embodiments include amino acid sequences that can be degraded by cathepsin B, such as, for example, Val-Cit, Ala-Leu-Ala-Leu- (SEQ ID NO: 260), Gly-Phe-Leu-Gly- (SEQ ID NO: 261), and Phe-Lys.

[0236] 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 more, or any intermediate half-life, at, for example, physiological pH, pH 7.4 such as body temperature, and 25°C (e.g., in vivo, in serum, in a given tissue). Those skilled in the art will recognize that the half-life of the HRS-Fc conjugate polypeptide can be finely tuned by using a particular releasable linker.

[0237] However, in some embodiments, any one or more of the peptide linkers are optional. For example, a linker sequence may not be required when the first and second polypeptides have non-essential N- and / or C-terminal amino acid regions that can separate the functional domains and prevent steric interference.

[0238] HRS polypeptides and polynucleotides, such as expressible polynucleotides, can be used in any of the compositions, methods and / or kits described herein.

[0239] Neuropilin-2 polypeptides and NRP2 ligands Embodiments of the present disclosure relate to the discovery that human histidyl-tRNA synthetase (HRS) polypeptides have unexpected biological properties suitable for the treatment of a wide range of diseases and conditions, certain of which properties are associated with the interaction of HRS with human neuropilin 2 (NRP2). Thus, HRS polypeptides can be used as a sole therapy in the treatment of diseases, such as NRP2-related diseases, and / or in combination with other agents that address many diseases and conditions associated with the biological activity of neuropilin-2.

[0240] NRP2 is a single-pass transmembrane receptor whose main extracellular region contains two CUB domains (a1 / a2 mixed domains), two factor V / factor VIII homology domains (b1 / b2 mixed domains), and a MAM domain (c domain) (see Figures 1A-1B). The a1a2 mixed domain interacts with the sema domain of semaphorins, and the b1 domain interacts with the PSI and Ig-like domains of semaphorins. NRP2 has high affinity for SEMA 3F and 3G. In contrast, SEMA 3A, 3B, and 3E preferentially interact with NRP1. Both NRP1 and NRP2 have similar affinity for SEMA 3C. The b1b2 mixed domain interacts with several growth factors containing heparin-binding domains, including VEGF C and D, placental growth factor (PIGF)-2, fibroblast growth factor (FGF), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF)-beta (see, e.g., Prud'homme et al., Oncotarget 3:921-939, 2012). NRP2 also interacts with various growth factor-specific receptors, and interactions with these receptors occur independently of binding to SEMA. In this context, integrins and growth factor receptors such as VEGF receptor, TGF-beta receptor, c-Met, EGFR, FGFR, and PDGFR have been shown to interact with NRPs, generally increasing the affinity of each ligand for its receptor and modulating downstream signaling. The c domain (Mam) is not believed to be required for ligand binding but is believed to be essential for signal transduction.

[0241] NRP2 can form homodimers, heterodimers with other cell surface receptors, and coreceptors, and is heavily glycosylated. Different splice variants of NRP2 exist, ranging in length from approximately 551 to 926 amino acids. The two major NRP2 variants are categorized as NRP2a and NRP2b. These variants differ in their intracellular C-terminal regions (see Figures 1A-1B). NRP2a contains a 42-amino acid C-terminal domain and a PDZ-binding domain with a C-terminal SEA amino acid sequence. NRP2b, on the other hand, contains a 46-amino acid C-terminal domain that shares approximately 11% of the intracellular and transmembrane sequences of NRP2a. An additional splice event can occur between the MAM and transmembrane domains, adding an additional five amino acids (GENFK) to either NRP2a or NRP2b. These variants are named based on 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). A soluble form, termed sNRP2b, may also be produced (see Figure 2). The terms "neuropilin-2" or "NRP2" or "NRP2 polypeptide" refer to all isoforms, splice variants, and native fragments of NRP2, unless a different, specific meaning is evident from the context. An example NRP2 polypeptide sequence is provided in Table N1 below. [Table N1-1] [Table N1-2] [Table N1-3] [Table N1-4] [Table N1-5] [Table N1-6] [Table N1-7]

[0242] Neuropilin-2 responds to multiple ligands through the recruitment of diverse coreceptors and can regulate a wide range of cellular functions through its role both as a basal cell surface receptor and as a coreceptor for various ligands (see, e.g., Guo and Vander Kooi, J. Cell. Biol. 290 No 49:29120-29126, 2015; Prud'homme et al., Oncotarget 3:921-939, 2012). For example, NRP2 functions during epithelial-mesenchymal transition (EMT) by promoting TGF-β1-mediated EMT in colorectal and other cancer cells (see, e.g., Grandclement et al., PLoS ONE 6(7)e20444, 2011), and by promoting fibrosis formation via EMT or endothelial-EMT in fibroblasts, myofibroblasts, and endothelial cells (see, e.g., Pardali et al., Int. J. Mol. Sci. 18 2157 2017).

[0243] Neuropilin-2 expression further promotes lymphangiogenesis and regulates vascular permeability (see, e.g., Doci et al., Cancer Res. 75(14)2937-2948, 2015; Mucka, et al., Am. J. Path 186(11)2803-2812 2016), and single nucleotide polymorphisms (SNPs) in NRP2 are associated with lymphedema (see, e.g., Miaskowski et al., PLoS ONE 8(4)e60164, 2013). NRP2 also regulates smooth muscle contractility (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559, 2012) and is highly expressed in developing skeletal muscle (see, e.g., Meye, et al., PLOS ONE DOI.10.1371 / journal.pone.0139520 2015).

[0244] Neuropilins are also multifunctional coreceptors involved in tumor initiation, growth, metastasis, lymphangiogenesis, lymphatic metastasis, and tumor immune surveillance, and thus directly contribute to tumor initiation, survival, and metastasis (e.g., 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. al., PLoS ONE 6(10)e23208, 2011).

[0245] Neuropilin 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 regulating immune cell activation and migration (see, e.g., Mendes-da-Cruz et al., PLoS ONE 9(7)e103405, 2014), including endosomal maturation, regulation of autophagy, and efferocytosis (see, e.g., Stanton et al., Cancer Res. 73:160-171, 2013; Schellenburg et al., Mol. Imm 90:239-244, 2017; Wang et al., Cancer Lett. 418 176-184 2018).

[0246] NRP2 is also expressed in other tissues and cell types, including pulmonary endothelial and epithelial cells, as well as osteoclasts and muscle cells (e.g., Bielenberg et al., Amer. J. Path. 181:548-559, 2012; Aung, et al., PLoS ONE 11(2)e0147358, 2016; and Wild et al., Int. J. Exp. Path. 93:81-103, 2012).

[0247] Neuropilin 2 plays an active role in the development of the nervous system, and in adults, NRP2 is actively involved in peripheral nerve growth and remodeling, as well as in pain perception in inflammatory conditions such as arthritis, osteoarthritis, and rheumatoid arthritis (see, e.g., Hamilton, J et al., J. Bone & Min. Res. 2016 31(5)911-924; Bannerman, P., et al., J. Neurosci. Res. 2008 86(14)3163-3169; Malykhina, A., et al., BMC Physiology 2012,12,15).

[0248] It is also becoming increasingly clear that neuropilin-2 plays a key role in endosome biogenesis and regulates late endosome maturation, processes that are important for phagocytosis and efferocytosis, which play important roles in the clearance of infected and apoptotic cells, respectively (e.g., Diaz-Vera et al., 2014). et al., J. Cell. Sci. 130, 697-711 2017; Dutta et al., Cancer Res. 76(2) 418-428 2016).

[0249] Neuropilin-2 is known to be a key player in the pathophysiology of many diseases ("NRP2-associated diseases") and interacts with a wide range of soluble ligands, including semaphorin 3F, VEGF-C and D, and TGF-beta, as well as many cellular receptors and cofactors ("NRP2 ligands") (see, e.g., Tables N2 and N3 below, and Figures 1A-1B). [Table N2-1] [Table N2-2] [Table N3-1] [Table N3-2]

[0250] NRP2 is also polysialylated on dendritic cells and actively interacts with the chemokine CCL21 to mediate immune cell migration via the CCR7 receptor. In this regard, single-nucleotide polymorphisms in NRP2 have been reported to be associated with ILD and RA (see, e.g., Rey-Gallardo et al., Glycobiology 20:1139-1146, 2010; Stahl et al., Nat. Genet. 42:508-514, 2013; Miller et al., Arthritis Rheum. 65:3239-3247). Therefore, a soluble circulating form of NRP2 is known (see, e.g., Parker et al., Structure 23(4)677-687, 2015). Our experiments have confirmed the existence of a circulating complex of HRS and NRP2 polypeptides in serum and other fluids.

[0251] Given the central role of NRP2 in pathophysiology, it is clear that the interaction between NRP2 and HRS polypeptides offers the potential for treating diseases, including NRP2-related diseases. Thus, by selectively modulating the interaction of NRP2 with one or more of the ligands listed in Tables N2 and N3, the HRS polypeptides described herein can be used to treat a wide range of diseases and conditions described herein.

[0252] Additional Therapeutic Agents and Compositions Immunotherapeutic Agents. Some embodiments employ one or more immunotherapeutic agents. In some instances, the immunotherapeutic agent modulates a subject's immune response, e.g., increasing or maintaining a cancer-associated or cancer-specific immune response, resulting in immune cell inhibition or increased immune suppression by cancer cells. Examples of immunotherapeutic agents include polypeptides, e.g., antibodies and antigen-binding fragments thereof, ligands, small peptides, and mixtures thereof. Also included as immunotherapeutic agents are small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapies, or other polynucleotide-based agents, including oncolytic viruses, and other agents known in the art. Thus, in some embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy.

[0253] In some embodiments, the cancer immunotherapeutic agent is an immune checkpoint modulator. Specific examples include "antagonists" of one or more inhibitory immune checkpoint molecules and "agonists" of one or more stimulatory immune checkpoint molecules. Generally, immune checkpoint molecules are components of the immune system that up-regulate (co-stimulatory molecules) or down-regulate (stimulatory molecules). Because cancer cells can disrupt the natural function of immune checkpoint molecules, targeting them has the potential for cancer therapy (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, an immune checkpoint modulator (e.g., antagonist, agonist) "binds" or "specifically binds" to one or more immune checkpoint molecules described herein.

[0254] In certain embodiments, the immune checkpoint modulator is a polypeptide or peptide. While the terms "peptide" and "polypeptide" are used interchangeably herein, in certain embodiments, the term "peptide" can refer to short polypeptides, e.g., polypeptides 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, including all integers and ranges therebetween (e.g., 5-10, 8-12, 10-15). Polypeptides and peptides can be composed of natural and / or unnatural amino acids, as described herein.

[0255] Antibodies are also included as polypeptides. Thus, in some embodiments, the immune checkpoint modulating polypeptide agent is an antibody, or an "antigen-binding fragment thereof," as otherwise described herein.

[0256] In some embodiments, an agent is or comprises a "ligand," e.g., the native ligand of an immune checkpoint molecule. "Ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) to serve a biological purpose, and includes "protein ligands" that generally bind to a site on a target molecule or target protein to generate a signal. Thus, certain agents are protein ligands that naturally bind to an immune checkpoint molecule to generate a signal. Also included are "modified ligands," e.g., protein ligands fused to a pharmacokinetic modifier, e.g., an Fc region derived from an immunoglobulin.

[0257] The binding activity of a polypeptide can be quantified using methods known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, the polypeptide specifically binds to a target molecule, such as an immune checkpoint molecule or an epitope thereof, with an equilibrium dissociation constant that is, or is in the range of, about ≦10 M to about 10 M. In some embodiments, the equilibrium dissociation constant is, or is in the range of about ≦10 M to about 10 M. In certain exemplary embodiments, the polypeptide has an affinity (Kd or EC) for (specifically binds to) a target described herein that is about, or at least about, 0.01, 0.05, 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, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM or less. 50 )

[0258] In some embodiments, the agent is a "small molecule," which refers to an organic compound that is synthetic or of biological origin (biomolecule), but is often not a polymer. Organic compounds refer to a large class of compounds whose molecules contain carbon, typically excluding those that contain only carbohydrates, simple oxides of carbon, or cyanides. "Biomolecules" generally refer to organic molecules produced by living organisms and include large macromolecules (biopolymers), such as peptides, polysaccharides, and nucleic acids, as well as small molecules, such as primary and secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones. "Polymer" generally refers to a macromolecule or macromolecule composed of repeating structural units, usually connected by covalent chemical bonds.

[0259] In certain embodiments, small molecules have a molecular weight of about 1000-2000 daltons or less, typically about 300-700 daltons, including about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, 1000, or 2000 daltons or less.

[0260] Certain small molecules may have the characteristic of "specific binding" as described herein with respect to polypeptides such as antibodies. For example, in some embodiments, a small molecule binds to a target, e.g., an immune checkpoint molecule, with a binding affinity (Kd or EC 50 ) and binds specifically to it.

[0261] In some embodiments, the 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 and mucin domain 3 (TIM-3), lymphocyte-activation gene-3 (LAG-3), V-domain Ig suppressor of T-cell activation (VISTA), B- and T-lymphocyte attenuator (BTLA), CD160, and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0262] In certain embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, the targeting of which has been shown to restore immune function in the tumor environment (see, e.g., Phillips et al., Int Immunol. 27:39-46, 2015). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functions as an inhibitory immune checkpoint molecule, for example, by reducing or interfering with T cell activation, thereby reducing autoimmunity and promoting self-tolerance. The inhibitory effect of PD-1 is achieved, at least in part, through a dual mechanism: it promotes apoptosis of antigen-specific T cells in lymph nodes while also reducing apoptosis of regulatory T cells (suppressor T cells). Some examples of PD-1 antagonists or inhibitors include 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 downstream signaling or 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 antigen-binding fragments thereof (see, e.g., U.S. Patent No. 8,008,444). 9, 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. Patent Applications 2012 / 0039906 and 2015 / 0203579.

[0263] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As mentioned above, PD-L1 is one of the natural ligands of the PD-1 receptor. General examples of PD-L1 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to PD-L1 and reduce one or more of its immunosuppressive activities, such as binding to the PD-1 receptor. Specific examples of PD-L1 antagonists include atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), as well as antigen-binding fragments thereof (see, e.g., U.S. Patent Nos. 9,102,725, 9,393,301, 9,402,899, and 9,439,962).

[0264] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As mentioned above, PD-L2 is one of the natural ligands of the PD-1 receptor. Common examples of PD-L2 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L2 and reduce one or more of its immunosuppressive activities, such as binding to the PD-1 receptor.

[0265] 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 (cluster of differentiation 152), is a protein receptor that functions as an inhibitory immune checkpoint molecule by transmitting an inhibitory signal to T cells upon binding to CD80 or CD86 on the surface of antigen-presenting cells, for example. Common examples of CTLA-4 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Specific examples include the antibodies ipilimumab and tremelimumab, and antigen-binding fragments thereof. At least part of the activity of ipilimumab is believed to be mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of CTLA-4-expressing inhibitory Tregs.

[0266] In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are tryptophan metabolic enzymes with immunosuppressive properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. Common examples of IDO and TDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDO or TDO and reduce or inhibit one or more immunosuppressive activities (see, for example, Platten et al., Front Immunol. 5:673, 2014). Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0267] 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 inducing cell death upon interaction with its ligand, galectin-9. TIM-3 contributes to a suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (see, e.g., Li et al., Acta Oncol. 54:1706-13, 2015). Common examples of TIM-3 antagonists or inhibitors include 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.

[0268] In some embodiments, the agent is an antagonist or inhibitor of LAG-3. 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 CTLA-4 and PD-1 (see, e.g., Workman and Vignali, European Journal of Clinical Oncology, 2014). of Immun. 33:970-9, 2003; and Workman et al., Journal of Immun. 172:5450-5, 2004), and has been reported to play an important role in the suppressive function of Tregs (see, for example, Huang et al., Immunity. 21:503-13, 2004). LAG3 also maintains CD8+ T-cells in a tolerogenic state and maintains CD8 T cell exhaustion by binding to PD-1. General examples of LAG-3 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016 and its antigen-binding fragments.

[0269] In some embodiments, the agent is a VISTA antagonist or inhibitor. V-domain Ig suppressor of T cell activation (VISTA) is an inhibitory immune checkpoint regulator that is primarily expressed on hematopoietic cells, suppresses T cell activation, induces Foxp3 expression, and is highly expressed in the tumor microenvironment where anti-tumor T cell responses are suppressed (see, e.g., Lines et al., Cancer Res. 74:1924-32, 2014). Common examples of VISTA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA and reduce one or more of its immunosuppressive activities.

[0270] In some embodiments, the agent is a BTLA antagonist or inhibitor. Expression of B- and T-lymphocyte attenuator (BTLA; CD272) is induced during T cell activation and inhibits T cells through interaction with tumor necrosis factor family receptors (TNF-R) and the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (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, e.g., Derre et al., J Clin Invest 120:157-67, 2009). Common examples of BTLA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

[0271] In some embodiments, the agent is an antagonist or inhibitor of HVEM, e.g., an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD 160. Common examples of HVEM antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM and optionally reduce the HVEM / BTLA and / or HVEM / CD 160 interaction, thereby reducing one or more of the immunosuppressive activities of HVEM.

[0272] In some embodiments, the agent is a CD160 antagonist or inhibitor, e.g., an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. Common examples of CD160 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CD160 and optionally reduce the CD160 / HVEM interaction, thereby reducing or inhibiting one or more of its immunosuppressive activities.

[0273] In some embodiments, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor present on the surface of various lymphoid cells that suppresses anti-tumor immunity, for example, via Tregs (see Kurtulus et al., J Clin Invest. 125:4053-4062, 2015). Common examples of TIGIT antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT and reduce one or more of its immunosuppressive activities (see, for example, Johnston et al., Cancer Cell. 26:923-37, 2014).

[0274] In certain embodiments, the immune checkpoint modulator is an agonist of one or more stimulatory immune checkpoint molecules, including OX40, CD40, glucocorticoid-inducible TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0275] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the expansion of effector and memory T cells and suppresses the differentiation and activity of T regulatory cells (see, e.g., Croft et al., Immunol Rev. 229:173-91, 2009). Its ligand is OX40L (CD252). OX40 signaling influences both T cell activation and survival, and therefore plays an important role in initiating antitumor immune responses in lymph nodes and maintaining antitumor immune responses in the tumor microenvironment. Common examples of OX40 agonists include 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 (murine OX4 agonist), and MEDI6383 (OX40 agonist), and antigen-binding fragments thereof.

[0276] In some embodiments, the agent is a CD40 agonist. CD40 is expressed on antigen-presenting cells (APCs) and some malignant tumors. Its ligand is CD40L (CD154). Ligation on APCs results in the upregulation of costimulatory molecules, potentially bypassing the need for T cell help in antitumor immune responses. CD40 agonist therapy plays an important role in APC maturation and migration from tumors to lymph nodes, resulting in increased antigen presentation and T cell activation. Anti-CD40 agonist antibodies have been shown to produce significant reactivity and long-lasting anticancer immunity in animal models, an effect mediated at least in part by cytotoxic T cells (see, e.g., Johnson et al., Clin Cancer Res. 21:1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). Common 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, dacetuzumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L, and antigen-binding fragments thereof.

[0277] In some embodiments, the agent is a GITR agonist. Glucocorticoid-Induced TNFR family Related gene (GITR) increases T cell expansion, inhibits the suppressive activity of Tregs, and prolongs the survival of T-effector cells. GITR agonists have been shown to promote anti-tumor responses through a decrease in the stability of the Treg lineage (see, e.g., Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms indicate that GITR plays an important role in initiating immune responses in lymph nodes and maintaining immune responses in tumor tissues. Its ligand is GITRL. Common examples of GITR agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to GITR and increase one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

[0278] 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. Common examples of CD137 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and increase one or more of its immunostimulatory activities. A specific example is the antibody utomilumab, which contains a CD137 (or 4-1BB) ligand (see, e.g., Shao and Schwarz, J. Leukoc. Biol. 89:21-9, 2011) and its antigen-binding fragment.

[0279] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific expansion of naive T cells, contributing to T cell memory and long-term maintenance of T cell immunity. Its ligand is CD70. Targeting human CD27 with agonistic antibodies stimulates T cell activation and antitumor immunity (see, e.g., Thomas et al., Oncoimmunology. 2014;3:e27255.doi:10.4161 / onci.27255; and He et al., J Immunol. 191:4174-83, 2013). Common examples of CD27 agonists include 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 antibodies containing its antigen-binding fragments, varlilumab and CDX-1127 (1F5).

[0280] In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed on CD4+ T cells and a subset of CD8+ T cells. Its ligands include CD80 and CD86, stimulation of which increases T cell expansion. Common 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 its immunostimulatory activities. Specific examples include CD80, CD86, the antibody TAB08, and antigen-binding fragments thereof.

[0281] In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT. In contrast to TIGIT, association with CD226 enhances T cell activation (see, e.g., Kurtulus et al., J Clin Invest. 125:4053-4062, 2015; Bottino et al. (See, e.g., J Exp Med. 1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538, 2004.) General examples of CD226 agonists include 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.

[0282] 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 expressed at high levels on resting T cells and is downregulated upon activation. HVEM signaling has been shown to play an important role in the early phase of T cell activation and during the expansion of tumor-specific lymphocyte populations in lymph nodes. Common examples of HVEM agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to HVEM and increase one or more of its immunostimulatory activities.

[0283] In certain embodiments, the cancer immunotherapeutic agent is a cancer vaccine.Exemplary cancer vaccines include Oncophage, human papillomavirus HPV vaccines, such as Gardasil or Cervarix, hepatitis B vaccines, such as Engerix-B, Recombivax HB, or Twinrix, and sipuleucel-T (Provenge).In some embodiments, the cancer vaccine comprises 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, CD33, CD37, CD40, CD44, CD51, CD5 2, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, 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 activation protein alpha (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), EGP40pan The cancer antigens include those selected from one or more of 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 (a disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0284] In certain embodiments, the cancer immunotherapeutic agent is a cancer vaccine. Oncolytic viruses are viruses that selectively infect and kill cancer cells. They include natural oncolytic viruses and artificial or genetically engineered oncolytic viruses. Most oncolytic viruses are genetically engineered for tumor selectivity, but there are also natural examples, such as reovirus and SVV-001 Seneca Valley virus. Common examples of oncolytic viruses include VSV, poliovirus, reovirus, Seneca virus, and RIGVIR, as well as genetically engineered versions thereof. Non-limiting examples of oncolytic viruses include herpes simplex virus (HSV) and genetically modified forms thereof, talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), 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, among others.

[0285] In some embodiments, the cancer immunotherapeutic agent is a cytokine. Examples of cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0286] In some embodiments, the cancer immunotherapeutic agent is a cell-based immunotherapy, such as, for example, T cell-based adoptive immunotherapy. In some embodiments, the cell-based immunotherapy comprises cancer antigen-specific T cells, optionally ex vivo induced T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells, 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 are targeted against CD-19 (see, for example, Maude et al., Blood. 125:4017-4023, 2015).

[0287] In certain instances, the cancer being treated is associated with a cancer antigen, i.e., cancer antigen-specific T cells are targeted or enriched for at least one antigen known to be associated with the cancer being treated. In some embodiments, the cancer antigen is selected from the group consisting of 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, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD 74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, 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 receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (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 Selected from one or more of 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 (a disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0288] Additional exemplary cancer antigens include 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4 / m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA15-3 / CA27-29, 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, coactosin-like protein, colla geXXIII, 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, GAGE-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-A1 1 / 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, MAGE-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-C2, 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, MCIR, 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, myosin class l / m, NA88-A, N-acetylglucosaminyltransferase-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, pi5, p190 minor, bcr-abl, p53, p53 / m, P AGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 kinase, Pin-1, Pml / PAR alpha, POTE, PRAME, PRDX5 / m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX-4These include STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF-beta, TGFbetaRII, 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, more preferably PAP, MAGE-A3, WT1, and MUC-1.

[0289] In some embodiments, the antigen is MAGE-A1 (e.g., MAGE-A1 with accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g., MAGE-A6 with accession number NM_005363), MAGE-C1, MAGE-C2, Melan-A (e.g., Melan-A with accession number NM_005511), GP100 (e.g., GP100 with accession number M77348), tyrosinase (e.g., tyrosinase with accession number NM_000372), survivin (e.g., survivin with accession number AF077350), CEA (e.g., CEA with accession number NM_004363), Her-2 / neu (e.g., Her-2 / neu with accession number M11730), WT1 (e.g., WT1 with accession number M11730), or a combination thereof. 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), SEC61G (e.g., SEC61G according to accession number NM_014302), hTERT (e.g., hTERT according to 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 (prostate six-transmembrane epithelial antigen 1), PSCA, PSA, PSMA, and the like.

[0290] In some embodiments, the cancer antigen is selected from PCA, PSA, PSMA, STEAP, including fragments, variants, and derivatives thereof, and optionally MUC-1, hi 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.

[0291] In some cases, cancer antigens include idiotypic antigens associated with cancer and tumor diseases, particularly lymphoma or lymphoma-related diseases, for example, the idiotypic antigen is an immunoglobulin idiotype of a lymphoid blood cell or a T-cell receptor idiotype of a lymphoid blood cell.

[0292] In some cases, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells (e.g., targeted to a cancer antigen) and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0293] Those skilled in the art will recognize that the various cancer immunotherapeutic agents described herein can be used in combination with any one or more of the various HRS polypeptides described herein and in accordance with any one or more of the methods or compositions described herein.

[0294] Chemotherapeutic Agents. Certain embodiments employ one or more chemotherapeutic agents, e.g., small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), microtubule inhibitors, among others.

[0295] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (e.g., procarbazine and hexamethylmelamine).

[0296] Examples of antifolates include (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine). Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin.

[0297] Examples of microtubule inhibitors include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

[0298] Those skilled in the art will recognize that the various chemotherapeutic agents described herein can be used in combination with any one or more of the various HRS polypeptides described herein and in accordance with any one or more of the methods or compositions described herein.

[0299] Hormonal Therapy Agents. Some embodiments employ at least one hormonal therapy agent. General examples of hormonal therapy agents include hormonal agonists and hormonal antagonists. Specific examples of hormonal agonists include progestogens (progestins), corticosteroids (e.g., prednisolone, methylprednisolone, or dexamethasone), insulin-like growth factors, VEGF-derived angiogenic and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factors (FGFs), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgens, estrogens, and somatostatin analogs. Examples of hormonal antagonists include hormone synthesis inhibitors, such as aromatase inhibitors and gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), including analogs thereof. 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).

[0300] Also included are hormonal pathway inhibitors, such as antibodies directed against hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and robatumumab; inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as alacizumab pegol; pegol, bevacizumab, icrucumab, ramucirumab, etc.; TGF-beta receptor R1, R2, or R3 inhibitors, such as frezolimumab and metelimumab; c-Met inhibitors, such as naxitamab; EGF receptor inhibitors, such as cetuximab, depatuximab mafodotin, futuximab, imgatuzumab, rapamycin, etc. FGF receptor inhibitors, such as laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, and zalutumumab; FGF receptor inhibitors, such as april tuzumab ixadotin and bemarituzumab; and PDGF receptor inhibitors, such as olaratumab and tobetumab.

[0301] Those skilled in the art will recognize that the various hormonal therapy agents described herein can be used in combination with any one or more of the various HRS polypeptides described herein and in accordance with any one or more of the methods or compositions described herein.

[0302] Kinase Inhibitors: Certain embodiments employ at least one kinase inhibitor, including a tyrosine kinase inhibitor and a phosphoinositide 3 (PI3) kinase inhibitor. Kinase inhibitors include, but are not limited to, one or more selected from adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozanib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib. Exemplary PI3 kinase inhibitors include 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.

[0303] Those skilled in the art will recognize that the various kinase inhibitors described herein can be combined with any one or more of the various HRS polypeptides described herein and used in accordance with any one or more of the methods or compositions described herein.

[0304] How to use Some embodiments include methods for treating, ameliorating symptoms, and / or slowing the progression of a disease or condition in a subject in need thereof, comprising administering an HRS polypeptide to the subject. In some examples, the HRS polypeptide specifically binds to a human neuropilin-2 (NRP2) polypeptide (see Table N1). In some examples, the HRS polypeptide interferes with the binding of a human NRP2 polypeptide to an NRP2 ligand. In some examples, the HRS polypeptide mimics one or more of the signaling activities induced by the binding of an NRP2 polypeptide to an NRP2 ligand, for example, by acting as an agonist, partial agonist, antagonist, partial agonist, or inverse agonist of the NRP2 ligand. Examples of NRP2 ligands are provided in Tables N2 and N3.

[0305] In certain embodiments, the subject in need thereof has a disease or condition associated with NRP2. Exemplary NRP2-related diseases and conditions include, but are not limited to, cancer and cancer-related diseases or conditions, such as cancer cell growth, cancer initiation, cancer migration, cancer cell adhesion, cancer cell invasion, cancer cell chemotherapy resistance, and cancer cell metastasis. Also included are diseases associated with inflammation and autoimmunity, such as inflammatory lung diseases, such as hypersensitivity pneumonitis and pulmonary inflammation, and related inflammatory diseases. Also included are 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). Additional examples include diseases associated with lymphatic vessel development, lymphangiogenesis, and lymphatic vessel damage, such as edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability. Also included are diseases associated with infection, including latent infection, and allergic disorders / diseases and diseases associated with allergic responses, such as chronic obstructive pulmonary disorder (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic lupus erythematosus, rheumatoid arthritis, inflammasome-associated diseases, and skin-related neutrophil-mediated diseases, such as pyoderma gangrenosum. Additional examples include diseases associated with granulomatous inflammatory diseases, such as sarcoidosis, other granulomatous lung diseases, and non-pulmonary granulomas. Further included are fibrotic diseases, such as endometriosis, fibrosis, endothelial-mesenchymal transition (EMT), and wound healing. Further included are diseases associated with inappropriate smooth muscle contractility and vascular smooth muscle cell migration and / or adhesion, and diseases associated with inappropriate autophagy, phagocytosis, and efferocytosis. Additional examples include neuronal disorders, including those associated with peripheral nervous system remodeling and pain sensation, as well as those associated with bone development and / or bone remodeling, and those associated with inappropriate migratory cell migration.

[0306] In some embodiments, a subject has, and / or is selected for treatment based on, increased extracellular fluid (e.g., circulating or serum) levels of soluble NRP2 polypeptide, either bound or free (e.g., selected from Table N1), or its encoding mRNA, e.g., compared to levels in a population of healthy or matched control or reference subjects. In some embodiments, extracellular fluid levels of NRP2 are detected in serum, plasma, lymph, interstitial fluid, and / or in specific tissue compartments associated with NRP2-related disorders, such as bronchoalveolar lavage fluid (BALF) and synovial fluid. In some embodiments, the level of soluble NRP2 polypeptide in the extracellular fluid is about or at least about 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000 pM soluble NRP2 polypeptide. The concentration of the NRP2 polypeptide is about 30-50, 50-100, 100-2000, 200-2000, 300-2000, 400-2000, 500-2000, 600-2000, 700-2000, 800-2000, 900-2000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 pM of soluble NRP2 polypeptide.

[0307] In some embodiments, the subject has, and / or is selected for treatment based on, an increased level of soluble NRP2 polypeptide, either bound or free (e.g., selected from Table N1), or its encoding mRNA, compared to cells or tissue of a non-disease control, e.g., cells or tissue of the same type as the cells or tissue of the NRP2-related disease. By way of example, in some embodiments, the level of soluble NRP2 polypeptide (or its encoding mRNA) in cells or tissue of an NRP2-related disease is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, compared to the level in cells or tissue of the non-disease control. Thus, some embodiments include a method of selecting a subject for treatment, the method comprising: (i) determining the extracellular fluid level of a soluble NRP2 polypeptide and / or the mRNA encoding it in the subject compared to a control or reference; and (ii) administering to the subject a therapeutic composition comprising at least one HRS polypeptide as described herein if the subject has an increased level of the soluble NRP2 polypeptide and / or the mRNA encoding it relative to the control or reference.

[0308] In some embodiments, the subject has, and / or is selected for treatment based on having, an increased extracellular fluid level of an NRP2 ligand, or an increased extracellular fluid level of an NRP2:NRP2 ligand complex (optionally selected from Table N1 and Table N2), compared to a population of healthy or matched control standard or reference subjects, e.g., a level that is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, of the control or reference level. Certain embodiments therefore include a method of selecting a subject for treatment, the method comprising: (i) determining the extracellular fluid level of NRP2:NRP2 ligand complex in the subject relative to a control or reference; and (ii) administering to the subject a therapeutic composition comprising at least one HRS-polypeptide as described herein if the subject has an increased level of NRP2:NRP2 ligand complex relative to the control or reference.

[0309] In some embodiments, the subject has, and / or is selected for treatment based on, an increased extracellular level of the HRS:NRP2 complex (optionally selected from Table H1 and Table N1) compared to a population of healthy or matched control standard or reference subjects, e.g., a level that is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, of the control or reference level. Certain embodiments therefore include a method of selecting a subject for treatment, comprising: (i) determining the extracellular level of the HRS:NRP2 complex in the subject relative to the control or reference; and (ii) administering to the subject a therapeutic composition comprising at least one HRS-polypeptide as described herein if the subject has an increased level of the HRS:NRP2 complex relative to the control or reference.

[0310] In some embodiments, a subject has, and / or is selected for treatment based on having, a single nucleotide polymorphism (SNP) in an NRP2 polypeptide or polynucleotide encoding NRP2 from the subject.

[0311] In some embodiments, the subject has, and / or is selected for treatment based on having, a disease associated with increased levels or expression of NRP2a and / or NRP2b, or an altered ratio of NRP2a:NRP2b expression, compared to a population of healthy or matched control standards or subjects. 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%, 1000% compared to a population of healthy or matched control standards or subjects.

[0312] In some embodiments, a population of healthy or matched controls or subjects comprises an average range of age-matched samples of cancerous or non-cancerous cells or tissues of the same type as the cancer, which samples contain specific characteristics, such as drug resistance, metastatic potential, invasiveness, genetic characteristics (optionally p53 mutation, PTEN deletion, IGFR expression), and / or expression patterns.

[0313] In some embodiments, the subject has and / or is selected for treatment based on having an infection. In some cases, the infection is a lymphedema-associated infection, such as erysipelas, cellulitis, lymphangitis, and / or sepsis.

[0314] In some embodiments, the HRS polypeptide is administered in combination with a second agent, such as an antibacterial agent, an antifungal agent, and / or an antihelminthic agent. In some embodiments, the HRS polypeptide and the second agent are administered together as part of the same therapeutic composition. In some embodiments, the HRS polypeptide and the second agent are administered as separate therapeutic compositions. In some embodiments, the second agent is an aminoglycoside, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin; a carbapenem, such as ertapenem, doripenem, imipenem / cilastatin, and meropenem; a cephalosporin, such as cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cephalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam , cefepime, ceftaroline fosamil, and ceftobiprole; glycopeptides, such as teicoplanin, vancomycin, telavancin, dalbavancin, and oritavancin; lincosamines, such as clindamycin and lincomycin; macrolides, such as azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, and spiramycin; penicillins, such as amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, and ticarcillin; polypeptides, such as bacitracin, colistin, and polymyxin B;Quinolones / fluoroquinolones, such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin; sulfonamides, such as mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (cotrimoxazole) (TMP-SMX) ), and sulfonamide chrysoidine; tetracyclines such as demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline; antimycobacterial agents such as clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin (rifampin), rifabutin, rifapentine, and streptomycin; chloramphenicol; metronidazole; mupirocin; tigecycline; tinidazole; and antihelminthic agents such as diethylcarbamazine and albendazole.

[0315] HRS Polypeptide-Mediated Treatment of Lymphedema Lymphedema is a chronic, debilitating disease that occurs most frequently in the United States and Western countries as a result of cancer surgery, obesity, congestive heart failure, hypertension, and / or peripheral vascular / venous disease. In the setting of cancer treatment, lymphedema occurs as a result of iatrogenic injury to the lymphatic system, most commonly after lymph node dissection, but also as a result of extensive skin excision and adjuvant therapy with radiation (e.g., Purushotham et al., J. Clin. Oncol. 23:4312-4321, 2005; Szuba et al., Cancer 95:2260-2267, 2002; Tsai (See, e.g., et al., Ann. Surg. Oncol. 16:1959-72, 2009).

[0316] It is estimated that one in three patients who undergo lymph node dissection develops lymphedema, with conservative estimates suggesting that as many as 50,000 new cases are diagnosed each year. (See, e.g., 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 affected individuals increases annually, currently estimated at 5-6 million Americans (Rockson et al., Ann. NY Acad. Sci. 1131:147-154, 2008) and 200 million worldwide. The incidence of lymphedema correlates nearly linearly with cancer patient survival, and the number of affected individuals is likely to continue to increase in the future. And the prevalence of known risk factors for lymphedema, such as obesity and radiation therapy, is also increasing (see, eg, Erickson et al., J. Natl. Cancer Inst. 93:96-111, 2001).

[0317] Secondary lymphedema can take years to develop, suggesting that its development is not solely due to lymphatic damage but may also be caused by an underlying chronic inflammatory state. + The cellular response, similar to other fibroproliferative disorders, is characterized by a mixed Th1 / Th2 cell population (Avraham et al., FASEB J. 27:1114-1126, 2013). +T cells, also known as T-helper cells or Th cells, patrol secondary lymphoid structures and, upon activation, differentiate into many different / overlapping cell types (e.g., Th1, Th2, Th17, T regulatory, etc.). The Th2 cell subset plays an important role in controlling responses to parasites and some autoimmune responses. These cells also contribute to the pathology of fibroproliferative diseases in multiple organ systems, including the heart, lungs, kidneys, and skin. Recent studies have shown that the number of Th2 cells is increased in tissue biopsies from patients with lymphedema, and that inhibiting Th2 differentiation reduces lymphedema pathology in mouse models.

[0318] CD4 + cells or macrophages (CD8 +Depletion of Th2 cells (but not other inflammatory cell types, including leukocytes) or inhibition of Th2 differentiation (but not systemic inflammation or interleukin-6) significantly reduced the degree of fibrosis, increased lymphangiogenesis and lymphatic transport, and effectively treated established lymphedema in preclinical mouse models (see, e.g., Avraham et al., FASEB J. 27:1114-1126, 2013; Zampell et al., PLoS ONE 7:e49940, 2012; Ghanta et al., Am. J. Physiol. Heart Circ. Physiol. 308:H1065-1077, 2015). These findings are supported by recent studies demonstrating that T cells potently inhibit lymphangiogenesis by producing anti-lymphangiogenic cytokines / growth factors, including interferon gamma (IFN-γ), interleukin (IL)-4, IL-13, and TGF-β1 (see, e.g., Kataru et al., Immunity 34:96-107, 2011; Shin et al., Nat. Commun. 6:6196, 2015; Shao et al., J. Interferon. Cytokine Res. 26:568-574, 2006; Oka et al., Blood 111:4571-4579, 2008; Corliss et al., Microcirculation 23(2)95-121, 2016). Collectively, these findings suggest that infiltrating CD4 + These findings suggest that lymphatic function is impaired in lymphedema tissues through multiple mechanisms, including the induction of structural changes in lymphatic vessels secondary to tissue fibrosis and the inhibition of collateral lymphatic vessel formation.

[0319] Lymphedema is disfiguring and debilitating; patients experience chronic swelling of the affected limb, recurrent infections, limited mobility, and a reduced quality of life. (See, e.g., Hayes et al., Cancer 118:2237-2249, 2012.) Furthermore, once lymphedema develops, it is usually progressive. Currently, there are no known pharmacological treatments that can halt the progression or promote resolution of lymphedema. (See, e.g., Cormier et al., Ann. Surg. Oncol. 19:642-651, 2012.) As a result, patients are required to wear tight, uncomfortable clothing for the rest of their lives, try to prevent lymphatic fluid accumulation in the affected limb, and undergo rigorous and time-consuming physical therapy procedures. (See, e.g., Koul et al., Int. J. Radiat. Oncol. Biol. Phys., 67:841-846, 2007.) Furthermore, despite receiving chronic follow-up care, some patients experience significant disease progression, increasing swelling, and frequent infections in the lymphedema-affected limbs. Therefore, the development of targeted treatments for lymphedema is an important goal and unmet biomedical need.

[0320] A direct role for NRP2 in regulating adult lymphatic remodeling has been demonstrated both in animal knockout systems, which demonstrate enhanced edema after inflammatory challenge in NRP2 knockout mice (see, e.g., Mucka et al., Am. J. Path 186(11)2803-2812, 2016), and in subjects with SNPs in neuropilin-2 (NP-2) who exhibit increased susceptibility to secondary lymphedema (see, e.g., Miaskowski et al., PLoS ONE 8(4)e60164, 2013). Furthermore, the expression of NRP2 on immune cells and the regulation of its activity by HRS polypeptides, along with potential NRP2-mediated effects on vascular biology, strongly suggest that HRS polypeptides represent a novel and promising therapeutic option for the treatment of lymphedema.

[0321] In some embodiments, the subject has, and / or is selected for treatment based on having, lymphedema at a stage selected from stage 1, stage 2, stage 3, stage 4, stage 5, stage 6, and stage 7, as set forth below.

[0322] Stage 1: Swelling increases over the course of a day and typically resolves overnight when the patient is lying in bed. The affected tissue is in the indentation stage, where the affected area indents when pressed with a fingertip and then rises back up.

[0323] Stage 2: The swelling does not subside overnight and does not resolve without proper management. The affected tissue is elastic and firm and is considered non-pitting. When pressed with a fingertip, the affected area bounces back without indenting. Fibrosis is found in stage 2, and lymphedema signals the beginning of stiffening and expansion in size of the limb.

[0324] Stage 3: The swelling is irreversible and the affected tissue (e.g., limbs) often becomes larger and larger. The affected tissue is stiff (fibrous) and non-responsive.

[0325] Stage 4: The size and circumference of the affected tissue (e.g., limbs) increases significantly. Humps, lumps, and / or protrusions (also called bumps) begin to appear on the skin.

[0326] Stage 5: The affected tissue (e.g., limbs) becomes quite large. In some cases, one or more deep skin folds expand at this stage.

[0327] Stage 6: Small, elongated bumps or small, rounded bumps appear in clusters, resulting in a mossy appearance on the affected tissue (e.g., limbs). The subject's mobility is significantly reduced.

[0328] Stage 7: The subject becomes disabled and unable to independently perform normal daily activities such as walking, bathing, and cooking. Assistance from family or the health care system is required.

[0329] In some embodiments, the subject has, and / or is selected for treatment based on, a grade of lymphedema selected from Grade 1 (mild edema), Grade 2 (moderate edema), Grade 3a (severe edema), Grade 3b (widespread edema), and Grade 4 (massive edema), as described below.

[0330] Grade 1 (mild edema) involves the extremities, such as the forearms and hands, or the lower legs and feet, with a circumference difference of less than 4 cm and no other tissue changes.

[0331] Grade 2 (moderate edema) involves the entire limb or the corresponding quadrant of the trunk. The difference in circumference is 4-6 cm. Histological changes, such as pitting, are present. The subject experiences a single infection of the upper dermis and superficial lymphatics, usually caused by beta-hemolytic group A streptococcus.

[0332] Grade 3a (severe edema) lymphedema presents in one limb and the associated quadrant of the torso, with a circumference difference of more than 6 centimeters. Significant skin changes such as keratinization or hyperkeratosis, cysts and / or fistulas present. Additionally, the subject suffers from repeated attacks of erysipelas.

[0333] Grade 3b (widespread edema) includes the same symptoms as grade 3a, except that two or more limbs are affected.

[0334] Grade 4 (massive edema): The affected limb is enlarged and is caused by almost complete obstruction of the lymphatic vessels.

[0335] Thus, certain embodiments include methods of treating, ameliorating the symptoms of, and / or slowing the progression of lymphedema in a subject in need thereof, comprising administering to the subject an HRS polypeptide or a therapeutic composition provided herein.

[0336] HRS polypeptide-mediated treatment of fibrosis Fibrosis encompasses a wide range of disease forms, including systemic fibrosis, such as systemic sclerosis, scleroderma graft-versus-host disease, nephrogenic systemic fibrosis, lymphedema-associated fibrosis, and IgG4-related sclerosis, as well as radiation-induced fibrosis and many organ-specific diseases, including cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, and renal fibrosis. While the underlying mechanisms are highly diverse, these diseases share a common feature: the uncontrolled and progressive accumulation of fibrotic tissue macromolecules in the affected organ, leading to functional impairment and ultimately failure. Numerous studies have identified myofibroblasts as the cells responsible for the establishment and progression of the fibrotic process. Fibrotic tissue myofibroblasts originate from several sources, including quiescent tissue fibroblasts, circulating CD34+ fibroblasts, and the phenotypic transformation of various cell types, including epithelial and endothelial cells, into activated myofibroblasts.

[0337] Transforming growth factor beta-1 (TGF-β1) is a key regulator of fibrosis in various organ systems, acting through direct mechanisms to increase collagen production by fibroblasts and decrease turnover of matrix products (see, e.g., Willis et al., Am. J. Pathol. 166:1321-1332, 2005; Sakai et al., Am. J. Pathol. 184:2611-2617, 2014; Qi et al., Am. J. Physiol. Renal Physiol. 288:F800-F809, 2005; Bonniaud et al., J. Immunol. 173:2099-2108, 2004). Furthermore, NRP2 plays a direct role in regulating TGF-β1-mediated EMT, which directly leads to fibrosis (see, e.g., Grandclement et al., PLoS ONE 6(7)e20444, 2011), and in promoting fibrosis through EMT or endothelial-EMT in fibroblasts, myofibroblasts, and endothelial cells (see, e.g., Pardali et al., Int. J. Mol. Sci. 18 2157 2017). Furthermore, 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).

[0338] Furthermore, TGF-β1 significantly increases lymphedematous tissue both in clinical settings and in animal models of lymphedema. Inhibition of TGF-β1 using immunotherapy significantly accelerated lymphatic regeneration, reduced fibrosis, decreased inflammation, and improved lymphatic function in a mouse tail model. (See, e.g., Avraham et al., Plast. Reconstr. Surg. 124:438-450, 2009; Clavin et al., Am. J. Physiol. Heart Circ. Physiol. 295:H2113-H2127, 2008; Avraham et al., Am. J. Pathol. 177:3202-3214, 2010.) Inhibiting the fibrotic response preserved the lymphatic system's ability to transport interstitial fluid and inflammatory cells.

[0339] Recent research has shown that CD4 + Cells and macrophages have been shown to play an important role in regulating fibrosis both clinically and in animal models of edema (e.g., Ogata et al., J. Invest. Derm. 136 706-714, 2016; Avraham et al., Am. J. Pathol. 177:3202-3214, 2010; Avraham et al., FASEB J. 27:1114-1126, 2013; Zampell et al., Am. J. Physiol. Cell Physiol. 302:C392-C404, 2012; Zampell et al., PLoS ONE 7:e49940, 2012). For example, in biopsy specimens of clinical lymphedema and in animal models of lymphedema, CD4 + The presence of infiltrating cells and the number of these cells have been found to correlate with the degree of fibrosis and the clinical severity of the disease (see, e.g., Avraham et al., FASEB J. 27:1114-1126, 2013). Patients with late-stage lymphedema generally have a significantly increased number of infiltrating T cells, particularly CD4 T cells, compared with those in early disease stages. +Clinical improvement of lymphedema after lymphovenous bypass, a procedure that shunts blocked lymphatic vessels to the venous circulation, is associated with a decrease in tissue fibrosis and an increase in CD4 + This is associated with a decrease in cell invasion. (Torrisi, et al., Lymphat. Res. Biol. 13:46-53, 2015)

[0340] Fibrosis is also a hallmark of many autoimmune diseases, including chronic graft-versus-host disease (GVHD). This is particularly relevant in allogeneic hematopoietic stem cell transplantation (allo-HSCT), a potentially curative treatment for many hematologic malignancies. Its antitumor activity relies largely on the immune-mediated graft-versus-tumor (GvT) effect. However, donor immune cells contained in the graft may also target healthy host tissues, resulting in graft-versus-host disease (GVHD). GVHD is classified into two syndromes: acute GVHD and chronic GVHD. Acute GVHD has historically been defined as GVHD occurring within the first 100 days after allo-HSCT, whereas chronic GVHD (cGVHD) typically develops beyond 100 days. Although cGVHD is associated with the graft-versus-tumor effect, it is also a major cause of long-term morbidity and mortality in transplant recipients.

[0341] Scleroderma cGVHD (scl-GVHD) is one of the most severe forms of cGVHD, affecting approximately 20% of cGVHD patients. While scl-GVHD shares common features with systemic fibrosis, the two syndromes differ in pathology (scl-GVHD typically begins in the superficial layers of the skin and then spreads to the deeper layers of the skin, whereas the reverse is true for systemic sclerosis) and clinical manifestations. Patients with systemic sclerosis often exhibit clinical features such as Raynaud's syndrome, pulmonary hypertension, and cardiac dysfunction, but these are rarely seen in patients with scl-GVHD.

[0342] Given the role of NRP2 in regulating both immune cell activation and migration, the TGF-β1-mediated EMT promoting fibrosis formation, and the ability of HRS polypeptides to modulate these processes, it is clear that HRS polypeptides may represent a novel and promising therapeutic option for the treatment of fibrosis in the setting of lymphedema and other fibrotic diseases and disorders.

[0343] Thus, certain embodiments include methods of treating, ameliorating the symptoms of, and / or slowing the progression of fibrosis in a subject in need thereof, comprising administering to the subject an HRS polypeptide or a therapeutic composition provided herein.

[0344] HRS polypeptide-mediated regulation of lymphangiogenesis The lymphatic system consists of an interconnected network of capillaries, collecting ducts, and lymph nodes that absorb, collect, and transport fluids and proteins filtered from the vascular system. In humans, this system allows lymphatic vessels to return more than 4 liters of fluid and large amounts of proteins per day to the large veins in the neck.

[0345] Dysfunction of lymphatic vessels (lymphedema) leads to the accumulation of excessive fluid in the interstitial space (edema). While lymphedema is not typically life-threatening, it can lead to serious health consequences, including pain, immobility, fibrosis, inflammation, fatty tissue accumulation, and tissue damage. Because the lymphatic system is also a critical component of the immune response, lymphedema is often accompanied by an increased risk of infection and other immune system problems.

[0346] Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing lymphatic vessels, is associated with a variety of pathologies, including metastatic dissemination, transplant rejection (e.g., cornea, kidney, and heart), type 2 diabetes, obesity, hypertension, and lymphedema (e.g., Alitalo, K., et al. Nature 438:946-953, 2005; Karaman, S., et al. J Clin Invest 124:922-928, 2014; Kim, H., et al. J Clin Invest 124:936-942, 2014; Maby-El Hajjami, H., et al. Histochem Cell Biol 130:1063-107, 2008; Machnik, A., et al. Nat Med 15:545-552; Mortimer, P.S., et al. 2014 J Clin Invest 124:915-921; Skobe, M., et al. 2009. Nat Med 15:993-994).

[0347] Compared with vascular invasion, lymphatic invasion in and around the primary tumor is a prognostic marker for aggressive cancers of various types. Lymphatic proliferation has also been implicated in transplant rejection (Dietrich, T., et al., J Immunol 184:535-539, 2010; Hall, FT, et al., Arch Otolaryngol Head Neck Surg 129:716-719,2003.;Maula,SM,et al.,Cancer Res 63:1920-1926,2003;Miyata,Y.,et al.,J Urol 176:348-353,2006;Saad,RS,et al.,Mod Pathol 19:1317-1323, 2006; Schoppmann, SF, et al., Ann Surg 240:306-312, 2004; Zeng, Y., et al., Prostate 65:222-230, 2005).

[0348] Despite the well-established importance of the lymphatic system in the pathogenesis of many diseases, little progress has been made in the development of anti-lymphangiogenic agents compared with the abundance of anti-angiogenic agents in clinical trials. Therefore, the development of additional lymphangiogenesis inhibitors is of interest for the treatment of a wide range of diseases, including lymphedema and cancer metastasis.

[0349] Anti-lymphangiogenic agents are useful, for example, in the treatment of debilitating ocular diseases, in which lymphatic vessel proliferation is a major cause of corneal graft rejection. It is also a major cause of the cardiovascular events associated with age-related macular degeneration (see, e.g., Dietrich et al., J Immunol 184:535-539, 2010). Penetrating keratoplasty, in particular, is the most common solid tissue transplant procedure, with approximately 40,000 corneal transplants performed annually in the United States. The success rate of penetrating keratoplasty is as high as 90% for uncomplicated initial transplants performed in low-risk, avascular areas. However, the rejection rate for corneal transplants in high-risk, vascular areas is very high (70%–90%). Therefore, there is a need for new, safe, targeted lymphangiogenesis-inhibiting regimens that promote graft survival and reduce or inhibit neovascularization.

[0350] Anti-lymphangiogenic drugs are also useful for the treatment of dry eye disease. Significant upregulation of pro-lymphangiogenic factors (e.g., VEGF-C, VEGF-D, and VEGFR-3) and selective proliferation of lymphatic vessels without concomitant vascular proliferation have been demonstrated in the cornea of ​​dry eye disease (Goyal, S., et al., Arch Ophthalmol 128:819-824, 2010). Dry eye disease is an immune-mediated disorder affecting approximately 5 million Americans. It significantly impacts vision-related life, and symptoms can be debilitating. Current treatment options for dry eye disease are limited, mostly palliative and expensive. Therefore, the development of lymphangiogenesis inhibitors for the treatment of dry eye disease would be of therapeutic value.

[0351] Currently, metastasis is estimated to be the leading cause of death from solid tumors, accounting for as much as 90% of deaths (Gupta and Massague, Cell 127, 679-695, 2006). The complex process of metastasis involves a series of discrete steps, including tumor cell detachment from the primary tumor, tumor cell intravasation into lymphatic or blood vessels, and tumor cell extravasation and proliferation at secondary sites. Analysis of regional lymph nodes in many tumor types suggests that the lymphatic system is an important pathway for the dissemination of human cancers. Furthermore, in almost all carcinomas, the presence of tumor cells in lymph nodes is the most important adverse prognostic factor. While such metastasis was previously thought to involve solely the migration of malignant cells along pre-existing lymphatic vessels near the tumor, recent experimental and clinicopathological studies (see, e.g., Achen et al., Br J Cancer 94, 1355-1360, 2006 and Nathanson, Cancer 98, 413-423, 2003) suggest that solid tumors can induce lymphangiogenesis and facilitate tumor spread. These and other recent studies suggest that targeting the lymphatic system and lymphangiogenesis may be a useful therapeutic strategy for limiting the development of cancer metastasis, which could provide significant benefit to many patients.

[0352] Thus, there is a need for methods and compositions that inhibit the activity of lymphangiogenic factors, as well as methods for preventing or treating transplant rejection, dry eye disease, tumor metastasis, lymphedema and other inflammatory conditions.

[0353] Given the role of HRS polypeptides in regulating the binding and activity of NRP2-interacting NRP2 ligands, HRS polypeptides may represent novel and powerful tools for the development of both pro- and anti-lymphangiogenic therapies. Such differential effects can be modulated, for example, by the use of different HRS polypeptide compositions, different dosages, different treatment durations, or the appropriate use of additional cofactors, such as VEGF-C or semaphorin 3F and / or 3G.

[0354] Thus, certain embodiments include methods of modulating (e.g., increasing, decreasing) lymphangiogenesis in a subject in need thereof, comprising administering to the subject an HRS polypeptide or a therapeutic composition provided herein. Some embodiments include methods of treating, ameliorating the symptoms of, and / or slowing the progression of lymphedema and / or neovascularization in a subject in need thereof, comprising administering to the subject an HRS polypeptide or a therapeutic composition provided herein. Some embodiments include methods of treating, ameliorating the symptoms of, and / or promoting the progression of, or restoring lymphangiogenesis in a subject in need thereof, comprising administering to the subject an HRS polypeptide or a therapeutic composition provided herein.

[0355] HRS polypeptide-controlled regulation of smooth muscle contraction. Decreased smooth muscle (SM) contractility in the bladder can result from many etiologies, including benign prostatic hyperplasia (BPH), posterior urethral valve obstruction, diabetes, multiple sclerosis, spinal cord injury, or obstruction secondary to idiopathic causes (see, e.g., Drake et al., Nat Rev Urol. 11(8):454-464, 2014). In conditions such as BPH or posterior urethral valve obstruction, the bladder contracts against an obstructed outlet. The initial response is adaptive, involving a compensatory phase of SM hypertrophy, which can generate high forces to overcome increased outlet resistance. When demands exceed the bladder's adaptive capacity, contractile performance becomes less efficient, residual volume increases, the bladder undergoes remodeling, and ultimately detrusor contractility is lost as the bladder decompensates (see, e.g., Zderic SA, et al., J Cell Mol Med. 16(2):203-217, 2012). The prevalence of underactive detrusor function has been reported to be as high as 48% in adults (Osman NI, et al., Eur Urol;65(2):389-398, 2014). Furthermore, existing pharmacological treatments for restoring SM contractility, such as muscarinic agonists or cholinesterase inhibitors, have been shown to have limited efficacy and even deleterious effects (Barendrecht et al., BJU Int.99(4):749-752, 2007).

[0356] Recent studies have identified bladder smooth muscle as a major site of Nrp2 expression, demonstrating RhoA inhibition and cytoskeletal muscle stiffness in primary bladder smooth muscle cells treated with the NRP2 ligand SEMA3F. Increased contractility has also been observed in bladder SM strips removed from mice with ubiquitous or smooth muscle-specific deletion of Nrp2 in vivo compared with tissue from Nrp2-intact littermate controls. (See, e.g., Bielenberg et al., Am. J. Pathol. 181 548-559, 2012; Vasquez et al., JCI Insight 2(3)e90617, 2017.)

[0357] Collectively, these findings suggest that downregulation of Nrp2 is an important component of the compensatory response to obstruction in experimental animals and humans and suggest that Nrp2 may represent a novel pharmacological target for maintaining and restoring detrusor contractility in the decompensated bladder.

[0358] Furthermore, recent studies have demonstrated that targeting Nrp2 in decompensating bladders can restore contractility despite ongoing obstruction (Vasquez et al., JCI Insight 2(3)e90617, 2017). These findings support the idea that the Np2 axis may be a novel pharmacological target for restoring SM contractility and provide an important platform for developing HRS polypeptide-based modulators of Nrp2 function.

[0359] To date, pharmacological management of decreased detrusor contractility has focused on stimulating parasympathetic activity to enhance bladder contraction and decrease outflow resistance, thereby facilitating bladder emptying (Chancellor et al., Urology 72(5)966-967, 2008). However, an analysis of a randomized clinical trial of 10 patients with decreased bladder contractility with parasympathomimetic drugs revealed either worsening of symptoms or no significant improvement (Barendrecht et al., BJU Int.99(4)749-752,2007).

[0360] The increased contractility after Nrp2 deletion in decompensated bladders suggests that Nrp2 may be a novel target for alleviating decreased detrusor contractility under chronic obstructive conditions. Given the role of HRS polypeptides in regulating the binding and activity of NRP2-interacting ligands, such HRS polypeptides may represent a powerful new tool for developing novel therapies that modulate smooth muscle (SM) contractility, including for treating decreased SM contractility in the bladder.

[0361] Thus, certain embodiments include methods of modulating (e.g., increasing, decreasing) smooth muscle contractility in a subject in need thereof, comprising administering to the subject an HRS polypeptide or a therapeutic composition provided herein. Certain embodiments include methods of treating, ameliorating the symptoms of, and / or slowing the progression of reduced smooth muscle contractility in a subject in need thereof, comprising administering to the subject an HRS polypeptide or a therapeutic composition provided herein.

[0362] HRS polypeptide-regulated regulation of sarcoidosis and related granulomatous inflammatory diseases Sarcoidosis is a multisystem granulomatous inflammatory disease that is typically characterized by the formation of small, granular inflammatory lesions or granulomas (e.g., caseous granulomas) in various organs and / or the presence of an immune response (e.g., CD4 + Granulomatous inflammation is characterized by the presence of monocytes, macrophages, a strong Th1 response, and the accumulation of activated T lymphocytes, resulting in increased production of TNFα, IL-2, IL-12, IFNγ, IL-1, IL-6, or IL-15. ...

Claims

1. 1. A therapeutic composition for use in a method of treating a neuropilin-2 (NRP2)-associated inflammatory lung disease in a subject in need thereof, the method comprising: determining the level of NRP2a and / or NRP2b in a sample from the subject; and administering the therapeutic composition to the subject if the sample has an increased level of NRP2a and / or NRP2b compared to a population of healthy controls or subjects. Including, A therapeutic composition for use, wherein the HRS polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

156.

2. 2. The therapeutic composition for use according to claim 1, wherein the inflammatory lung disease is selected from pulmonary sarcoidosis, RA-ILD, chronic hypersensitivity pneumonitis, pulmonary inflammation, granulomatous lung disease, neutrophilic asthma, and pulmonary fibrosis.

3. The therapeutic composition for use according to claim 2, wherein the inflammatory lung disease is pulmonary sarcoidosis.

4. The therapeutic composition for use according to claim 1, wherein the subject has systemic sclerosis.

5. The therapeutic composition for use according to any one of claims 1 to 4, wherein said HRS-Fc polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO:

156.

6. The therapeutic composition for use according to claim 5, wherein said HRS-Fc polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 156.

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