Biomarkers for assessing risk of developing acute and post-acute COVID-19

Abstract

Disclosed herein are compositions, kits, and methods for determining the concentration of fluid-phase MASP-2 / C1-INH complexes in biological fluids, such as biological fluids obtained from subjects infected with SARS-CoV-2. Also disclosed are methods of using said compositions, methods, and kits to detect MASP-2 / C1-INH complexes for the purpose of determining the status of lectin pathway activation in mammalian subjects, thereby assessing the risk of developing COVID-19-associated ARDS or other poor outcomes in subjects who are or have been infected with SARS-CoV-2, or determining the need for or efficacy of treatment with complement inhibitors, such as MASP-2 inhibitors, in subjects in need thereof. TIFF2024509702000097.tif106158

Description

[Technical field]

[0001] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of hardcopy and is incorporated herein by reference. The name of the text file containing the sequence listing is MP_1_0319_PCT_Sequence_Listing_20220131_ST25.txt. This text file is 147KB, was created on February 1, 2022, and has been submitted via EFS-Web with the application herein. [Background technology]

[0002] background The complement system provides an early-acting mechanism for initiating, amplifying, and orchestrating immune responses to microbial infections and other acute insults in humans and other vertebrates (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd., New York). Although complement activation provides a beneficial first line of defense against potential pathogens, complement activity that promotes a protective immune response can also pose a potential threat to the host (K.R. Calli,et al., Springer Semin. Immunopathol.15:417-431, 1994; B.P. Morgan, Eur.J. Clinical Investig.24:219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Activated neutrophils are essential for host defense but can indiscriminately release destructive enzymes, causing organ damage. Furthermore, complement activation can result in the deposition of lytic complement components on the surfaces of nearby host cells as well as microbial targets, resulting in host cell lysis.

[0003] It is now widely accepted that the complement system can be activated through three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by a complex consisting of a host antibody bound to a foreign particle (i.e., an antigen) and therefore requires prior exposure to the antigen to generate a specific antibody response. Since activation of the classical pathway depends on a previous adaptive immune response by the host, the classical pathway is part of the acquired immune system. In contrast, both the lectin pathway and the alternative pathway are independent of adaptive immunity and are part of the innate immune system.

[0004] The lectin pathway is widely believed to have a major role in host defense against infection in naive hosts. Strong evidence for the involvement of MBL in host defense comes from the analysis of patients with low serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572:401-413, (2002)). Such patients are susceptible to recurrent bacterial and fungal infections. These symptoms usually appear at an early age, when maternal antibody titers are declining, but during a period of apparent vulnerability before the full repertoire of antibody responses has developed. This syndrome is often due to mutations at several sites in the collagen moiety of MBL that prevent proper formation of MBL oligomers. However, since MBL can function as an opsonin independent of complement, it is not known to what extent the increased susceptibility to infection is due to impaired complement activation.

[0005] All three pathways (i.e., classical, lectin, and alternative) have been thought to converge at C5. C5 is cleaved to form products with multiple proinflammatory actions. The convergent pathway has been called the terminal complement pathway. C5a is the most potent anaphylatoxin that induces changes in smooth muscle tone and vascular tone as well as vascular permeability. C5a is also a potent chemotaxin and activator of neutrophils and monocytes. Cell activation via C5a can significantly amplify the inflammatory response by inducing the release of multiple additional inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites, and reactive oxygen species. Once C5 is cleaved, C5b-9, also known as the membrane attack complex (MAC), is formed. There is now strong evidence that MAC deposition, which is insufficient to cause lysis, may play an important role in inflammation in addition to its role as a lytic pore-forming complex.

[0006] In addition to its essential role in immune defense, the complement system contributes to tissue injury in many clinical conditions. Although there is extensive evidence linking the classical and alternative complement pathways to the development of non-infectious human diseases, the role of the lectin pathway is only beginning to be evaluated. Recent studies have provided evidence that activation of the lectin pathway may be responsible for complement activation and associated inflammation in ischemia / reperfusion injury. Collard et al., (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and exhibit C3 deposition when exposed to human serum (Collard et al., Am. J. Pathol 156:1549-1556, (2000)). Furthermore, treatment of human serum with a blocking anti-MBL monoclonal antibody inhibited MBL binding and complement activation. These findings were extended to a rat myocardial ischemia-reperfusion model. In this model, rats treated with blocking antibodies against rat MBL showed significantly less myocardial damage during coronary artery occlusion than control antibody-treated rats (Jordan et al., Circulation, 104:1413-1418,(2001)). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unclear. Recent studies suggest that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothelial cytokeratins but not glycoconjugates (Collard e al., Am. J. Pathol. 159:1045-1054,(2001)). Other studies have linked classical and alternative pathways to the development of ischemia / reperfusion injury, and the role of the lectin pathway in this disease remains controversial (Riedermann, NC et al., Am. J. Pathol. 162:363-367, 2003).

[0007] Fibrosis is the formation of excess connective tissue in an organ or tissue, generally in response to injury or damage. A hallmark of fibrosis is the production of excess extracellular matrix following local trauma. Although the normal physiological response to injury results in the deposition of connective tissue, this initially beneficial repair process may persist and cause disease, thereby altering tissue structure and function. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased stiffness, microvascular compression, and hypoxia. The influx of inflammatory cells, including macrophages and lymphocytes, releases cytokines that amplify the deposition of collagen, fibronectin, and other molecular markers of fibrosis. Traditional therapeutic approaches have been primarily directed at the inflammatory process of fibrosis with corticosteroids and immunosuppressive drugs. Unfortunately, these anti-inflammatory agents have had little or no clinical efficacy. Currently, there are no effective treatments or cures for fibrosis, although both animal studies and human anecdotal reports suggest that fibrotic tissue damage may be reversed (Tampe and Zeisberg, Nat Rev Nephrol, Vol 10:226-237, 2014).

[0008] The kidney has limited ability to recover from injury. Various renal pathologies cause local inflammation leading to scarring and fibrosis of renal tissue. Persistence of inflammatory stimuli drives tubulointerstitial inflammation and fibrosis and progressive renal dysfunction in chronic kidney disease. Progression to end-stage renal failure is associated with significant morbidity and mortality. As tubulointerstitial fibrosis is a common endpoint of multiple renal pathologies, it is an important target for therapies aimed at preventing renal failure. Risk factors independent of primary renal disease (e.g., proteinuria) drive local inflammation, contributing to the development of renal fibrosis and loss of renal excretory function, thereby hastening disease progression.

[0009] Considering the role of fibrosis in many diseases and disorders, such as tubulointerstitial fibrosis that leads to chronic kidney disease, there is an urgent need to develop therapeutically effective agents to treat diseases and conditions caused or exacerbated by fibrosis.Furthermore, considering the scarcity of new and existing therapies that target profibrotic inflammatory pathways in kidney disease, there is a need to develop therapeutically effective agents to treat, inhibit, prevent, and / or reverse kidney fibrosis, thereby preventing progressive chronic kidney disease.

[0010] Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-coronavirus 2 or SARS-CoV-2), a virus closely related to the SARS virus (World Health Organization, 2 / 11 / 2020, Novel Coronavirus Situation Report 22). People affected by COVID-19 may develop fever, dry cough, fatigue, and shortness of breath. Cases may progress to pneumonia, respiratory decline including severe acute respiratory distress syndrome, and death in the most vulnerable individuals (see, for example, Hui DS et al., Int J Infect Dis 91:264-266, Jan 14, 2020). There is no vaccine or specific antiviral treatment, and management involves treating symptoms and supportive care.

[0011] Influenza (also known as the "flu") is an infectious disease caused by an RNA influenza virus. Symptoms of influenza virus infection can range from mild to severe and include high fever, runny nose, sore throat, muscle and joint pain, headache, cough, and fatigue. These symptoms typically begin two days after exposure to the virus and last less than a week at most, although coughing can last for more than two weeks (see "Influenza Seasonal, World Health Organization 6 November 2018). Complications of influenza can include viral pneumonia, acute respiratory distress syndrome (ARDS), secondary bacterial pneumonia, sinus infections, and worsening of pre-existing health problems such as asthma or heart failure (see "Key Facts About Influenza (Flu)" Centers for Disease Control and Prevention (CDC), September 9, 2014). The effects of influenza are much more severe and last longer than those of the common cold. Most people recover completely in about one to two weeks, but others develop life-threatening complications such as pneumonia. Thus, influenza can be deadly, especially for those who are infirm, young and old, have compromised immune systems, or have chronic illnesses. See Hilleman MR, Vaccine. 20 (25-26): 3068-87 (2002).

[0012] Three of the four influenza viruses affect humans: types A, B, and C (see “Types of Influenza Viruses Seasonal Influenza (Flu), Centers for Disease Control and Prevention (CDC). 27 September 2017). Type D is not known to infect humans, but it is thought to have the potential to do so (see “Novel Influenza D virus: Epidemiology, pathology, evolution and biological characteristics”, Virulence. 8 (8): 1580-91, 2017). Influenza A serotypes identified in humans are H1N1 (which caused the "Spanish Flu" in 1918 and "Swine Flu" in 2009); H2N2 (which caused the "Asian Flu" in 1957), H3N2 (which caused the Hong Kong Flu in 1968), H5N1 (which caused the "Bird Flu" in 2004), H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and H6N1. World Health Organization (30 June 2006). Epidemiology of WHO-confirmed human cases of avian influenza A (H5N1) infection, Wkly Epidemiol Rec. 81 (26): 249-57.; Fouchier RA, et al. (2004) PNAS 101 (5): 1356-61; Wkly Epidemiol Rec. 83 (46): 415-20, Asian Lineage Avian Influenza A(H7N9) Virus, Centers for Disease Control and Prevention (CDC), 7 December 2018).

[0013] Common symptoms of influenza virus (also known as the flu), such as fever, headache, and fatigue, are the result of large amounts of proinflammatory cytokines and chemokines (e.g., interferon or tumor necrosis factor) produced by influenza-infected cells. See Eccles R. et al., Lancet Infect Dis 5(11):718-25 (2005); Schmitz N, et al., Journal of Virology. 79 (10): 6441-8 (2005). This powerful immune response can lead to a life-threatening cytokine storm. This effect has been proposed to be responsible for the unusual mortality of H5N1 avian influenza and the 1918 pandemic strain. Cheung CY, et al., Lancet. 360 (9348): 1831-37 (2002); Kash JC, et al., Nature. 443 (7111): 578-81 (2006). Influenza also appears to induce programmed cell death (apoptosis). See Spiro SG, et al., Clinical Respiratory Medicine, Elsevier Health Sciences. p. 311 (2012).

[0014] Thus, there is an urgent need to develop therapeutically effective agents to treat, inhibit, and / or prevent coronavirus-induced pneumonia and acute respiratory distress syndrome and influenza virus-induced pneumonia and acute respiratory distress syndrome.

[0015] Additionally, to maximize success in treating and protecting people from COVID-19, biomarkers and highly accurate tests are urgently needed to identify individuals at risk for developing acute COVID-19 and / or long-term disease (post-acute COVID-19, otherwise known as Long COVID-19 syndrome) or who have mounted a protective immune response to the COVID-19 disease response. Tests are also needed to determine the efficacy of therapeutic agents to treat and / or prevent COVID-19-related complications in subjects infected with SARS-CoV-2, including those with or at risk for developing Long COVID-19. Summary of the Invention

[0016] overview This Summary is provided to introduce various concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0017] In one aspect, the present invention provides a method for treating, inhibiting, alleviating, or preventing acute respiratory distress syndrome, pneumonia, or any other pulmonary or other acute manifestation of COVID-19, such as thrombosis, in a mammalian subject infected with SARS-CoV-2, comprising the steps of: (i) determining the level of MASP-2 / C1-INH complex in a biological sample obtained from the subject, where an increased level of MASP-2 / C1-INH complex compared to a healthy control sample or other reference standard indicates an increased risk of developing one or more acute manifestations of COVID-19; and (ii) administering to a subject having an increased level of MASP-2 / C1-INH complex an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject suffers from one or more respiratory symptoms, and the method comprises administering to the subject an amount of a MASP-2 inhibitor effective to improve at least one respiratory symptom (i.e., improve respiratory function). In one embodiment, the MASP-2 inhibitor is a MASP-2 antibody or an antigen-binding fragment thereof. In one embodiment, the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitor selectively inhibits lectin pathway complement activation without substantially inhibiting C1q-dependent complement activation. In one embodiment, the MASP-2 inhibitor is a small molecule, e.g., a synthetic small molecule or a semi-synthetic small molecule, that inhibits MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitor is an expression inhibitor of MASP-2. In one embodiment, the MASP-2 inhibitor antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitor antibody or a fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitor antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:69. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:69.

[0018] In another aspect, the present invention provides a method for treating, ameliorating, preventing, or reducing the risk of developing one or more COVID-19-associated long-term sequelae in a mammalian subject that has been infected with SARS-CoV-2, comprising the steps of: (i) determining the level of MASP-2 / C1-INH complex in a biological sample obtained from the subject, wherein an increased level of MASP-2 / C1-INH complex compared to a healthy control sample indicates an increased risk of developing one or more COVID-19-associated long-term sequelae; and (ii) administering to a subject having an increased level of MASP-2 / C1-INH complex an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitor is a MASP-2 antibody or an antigen-binding fragment thereof. In one embodiment, the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitor selectively inhibits lectin pathway complement activation without substantially inhibiting C1q-dependent complement activation. In one embodiment, the MASP-2 inhibitor is a small molecule, e.g., a synthetic small molecule or semi-synthetic small molecule, that inhibits MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitor is an expression inhibitor of MASP-2. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or a fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:69. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:69.

[0019] In another aspect, the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 complexed with C1-INH, the antibody comprising a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:87 and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:88, the CDRs being numbered according to the Kabat numbering system. In one embodiment, the MASP-2-specific antibody comprises a heavy chain variable region having at least 95% identity to the amino acid sequence shown in SEQ ID NO:87 and a light chain variable region having at least 95% identity to the amino acid sequence shown in SEQ ID NO:88. In one embodiment, the MASP-2-specific antibody or antigen-binding fragment thereof is labeled with a detectable moiety, for example a detectable moiety suitable for use in an immunoassay as further described herein. In one embodiment, the MASP-2-specific antibody or fragment thereof is immobilized on a support, e.g., on a support suitable for use in an immunoassay, e.g., an immunoassay for detecting the MASP-2 / C1-INH complex.

[0020] In another aspect, the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 complexed with C1-INH, the antibody comprising a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:97 and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:98, the CDRs being numbered according to the Kabat numbering system. In one embodiment, the MASP-2-specific antibody comprises a heavy chain variable region having at least 95% identity to the amino acid sequence shown in SEQ ID NO:97 and a light chain variable region having at least 95% identity to the amino acid sequence shown in SEQ ID NO:98. In one embodiment, the MASP-2-specific antibody or antigen-binding fragment thereof is labeled with a detectable moiety, for example a detectable moiety suitable for use in an immunoassay as further described herein. In one embodiment, the MASP-2-specific antibody or fragment thereof is immobilized on a support, e.g., on a support suitable for use in an immunoassay, e.g., an immunoassay for detecting the MASP-2 / C1-INH complex.

[0021] In another aspect, the present disclosure provides a method for measuring the amount of MASP-2 / C1-INH in a biological sample, comprising the steps of: (a) preparing a test biological sample from a human subject; (b) performing an immunoassay comprising capturing and detecting MASP-2 / C1-INH complex in the test sample, wherein the MASP-2 / C1-INH complex is captured by a monoclonal antibody that specifically binds to human MASP-2, and the MASP-2 / C1-INH complex is directly or indirectly detected by an antibody that specifically binds to C1-INH; and (c) comparing the level of MASP-2 / C1-INH complex detected according to (b) with a predetermined level or a control sample, wherein the level of MASP-2 / C1-INH complex detected in the test sample indicates the degree of lectin pathway complement activation. In some embodiments, the biological sample is a fluid sample from a human subject selected from the group consisting of whole blood, serum, plasma, urine, and cerebrospinal fluid. In some embodiments, the human subject is currently infected with SARS-CoV-2 or has previously been infected with SARS-CoV-2. In some embodiments, the antibody that specifically binds to MASP-2 comprises a binding domain that comprises HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:87 and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:88, where the CDRs are numbered according to the Kabat numbering system. In some embodiments, the antibody that specifically binds to MASP-2 comprises a binding domain that comprises HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:97 and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:98, where the CDRs are numbered according to the Kabat numbering system.

[0022] In another aspect, the disclosure provides a method for determining a risk of a subject who is or has been infected with SARS-CoV-2, of developing COVID-19 associated ARDS or long term sequelae associated with COVID-19, comprising the steps of: (a) obtaining a biological sample from the subject; (b) measuring the level of MASP-2 / C1-INH complex in the sample; (c) comparing the measured level with a predetermined level or reference standard of MASP-2 / C1-INH complex to assess the risk of developing COVID-19 associated ARDS and / or long term sequelae associated with COVID-19; and (d) determining the subject's risk of developing COVID-19 associated ARDS and / or long term sequelae associated with COVID-19 and reporting the result to a patient, a physician, or a database; and (e) optionally administering a treatment to the subject determined to be likely to develop acute disease and / or long term sequelae associated with COVID-19 infection. In some embodiments, the level of MASP-2 / C1-INH complex is measured in an immunoassay. In some embodiments, step (b) comprises performing an immunoassay such as an ELISA assay to measure the level of MASP-2 / C1-INH complex in the biological sample. In some embodiments, the immunoassay comprises the use of an antibody that specifically binds to MASP-2, comprising a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:87 and comprising LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:88, the CDRs being numbered according to the Kabat numbering system. In some embodiments, the immunoassay involves the use of an antibody that specifically binds to MASP-2, comprising a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:97 and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system.

[0023] In another aspect, the present disclosure provides a method for monitoring the effectiveness of treatment with a MASP-2 inhibitory antibody or antigen-binding fragment thereof in a mammalian subject in need thereof, comprising the following steps: (a) administering a dose of a MASP-2 inhibitory antibody or antigen-binding fragment thereof to the mammalian subject at a first time point; (b) evaluating a first level of MASP-2 / C1-INH complex in a biological sample obtained from the subject after step (a); (c) treating the subject with the MASP-2 inhibitory antibody or antigen-binding fragment thereof at a second time point; (d) evaluating a second level of MASP-2 / C1-INH complex in a biological sample obtained from the subject after step (c); and (e) comparing the level of MASP-2 / C1-INH complex evaluated in step (b) with the level of MASP-2 / C1-INH complex evaluated in step (d) to determine the effectiveness of the MASP-2 inhibitory antibody or antigen-binding fragment thereof in the mammalian subject. In some embodiments, the subject is a human subject suffering from or at risk of developing COVID-19 or long-term sequelae associated with COVID-19. In some embodiments, the subject is a human subject suffering from or at risk of developing a disease or disorder selected from the group consisting of HSCT-TMA, IgAN, lupus nephritis, and graft-versus-host disease, or any other lectin pathway disease or disorder. In some embodiments, the level of MASP-2 / C1-INH complex is measured in an immunoassay. In some embodiments, step (b) comprises performing an immunoassay, such as an ELISA assay, to measure the level of MASP-2 / C1-INH complex in the biological sample. In some embodiments, the immunoassay involves the use of an antibody that specifically binds to MASP-2, comprising a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:87 and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:88, wherein the CDRs are numbered according to the Kabat numbering system.In some embodiments, the immunoassay involves the use of an antibody that specifically binds to MASP-2, comprising a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:97 and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system. [Brief description of the drawings]

[0024] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, in which:

[0025] [Figure 1] This is a diagram showing the genomic structure of human MASP-2. [Diagram 2] Figure 2A is a schematic diagram showing the domain structure of human MASP-2 protein, and Figure 2B is a schematic diagram showing the domain structure of human MAp19 protein. [Diagram 3] FIG. 1 shows the mouse MASP-2 knockout strategy. [Figure 4] FIG. 1 shows the human MASP-2 minigene construct. [Figure 5A] As described in Example 2, the results are illustrated demonstrating that MASP-2 deficiency leads to a loss of C4 activation mediated by the lectin pathway, as measured by the lack of C4b deposition on mannan. [Figure 5B] As described in Example 2, the results are illustrated demonstrating that MASP-2 deficiency leads to abolishment of C4 activation mediated by the lectin pathway, as measured by the lack of C4b deposition on zymosan. [Figure 5C]The figures show results demonstrating relative C4 activation levels in serum samples obtained from MASP-2+ / -; MASP-2- / - and wild-type strains, as measured by C4b deposition on mannan and zymosan, as described in Example 2. [Figure 6] The figures show results demonstrating that addition of mouse recombinant MASP-2 to MASP-2- / - serum samples restored lectin pathway-mediated C4 activation in a protein concentration-dependent manner, as measured by C4b deposition on mannan, as described in Example 2. [Figure 7] As described in Example 8, the results demonstrating that the classical pathway functions in MASP-2- / - lines are shown. [Figure 8A] As described in Example 10, the results showing that anti-MASP-2 Fab2 antibody #11 inhibits C3 convertase formation are shown. [Figure 8B] As described in Example 10, the results showing that anti-MASP-2 Fab2 antibody #11 binds to native rat MASP-2 are shown. [Figure 8C] The figures show results demonstrating that anti-MASP-2 Fab2 antibody #41 inhibits C4 cleavage, as described in Example 10. [Figure 9] As described in Example 10, the results are shown demonstrating that all of the anti-MASP-2 Fab2 antibodies tested that inhibited C3 convertase formation were found to also inhibit C4 cleavage. [Figure 10] This figure shows the rat MASP-2-derived recombinant polypeptide used for epitope mapping of the MASP-2-blocking Fab2 antibody, as described in Example 11. [Figure 11] As described in Example 11, the results demonstrating the binding of anti-MASP-2 Fab2 #40 and #60 to rat MASP-2 polypeptide are shown. [Figure 12A]Illustrated are MAC deposition levels in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under lectin pathway-specific assay conditions as described in Example 12. This demonstrates that OMS646 inhibits lectin-mediated MAC deposition with an IC50 value of approximately 1 nM. [Figure 12B] Illustrated are MAC deposition levels in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under classical pathway-specific assay conditions as described in Example 12. This demonstrates that OMS646 does not inhibit MAC deposition via the classical pathway. [Figure 12C] Illustrated are MAC deposition levels in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under alternative pathway-specific assay conditions as described in Example 12. This demonstrates that OMS646 does not inhibit MAC deposition via the alternative pathway. [Figure 13] Illustrates the pharmacokinetic (PK) profile of human MASP-2 monoclonal antibody (OMS646) in mice, as described in Example 12. Shown is OMS646 concentration (mean of n=3 animals / group) as a function of time after administration at the indicated doses. [Figure 14A] This figure illustrates the pharmacodynamic (PD) response of a human MASP-2 monoclonal antibody (OMS646), measured as a reduction in systemic lectin pathway activity in mice after intravenous administration, as described in Example 12. [Figure 14B] This figure illustrates the pharmacodynamic (PD) response of a human MASP-2 monoclonal antibody (OMS646), measured as a reduction in systemic lectin pathway activity in mice after subcutaneous administration, as described in Example 12. [Figure 15]The results of computerized image analysis of Sirius Red stained kidney tissue sections are shown, in which the tissue sections were obtained from wild-type and MASP-2- / - mice after 7 days of unilateral ureteric obstruction (UUO) and from sham-operated wild-type and MASP-2- / - mice, as described in Example 14. [Figure 16] The figures show the results of computerized image analysis of kidney tissue sections stained with F4 / 80 macrophage-specific antibody, in which the tissue sections were obtained from wild-type and MASP-2- / - mice after 7 days of unilateral ureteral ligation (UUO) and from sham-operated wild-type and MASP-2- / - mice, as described in Example 14. [Figure 17] This figure shows the relative mRNA expression levels of collagen-4 as measured by quantitative PCR (qPCR) in kidney tissue sections from wild-type and MASP-2- / - mice and sham-operated wild-type and MASP-2- / - mice after 7 days of unilateral ureteral ligation (UUO), as described in Example 14. [Figure 18] This figure shows the relative mRNA expression levels of transforming growth factor beta-1 (TGFβ-1) as measured by qPCR in kidney tissue sections from wild-type and MASP-2- / - mice and sham-operated wild-type and MASP-2- / - mice after 7 days of unilateral ureteral ligation (UUO), as described in Example 14. [Figure 19] This figure shows the relative mRNA expression levels of interleukin-6 (IL-6) as measured by qPCR in kidney tissue sections from wild-type and MASP-2- / - mice and sham-operated wild-type and MASP-2- / - mice after 7 days of unilateral ureteral ligation (UUO), as described in Example 14. [Figure 20]The figures show relative mRNA expression levels of interferon-gamma as measured by qPCR in kidney tissue sections from wild-type and MASP-2- / - mice and sham-operated wild-type and MASP-2- / - mice after 7 days of unilateral ureteral ligation (UUO), as described in Example 14. [Figure 21] Illustrated are the results of computerized image analysis of Sirius Red stained kidney tissue sections obtained after 7 days of unilateral ureteral ligation (UUO) from wild-type mice treated with MASP-2 inhibitory antibodies and wild-type mice treated with isotype control antibodies, as described in Example 15. [Figure 22] This figure illustrates the hydroxylproline content from kidneys taken after 7 days of unilateral ureteral ligation (UUO) from wild-type mice treated with MASP-2 inhibitory antibodies, compared to hydroxylproline levels in tissue derived from ligated kidneys from wild-type mice treated with an IgG4 isotype control, as described in Example 15. [Diagram 23] The total serum protein (mg / ml) measured on day 15 of a protein overload study in wild-type control mice given saline only (n=2), wild-type mice given BSA (n=6), and MASP-2- / - mice given BSA (n=6), as described in Example 16, is shown. [Figure 24] The total amount of protein excreted (mg) in urine collected over a 24-hour period on day 15 of a protein overload study from wild-type control mice given saline only (n=2), wild-type mice given BSA (n=6), and MASP-2- / - mice given BSA (n=6), as described in Example 16. [Diagram 25]Representative hematoxylin and eosin (H&E) stained kidney tissue sections are shown from the following groups of mice on day 15 of the protein overload study, as described in Example 16: (Panel A) wild-type control mice; (Panel B) MASP-2- / - control mice, (Panel C) wild-type mice treated with BSA; and (Panel D) MASP-2- / - mice treated with bovine serum albumin (BSA). [Figure 26] Illustrated are the results of computerized image analysis of kidney tissue sections stained with the macrophage-specific antibody F4 / 80, showing the average macrophage stained area (%). Here, tissue sections were obtained from wild-type control mice (n=2), wild-type mice treated with BSA (n=6), and MASP-2- / - mice treated with BSA (n=5) on day 15 of the protein overload study, as described in Example 16. [Figure 27A] FIG. 1 illustrates an analysis of whether a macrophage-proteinuria correlation exists in each wild-type mouse (n=6) treated with BSA by plotting total excreted protein measured in urine from 24-hour samples versus macrophage infiltration (mean % stained area), as described in Example 16. [Figure 27B] As described in Example 16, an analysis of whether a macrophage-proteinuria correlation exists in each MASP-2- / - mouse (n=5) treated with BSA is shown by plotting total urinary excreted protein versus macrophage infiltration (mean % stained area) in 24-hour samples. [Figure 28] The results of computerized image analysis of tissue sections stained with anti-TGFβ antibody (measured as %TGFβ antibody stained area) in wild-type mice treated with BSA (n=4) and MASP-2- / - mice treated with BSA (n=5) as described in Example 16 are shown. [Figure 29] The results of computerized image analysis of tissue sections stained with anti-TNFα antibody (measured as % TNFα antibody stained area) in BSA-treated wild-type mice (n=4) and BSA-treated MASP-2- / - mice (n=5) as described in Example 16 are shown. [Diagram 30] As described in Example 16, the results of computerized image analysis of tissue sections stained with anti-IL-6 antibody (measured as % IL-6 antibody stained area) in wild-type control mice, MASP-2- / - control mice, wild-type mice treated with BSA (n=7), and MASP-2- / - mice treated with BSA (n=7) are shown. [Diagram 31] As described in Example 16, the frequency of TUNEL apoptotic cells counted in 20 consecutively selected high power fields (HPF) from tissue sections derived from the renal cortex of a wild-type control mouse (n=1), a MASP-2- / - control mouse (n=1), a wild-type mouse treated with BSA (n=6), and a MASP-2- / - mouse treated with BSA (n=7) are shown. [Diagram 32] Representative H&E stained tissue sections from the following groups of mice on day 15 after treatment with BSA as described in Example 17: (Panel A) saline-treated wild-type control mice, (Panel B) isotype antibody-treated control mice, and (Panel C) wild-type mice treated with MASP-2 inhibitory antibody. [Diagram 33] As described in Example 17, the frequency of TUNEL apoptotic cells counted in 20 consecutively selected high power fields (HPF) from tissue sections derived from the renal cortex of wild-type mice treated with saline control and BSA (n=8), wild-type mice treated with isotype control antibody and BSA (n=8), and wild-type mice treated with MASP-2 inhibitor antibody and BSA (n=7) are shown. [Diagram 34] As described in Example 17, the results of computerized image analysis of tissue sections stained with anti-TGFβ antibody (measured as %TGFβ antibody stained area) are shown in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). [Diagram 35]As described in Example 17, the results of computerized image analysis of tissue sections stained with anti-TNFα antibody (measured as % TNFα antibody stained area) are shown in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). [Diagram 36] As described in Example 17, the results of computerized image analysis of tissue sections stained with anti-IL-6 antibody (measured as % IL-6 antibody stained area) are shown in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). [Figure 37] Representative H&E stained tissue sections from the following groups of mice are shown on day 14 after treatment with adriamycin or saline alone (control), as described in Example 18: (Panels A-1, A-2, A-3) wild-type control mice treated with saline alone; (Panels B-1, B-2, B-3) wild-type mice treated with adriamycin; and (Panels C-1, C-2, C-3) MASP-2- / - mice treated with adriamycin. [Figure 38] The figures show the results of computerized image analysis of kidney tissue sections stained with the macrophage-specific antibody F4 / 80, showing the average area (%) of macrophages stained from the following groups of mice on day 14 after treatment with adriamycin or saline alone (wild-type control): wild-type control mice treated with saline alone; wild-type mice treated with adriamycin; MASP-2- / - mice treated with saline alone, and MASP-2- / - mice treated with adriamycin, as described in Example 18. **p=0.007. [Figure 39]The figures show the results of computerized image analysis of Sirius red stained kidney tissue sections showing the area of ​​collagen deposition staining (%) from the following mouse groups on day 14 after treatment with adriamycin or saline alone (wild-type control): wild-type control mice treated with saline alone; wild-type mice treated with adriamycin; MASP-2- / - mice treated with saline alone, and MASP-2- / - mice treated with adriamycin, as described in Example 18. **p=0.005. [Diagram 40] This figure illustrates the urinary albumin / creatinine ratio (uACR) in two IgA patients treated weekly with a MASP-2 inhibitory antibody (OMS646) during a 12-week study, as described in Example 19. [Figure 41A] Illustrated are representative images (H&E, 400x) of immunohistochemistry analysis of tissue sections of septal vessels from COVID-19 patient lungs, as described in Example 21. [Figure 41B] Illustrated are representative images (H&E, 400x) of immunohistochemistry analysis of tissue sections of septal vessels from COVID-19 patient lungs, as described in Example 21. [Figure 41C] Illustrated are representative images of immunohistochemical analysis of tissue sections of medium diameter pulmonary septal vessels from COVID-19 patients, as described in Example 21. [Figure 41D] Illustrated are representative images (H&E, 400x) of immunohistochemistry analysis of tissue sections of liver parenchyma from COVID-19 patients as described in Example 21. [Figure 42A] Illustrates the number of circulating endothelial cells (CECs) / ml in peripheral blood of normal healthy controls (n-6) compared to the number of CECs / ml in COVID-19 patients (n=33) who are not part of this study, as described in Example 21. [Figure 42B]Illustrates CEC / ml counts in six patients selected for this study before (baseline) and after narsoplimab treatment, as described in Example 21. Boxes represent values ​​from 1st to 3rd quartiles, horizontal lines indicate medians, and whiskers indicate minimum and maximum values. [Diagram 43] FIG. 1 illustrates serum levels of C-reactive protein (CRP) (median; interquartile range (IQR)) in six COVID-19 patients at baseline before treatment (day 0) and at various time points after narsoplimab treatment, as described in Example 21. [Diagram 44] FIG. 1 illustrates serum levels (median; IQR) of lactate dehydrogenase (LDH) in six COVID-19 patients at baseline (day 0) before treatment and at various time points after narsoplimab treatment, as described in Example 21. [Diagram 45] FIG. 1 illustrates serum levels of interleukin-6 (IL-6) (median; interquartile range (IQR)) in six COVID-19 patients at baseline (day 0) before treatment and at various time points after narsoplimab treatment, as described in Example 21. [Figure 46] FIG. 1 illustrates serum levels of interleukin-8 (IL-8) (median; interquartile range (IQR)) in six COVID-19 patients at baseline (day 0) before treatment and at various time points after narsoplimab treatment, as described in Example 21. [Figure 47] Figure 47A illustrates a CT scan of patient #4 on the 5th day after enrollment (i.e., after narsoplimab treatment), as described in Example 21. In this CT scan, the patient was observed to have severe interstitial pneumonia and diffuse ground-glass opacities involving both peripheral and central regions, consolidation of the lower lung lobes, particularly the lower lobe of the left lung, as well as extensive bilateral pulmonary embolism and opacification defects (not shown) in the interlobar and segmental arteries. Figure 47B illustrates a CT scan of patient #4 on the 16th day after enrollment (i.e., after narsoplimab treatment), as described in Example 21, with significantly less ground-glass opacities and almost complete resolution of parenchymal consolidation. [Figure 48]FIG. 1 illustrates serum levels of IL-6 (pg / mL) at baseline and at different time points after narsoplimab treatment (after 2 doses, after 4 doses) in patients treated with narsoplimab as described in Example 21. Boxes represent values ​​from the 1st to 3rd quartiles, horizontal lines indicate medians, and dots illustrate values ​​for all patients. [Figure 49] FIG. 1 illustrates serum levels of IL-8 (pg / mL) at baseline and at different time points after narsoplimab treatment (after 2 doses, after 4 doses) in patients treated with narsoplimab as described in Example 21. Boxes represent values ​​from the 1st to 3rd quartiles, horizontal lines indicate medians, and dots illustrate values ​​for all patients. [Figure 50] FIG. 2 illustrates the clinical outcomes of six COVID-19 infected patients treated with narsoplimab as described in Example 21. [Figure 51A] Illustrates serum levels (units per liter, U / L) of aspartate aminotransferase (AST) before and after narsoplimab treatment as described in Example 21. Black lines represent median and interquartile range (IQR). Red lines represent normality levels. Points represent values ​​for all patients. [Figure 51B] Figure 2 illustrates the serum levels (ng / ml) of D-dimer in four patients. Baseline values ​​were available before the start of narsoplimab treatment. Black circles indicate the start of steroid treatment. Red lines represent normalized levels as described in Example 21. [Figure 52A] FIG. 1 illustrates serum levels (ng / ml) of D-dimer values ​​in a seventh COVID-19 infected patient (Patient #7) treated with narsoplimab at baseline (day 0) before treatment and at different time points after narsoplimab treatment, as described in Example 22. In this figure, narsoplimab dosing is indicated by vertical arrows and the horizontal line represents normality levels. [Figure 52B]FIG. 1 illustrates serum levels of C-reactive protein (CRP) in COVID-19 infected patient #7 at baseline (day 0) before treatment and at different time points after narsoplimab treatment, as described in Example 22. In this figure, narsoplimab dosing is indicated by vertical arrows and the horizontal line represents normality levels. [Figure 52C] FIGURE 2 illustrates serum levels of aspartate aminotransferase (AST) (units per liter, U / L) in COVID-19-infected patient #7 at baseline (day 0) before treatment and at different time points after narsoplimab treatment, as described in Example 22. In this figure, narsoplimab dosing is indicated by vertical arrows and the horizontal line represents normality levels. [Fig. 52D] FIGURE 2 illustrates serum levels of alanine transaminase (ALT) (units per liter, U / L) in COVID-19-infected patient #7 at baseline (day 0) before treatment and at different time points after narsoplimab treatment, as described in Example 22. In this figure, dosing with narsoplimab is indicated by vertical arrows, and the horizontal line represents normality levels. [Figure 52E] FIGURE 2 illustrates serum levels of lactate dehydrogenase (LDH) in COVID-19 patient #7 at baseline (day 0) before treatment and at different time points after narsoplimab treatment, as described in Example 22. In this figure, narsoplimab dosing is indicated by vertical arrows and horizontal lines represent normality levels. [Figure 53] Illustrates anti-SARS-CoV-2 antibody titers over time in patient #7 as described in Example 22. This figure shows that narsoplimab treatment does not interfere with effector functions of the adaptive immune response. [Figure 54] Illustrates concentration-dependent binding of recombinant MASP-2 to SARS-Cov-2 nucleocapsid protein (NP2) compared to BSA control, as described in Example 23. [Figure 55]As described in Example 23, an SDS-PAGE Western blot gel is shown which shows that MASP-2 directly binds to NP and cleaves C4, and that addition of the MASP-2 inhibitory antibody HG4 inhibits NP / MASP-2-mediated C4 cleavage. [Figure 56] FIG. 1 illustrates CH50 values ​​for various subject populations in a longitudinal study as described in Example 24. Each "x" symbol on the graph represents an individual subject. [Figure 57] FIG. 1 illustrates C5a levels (ng / ml) in plasma samples obtained from various subject populations in a longitudinal study as described in Example 24. Each "x" symbol on the graph represents an individual subject. [Figure 58] FIG. 1 illustrates plasma Bb levels (μg / mL) from various subject populations in a longitudinal study as described in Example 24. Each "x" symbol on the graph represents an individual subject. [Figure 59] The figure shows the amount of MASP-2 / C1-INH complex detected based on OD450 values ​​using each of four candidate anti-MASP-2 mAbs (clones C1, C7, D8, and H1) at various activation serum concentrations as described in Example 25. [Figure 60] Illustrated are the results of an ELISA assay measuring MASP-2 / C1-INH complexes in 5% serum from acute COVID patients (16 samples from 3 patients <14 days after hospitalization), convalescent patients (n=15), seropositive staff (n=15), and seronegative staff (n=34), as described in Example 25. [Figure 61] Illustrates the amount of MASP-2 / C1-INH complexes present in three acute COVID-19 patients (#2, #3, and #4) upon admission to the hospital and over time up to 14 days after admission, as described in Example 25. The line below the graph shows the amount of MASP-2 / C1-INH detected in pooled normal seronegative healthcare workers. [Figure 62]Schematic diagram illustrating the steps involved in a bead-based immunofluorescence assay, as described in Example 26, using anti-C1s or anti-MASP-2 antibodies immobilized on polystyrene microspheres or on magnetic polystyrene microspheres (i.e., beads) to capture serine protease / C1-INH complexes (i.e., analytes) from human serum or plasma, and using anti-C1INH antibodies as detection antibodies to detect the captured complexes. [Figure 63] Illustrates detection of MASP-2 / C1-INH complexes in pooled human serum from acute COVID-19 patients in a bead-based assay using anti-MASP-2 mAb #C8 ​​as the capture antibody compared to BSA-coated control beads, as described in Example 26. [Figure 64] This is a photograph of a non-reducing gel loaded with a 6 μg sample obtained during SEC purification of recombinant MASP-2 / C1-INH complex as described in Example 27. [Figure 65] FIG. 1 illustrates MASP-2 / C1-INH complex levels in acute COVID-19 patients as determined in a duplexed bead-based assay as described in Example 28. [Figure 66] FIG. 1 illustrates C1s / C1-INH complex levels in acute COVID-19 patients as determined in a dual bead-based assay as described in Example 28. [Figure 67] FIG. 1 illustrates CH50 values ​​for acute COVID-19 patients, convalescent patients, seropositive staff, and seronegative staff in a longitudinal study as described in Example 28. [Figure 68] FIG. 1 illustrates C5a levels in acute COVID-19 patients, convalescent patients, seropositive staff, and seronegative staff in a longitudinal study as described in Example 28. [Figure 69]Illustrates levels of MASP-2 / C1-INH complexes in samples from eight acute COVID-19 patients at the time of admission (before narsoplimab treatment) and after narsoplimab treatment (days 3-4 after treatment initiation; days 7-8, day 9 - discharge) compared to 16 healthy controls as described in Example 29. [Figure 70] FIG. 70A illustrates CH50 values ​​in samples from eight acute COVID-19 patients at admission (before narsoplimab treatment) and after narsoplimab treatment (days 3-4 after treatment initiation; days 7-8, day 9 - discharge) compared to 16 healthy controls, as described in Example 29. FIG. 70B illustrates C5a values ​​in samples from eight acute COVID-19 patients at admission (before narsoplimab treatment) and after narsoplimab treatment (days 3-4 after treatment initiation; days 7-8, day 9 - discharge) compared to 16 healthy controls, as described in Example 29. [Figure 71] Illustrates MASP-2 / C1-INH complex levels in samples from seven COVID-19 patients at admission (day 0, prior to narsoplimab treatment) and after narsoplimab treatment (days 2-4 after treatment initiation; days 6-8, and day 9 - discharge) compared to samples obtained from nine COVID-19 patients not treated with narsoplimab during the same time period (untreated controls) and a pool of healthy control subjects (healthy controls) as described in Example 30. [Figure 72]FIG. 72A illustrates CH50 values ​​in samples from seven COVID-19 patients at admission (day 0, before narsoplimab treatment) and after narsoplimab treatment (days 2-4 after treatment initiation; days 6-8, and days 9-discharge) compared to samples from nine COVID-19 patients not treated with narsoplimab during the same time period (naïve control) and a pool of healthy control subjects (healthy control), as described in Example 30. FIG. 72B illustrates C5a values ​​in samples from seven COVID-19 patients at admission (day 0, before narsoplimab treatment) and after narsoplimab treatment (days 2-4 after treatment initiation; days 6-8, and days 9-discharge) compared to samples from nine COVID-19 patients not treated with narsoplimab during the same time period (naïve control) and a pool of healthy control subjects (healthy control), as described in Example 30. [Figure 73] 1 illustrates the viable bacterial counts of K. pneumoniae following incubation of serum from COVID-19 patients before treatment with narsoplimab (pre-treatment) and after treatment with narsoplimab, compared to normal healthy serum (NHS) and heat-inactivated normal healthy serum (HI-NHS), compared to serum from COVID-19 patients not treated with narsoplimab, as described in Example 30. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Description of sequence listing SEQ ID NO:1 Human MAp19 cDNA SEQ ID NO:2 Human MAp19 protein (with leader) SEQ ID NO:3 Human MAp19 protein (mature) SEQ ID NO:4 Human MASP-2 cDNA SEQ ID NO:5 Human MASP-2 protein (with leader) SEQ ID NO:6 Human MASP-2 protein (mature) SEQ ID NO:7 Human MASP-2 gDNA (exons 1-6) Antigen: (based on MASP-2 mature protein) SEQ ID NO:8 CUBI sequence (aa1-121) SEQ ID NO:9 CUBEGF sequence (aa1-166) SEQ ID NO:10 CUBEGFCUBII(aa1~293) SEQ ID NO:11 EGF region (aa122-166) SEQ ID NO:12 Serine protease domain (aa429-671) SEQ ID NO:13 Serine protease domain inactive (aa610-625 with Ser618→Ala mutation) TIFF2024509702000002.tif63128 Detailed Description Peptide inhibitors: SEQ ID NO:20 MBL full length cDNA SEQ ID NO:21 MBL full-length protein SEQ ID NO:22 OGK-X-GP (consensus combination) TIFF2024509702000003.tif70128 Expression inhibitor: SEQ ID NO:30 CUBI-EGF domain cDNA (nucleotides 22 to 680 of SEQ ID NO:4) TIFF2024509702000004.tif11128 Nucleotides 12 to 45 (sense) of SEQ ID NO:4, including the MASP-2 translation start site TIFF2024509702000005.tif11128 Nucleotides 361 to 396 (sense) of SEQ ID NO:4, which encode the region containing the MASP-2 MBL binding site TIFF2024509702000006.tif11128 Nucleotides 610 to 642 of SEQ ID NO:4 encoding the region containing the CUBII domain Cloning primers: TIFF2024509702000007.tif41136SEQ ID NO:38-47 are cloning primers for humanized antibodies. SEQ ID NO:48 is a 9 aa peptide bond. Expression Vector: SEQ ID NO:49 is the MASP-2 minigene insert. SEQ ID NO:50 is mouse MASP-2 cDNA. SEQ ID NO:51 is mouse MASP-2 protein (with leader). SEQ ID NO:52 is the mature mouse MASP-2 protein. SEQ ID NO:53 is rat MASP-2 cDNA. SEQ ID NO:54 is rat MASP-2 protein (with leader). SEQ ID NO:55 is the mature rat MASP-2 protein. SEQ ID NOs:56-59 are oligonucleotides for site-directed mutagenesis of human MASP-2 used to generate human MASP-2A. SEQ ID NO:60-63 are oligonucleotides for site-directed mutagenesis of mouse MASP-2 used to generate mouse MASP-2A. SEQ ID NO:64-65 are oligonucleotides for site-directed mutagenesis of rat MASP-2 used to generate rat MASP-2A. SEQ ID NO:66 17D20_dc35VH21N11VL (OMS646) DNA encoding the heavy chain variable region (VH) (without signal peptide) SEQ ID NO:67 17D20_dc35VH21N11VL (OMS646) heavy chain variable region (VH) polypeptide SEQ ID NO:68 17N16mc heavy chain variable region (VH) polypeptide SEQ ID NO:69 17D20_dc35VH21N11VL (OMS646) light chain variable region (VL) polypeptide SEQ ID NO:70 17D20_dc35VH21N11VL (OMS646) DNA encoding the light chain variable region (VL) SEQ ID NO:71 17N16_dc17N9 light chain variable region (VL) polypeptide SEQ ID NO:72: SGMI-2L (full length) SEQ ID NO:73: SGMI-2M (medium truncated version) SEQ ID NO:74: SGMI-2S (short truncated version) SEQ ID NO:75: Mature polypeptide comprising VH-M2ab6-SGMI-2-N and a human IgG4 constant region with a hinge mutation SEQ ID NO:76: Mature polypeptide comprising VH-M2ab6-SGMI-2-C and a human IgG4 constant region with a hinge mutation SEQ ID NO:77: Mature polypeptide comprising VL-M2ab6-SGMI-2-N and a human Ig λ constant region SEQ ID NO:78: Mature polypeptide comprising VL-M2ab6-SGMI-2-C and a human Ig λ constant region SEQ ID NO:79: Peptide linker (10aa) SEQ ID NO:80: Peptide linker (6aa) SEQ ID NO:81: Peptide linker (4aa) SEQ ID NO:82: A polynucleotide encoding a polypeptide comprising VH-M2ab6-SGMI-2-N and a human IgG4 constant region with a hinge mutation SEQ ID NO:83: A polynucleotide encoding a polypeptide comprising VH-M2ab 6-SGMI-2-C and a human IgG4 constant region with a hinge mutation SEQ ID NO:84: A polynucleotide encoding a polypeptide comprising VL-M2ab6-SGMI-2-N and a human Ig λ constant region SEQ ID NO:85: A polynucleotide encoding a polypeptide comprising VL-M2ab6-SGMI-2-C and a human Ig λ constant region SEQ ID NO:86: C1 inhibitory factor (C1-INH) human (homo sapiens) SEQ ID NO:87: MASP-2 mAb C7 heavy chain variable region SEQ ID NO:88: MASP-2 mAb C7 light chain variable region SEQ ID NO:89: MASP-2 mAb C7 HC-CDR1 SEQ ID NO:90: ​​MASP-2 mAb C7 HC-CDR2 SEQ ID NO:91: MASP-2 mAb C7 HC-CDR3 SEQ ID NO:92: MASP-2 mAb C7 LC-CDR1 SEQ ID NO:93: MASP-2 mAb C7 LC-CDR2 SEQ ID NO:94: MASP-2 mAb C7 LC-CDR3 SEQ ID NO:95: MASP-2 mAb C7 cDNA encoding the heavy chain variable region SEQ ID NO:96: MASP-2 mAb C7 cDNA encoding the light chain variable region SEQ ID NO:97: MASP-2 mAb C8 heavy chain variable region SEQ ID NO:98: MASP-2 mAb C8 light chain variable region

[0027] Detailed Description As described herein, the inventors have observed that the concentration of MASP-2 / C1-INH in the blood (e.g., serum and / or plasma) is abnormally high in severe COVID-19 patients, and also in subjects previously infected with COVID-19 and suffering from long-term sequelae. The inventors have also observed that after recovery, the concentration of MASP-2 / C1-INH complexes decreases to normal levels in most cases. The inventors believe that monitoring patients infected with SARS-CoV-2 for increased concentrations of MASP-2 / C1-INH complexes is useful for diagnosing patients as having or at risk of developing acute COVID-19, and also for diagnosing patients as having or at risk of developing post-acute COVID-19 (also called long COVID-19), and optionally treating subjects identified as being at risk with a complement inhibitor, e.g., a MASP-2 inhibitor. As further described herein, the use of MASP-2 inhibitors is also useful for treating, inhibiting, alleviating, or preventing acute respiratory distress syndrome in subjects infected with a coronavirus, e.g., COVID-19, and for treating, inhibiting, alleviating, or preventing acute respiratory distress in subjects infected with influenza virus. Thus, monitoring the status of the MASP-2 / C1-INH complex may also be useful for determining whether a COVID-19 patient is responding to therapy with a complement inhibitor, e.g., a MASP-2 inhibitor, and, optionally, for adjusting the dosage of the MASP-2 inhibitor as needed to bring the level of MASP-2 / C1-INH into the normal range.

[0028] The present disclosure also provides assay methods for measuring fluid-phase MASP-2 / C1-INH complexes in biological samples. Compositions, kits, and methods are also provided for determining the concentration of fluid-phase MASP-2 / C1-INH complexes in biological samples, such as biological fluids obtained from subjects infected with SARS-CoV-2.

[0029] I. Definition Unless otherwise defined herein, all terms used herein have the same meaning as would be understood by a person skilled in the art of the present invention. The following definitions are provided to facilitate understanding of the terms used in the specification and claims to describe the present invention.

[0030] As used herein, the term "MASP-2-dependent complement activation" refers to the activation of complement by the lectin pathway under physiological conditions (i.e., Ca+, Ca-dependent complement activation) that leads to the formation of the lectin pathway C3 convertase C4b2a and, subsequently, the formation of the C5 convertase C4b2a (C3b)n upon accumulation of the C3 cleavage product C3b. ++ It has been shown that this involves MASP-2-dependent activation of the lectin pathway, which occurs in the presence of IgG4 (in the presence of IgG4), primarily resulting in opsonization.

[0031] As used herein, the term "alternative pathway" refers to complement activation induced by, for example, zymosan derived from fungal and yeast cell walls, lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria, and rabbit erythrocytes, as well as many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites, and damaged cells, and which has traditionally been thought to result from the spontaneous proteolytic generation of complement factors C3 to C3b.

[0032] As used herein, the term "lectin pathway" refers to complement activation that occurs via the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, M-ficolin, or L-ficolin).

[0033] As used herein, the term "classical pathway" refers to complement activation that is triggered by antibodies bound to foreign particles and requires the binding of the recognition molecule C1q.

[0034] The term "MASP-2 inhibitor" as used herein refers to any agent that binds to or directly interacts with MASP-2 to effectively inhibit MASP-2-dependent complement activation, including anti-MASP-2 antibodies and their MASP-2-binding fragments, natural and synthetic peptides, small molecules, soluble MASP-2 receptors, expression inhibitors, and isolated natural inhibitors, and also includes peptides that compete with MASP-2 in binding to another recognition molecule (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, but does not include antibodies that bind to such other recognition molecules. MASP-2 inhibitors useful in the methods of the present invention can reduce MASP-2-dependent complement activation by more than 20%, for example, more than 50%, for example, more than 90%. In one embodiment, the MASP-2 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., results in only 10% or less MASP-2 complement activation).

[0035] The term "fibrosis" as used herein refers to the formation or presence of excess connective tissue in an organ or tissue. Fibrosis may occur as a repair or replacement response to stimuli such as tissue injury or inflammation. A hallmark of fibrosis is the production of excess extracellular matrix. The normal physiological response to injury results in the deposition of connective tissue as part of the healing process, but this connective tissue deposition may persist and cause disease, thereby altering tissue structure and function. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased stiffness, microvascular compression, and hypoxia.

[0036] As used herein, the term "treating fibrosis in a mammalian subject suffering from or at risk of developing a disease or disorder caused or exacerbated by fibrosis and / or inflammation" refers to reversing, reducing, ameliorating, or inhibiting fibrosis in said mammalian subject.

[0037] As used herein, the term "proteinuria" refers to the presence of an abnormal amount of protein in the urine, e.g., greater than 0.3 g protein in a 24-hour urine collection from a human subject, or greater than 1 g per liter in a human subject.

[0038] As used herein, the term "improving proteinuria" or "reducing proteinuria" refers to reducing 24-hour urinary protein excretion in a subject suffering from proteinuria by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, or more, compared to the baseline 24-hour urinary protein excretion in the subject prior to treatment with a MASP-2 inhibitor. In one embodiment, treatment with a MASP-2 inhibitor according to the method of the present invention is effective to reduce proteinuria in a human subject, for example, to achieve a reduction in 24-hour urinary protein excretion of more than 20 percent, or, for example, a reduction in 24-hour urinary protein excretion of more than 30 percent, or, for example, a reduction in 24-hour urinary protein excretion of more than 40 percent, or, for example, a reduction in 24-hour urinary protein excretion of more than 50 percent.

[0039] As used herein, the terms "small molecule," "small organic molecule," and "small inorganic molecule" refer to molecules (either organic, organometallic, or inorganic), organic molecules, and inorganic molecules, respectively, that are natural or synthetic and have a molecular weight greater than about 50 Da and less than about 2500 Da. Organic (for example) small molecules can be less than about 2000 Da, between about 100 Da and about 1000 Da, or between about 100 and about 600 Da, or between about 200 and 500 Da.

[0040] The term "antibody" as used herein includes antibodies and antibody fragments thereof derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate, including human) or derived from hybridoma, phage selection, recombinant expression, or transgenic animals (or other methods of producing antibodies or antibody fragments) that specifically bind to a target polypeptide, e.g., a MASP-2 polypeptide or a portion thereof. The term "antibody" is not intended to be limited in terms of the source of the antibody or the way in which the antibody is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal, and recombinant antibodies; pan-specific, multispecific antibodies (e.g., bispecific, trispecific); humanized antibodies: mouse antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotypic antibodies, and may be any intact antibody or fragment thereof. As used herein, the term "antibody" refers to an intact polyclonal or monoclonal antibody as well as a fragment thereof (e.g., dAb, Fab, Fab', F(ab')). 2 , Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins containing antibody portions and antigen-binding fragments of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule which contains an antigen-binding site or fragment (epitope recognition site) of the required specificity.

[0041] "Monoclonal antibody" refers to a homogenous antibody population, which consists of amino acids (natural and non-natural) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific for a target antigen. The term "monoclonal antibody" includes intact and full-length monoclonal antibodies as well as fragments thereof (e.g., Fab, Fab', F(ab')). 2The term also encompasses monoclonal antibodies, including whole chain (Fv), single chain (ScFv), variants thereof, fusion proteins containing an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) of the required specificity and ability to bind to an epitope. The term is not intended to be limited in terms of the source of the antibody or the manner in which the antibody is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins and fragments, etc., as set forth above in the definition of "antibody."

[0042] As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length antibody, such as an anti-MASP-2 antibody, generally including the antigen-binding or variable region thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab) and / or Fab' fragments. 2 , F(ab') 2 , and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0043] As used herein, a "single-chain Fv" or "scFv" antibody fragment is a fragment of an antibody H Domain or V L Fv polypeptides generally comprise a V domain, and these domains are present in a single polypeptide chain. H Domain and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding.

[0044] As used herein, a "chimeric antibody" is a recombinant protein that contains the variable domains and complementarity determining regions derived from a non-human species (e.g., rodent) antibody, while the remainder of the antibody molecule is derived from a human antibody.

[0045] As used herein, a "humanized antibody" is a chimeric antibody that contains minimal sequences matching specific complementarity determining regions derived from a non-human immunoglobulin grafted onto a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity determining regions are of non-human origin.

[0046] As used herein, the term "mannan-binding lectin" ("MBL") is synonymous with mannan-binding protein ("MBP").

[0047] As used herein, "membrane attack complex" ("MAC") refers to a complex of five terminal complement components (C5b in combination with C6, C7, C8, and C9) (also called C5b-9) that inserts into and destroys membranes.

[0048] As used herein, a "subject" includes all mammals, including, but not limited to, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents.

[0049] The abbreviations for amino acid residues used herein are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0050] In the broadest sense, the natural amino acids can be divided into groups based on the chemical properties of the side chain of each amino acid. By "hydrophobic" amino acid we mean Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By "hydrophilic" amino acid we mean Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This group of amino acids can be further divided into subgroups as follows: By "uncharged hydrophilic" amino acid we mean Ser, Thr, Asn or Gln. By "acidic" amino acid we mean Glu or Asp. By "basic" amino acid we mean Lys, Arg or His.

[0051] As used herein, the term "conservative amino acid substitutions" is exemplified by substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartic acid and glutamic acid, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

[0052] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term also covers oligonucleobases composed of naturally occurring nucleotides, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-natural modifications.

[0053] As used herein, "epitope" refers to a site on a protein (e.g., human MASP-2 protein) to which an antibody binds. "Overlapping epitopes" contain at least one (e.g., two, three, four, five, or six) common amino acid residues, including linear and non-linear epitopes.

[0054] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to any peptide-linked amino acid chain, regardless of length or post-translational modification. The MASP-2 protein described herein may contain a wild-type protein, or may be a wild-type protein or a variant having 50 or less conservative amino acid substitutions (e.g., 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 12 or less, 15 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, or 50 or less). Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.

[0055] In some embodiments, the human MASP-2 protein may have an amino acid sequence that is 70 (e.g., 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, or 100)% identical, or more than 70 (e.g., 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, or 100)% identical to the human MASP-2 protein having the amino acid sequence shown in SEQ ID NO:5.

[0056] In some embodiments, the peptide fragments are at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more amino acid residues (e.g., at least 6 consecutive amino acid residues of SEQ ID NO:5). In some embodiments, the antigenic peptide fragment of the human MASP-2 protein is less than 500 amino acids in length (e.g., less than 450 amino acids, less than 400 amino acids, less than 350 amino acids, less than 325 amino acids, less than 300 amino acids, less than 275 amino acids, less than 250 amino acids, less than 225 amino acids, less than 200 amino acids, less than 190 amino acids, less than 180 amino acids, less than 170 amino acids, less than 160 amino acids, less than 150 amino acids, less than 140 amino acids, less than 130 amino acids, less than 120 amino acids, less than 110 amino acids, less than 100 amino acids, less than 95 amino acids, less than 90 amino acids, less than 85 amino acids, less than 80 amino acids, less than 75 amino acids, less than 70 amino acids, less than 65 amino acids, less than 60 amino acids, less than 55 amino acids, less than 50 amino acids, less than 49 amino acids, less than 48 amino acids, less than 47 amino acids, less than 46, less than 45, less than 44, less than 43, less than 42, less than 41, less than 40, less than 39, less than 38, less than 37, less than 36, less than 35, less than 34, less than 33, less than 32, less than 31, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, or less than 6 amino acid residues (e.g., less than 500 contiguous amino acid residues in any one of SEQ ID NOs:5).

[0057] Amino acid sequence identity percentage (%) is defined as the percentage of amino acids in the candidate sequence that are identical to the amino acids in the reference sequence, after aligning the sequences to achieve maximum sequence identity percentage and introducing gaps if necessary.For the purpose of determining sequence identity percentage, alignment can be achieved in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment can be determined by known methods, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared.

[0058] II. Overview of the Invention As described herein, the inventors have identified the central role of lectin pathway in the initiation and disease progression of tubular kidney pathology. This implies the important role of lectin pathway activation in the pathophysiology of various kidney diseases, including IgA nephropathy, C3 glomerulopathy, and other glomerulonephritis. As further described herein, the inventors have discovered that by inhibiting mannan-binding lectin-associated serine protease-2 (MASP-2), a key regulator of the lectin pathway of the complement system, inflammation and fibrosis are significantly reduced in various animal models of fibrotic disease, including the unilateral ureteral ligation (UUO) model of renal fibrosis, the protein overload model, and the adriamycin-induced kidney disease model. Thus, the inventors have demonstrated that inhibition of MASP-2-mediated lectin pathway activation is an effective therapeutic approach for ameliorating, treating, or preventing kidney fibrosis, such as tubulointerstitial fibrosis, regardless of the underlying cause. As further described herein, the use of MASP-2 inhibitors is also useful for treating, inhibiting, alleviating or preventing acute respiratory distress syndrome in subjects infected with a coronavirus, such as COVID-19.

[0059] Lectins (MBL, M-ficolin, H-ficolin, L-ficolin, and CL-11) are specific recognition molecules that trigger the innate complement system, which includes the lectin initiation pathway and an associated terminal pathway amplification loop that amplifies terminal complement effector molecule activation initiated by lectins. C1q is a specific recognition molecule that triggers the acquired complement system, which includes the classical initiation pathway and an associated terminal pathway amplification loop that amplifies terminal complement effector molecule activation initiated by C1q. We refer to these two major complement activation systems as the lectin-dependent and C1q-dependent complement systems, respectively.

[0060] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these side effects. With the realization that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact, it was realized that it would be highly desirable to specifically inhibit only the complement activation system responsible for a particular pathology, without completely blocking the immune defense capabilities of complement. For example, in disease states where complement activation is primarily mediated by the lectin-dependent complement system, it would be advantageous to specifically inhibit only this system. To this end, the C1q-dependent complement activation system would remain intact to handle immune complex processing and aid in host defense against infection.

[0061] A preferred protein component of interest in the development of therapeutic agents that specifically inhibit the lectin-dependent complement system is MASP-2. Of all the known protein components of the lectin-dependent complement system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, factor B, factor D, and properdin), only MASP-2 is unique to the lectin-dependent complement system and is required for the system to function. The lectins (MBL, H-ficolin, M-ficolin, L-ficolin, and CL-11) are also unique components in the lectin-dependent complement system. However, loss of any one of these lectin components would not necessarily inhibit activation of the system due to lectin redundancy. Inhibition of all five lectins would be necessary to ensure inhibition of the lectin-dependent complement activation system. Furthermore, since MBL and ficolin are known to also have opsonic activity independent of complement, inhibition of lectin function would eliminate this beneficial host defense mechanism against infection. In contrast, this complement-independent lectin opsonic activity appears to remain intact when MASP-2 is the inhibitory target. An added benefit of MASP-2 as a therapeutic target for inhibiting the lectin-dependent complement activation system is that the plasma concentration of MASP-2 (approximately 500 ng / ml) is among the lowest plasma concentrations of complement proteins. Therefore, a correspondingly low concentration of a high-affinity MASP-2 inhibitor may be sufficient to obtain complete inhibition (Moller-Kristensen, M., et al., J. Immunol Methods 282:159-167, 2003).

[0062] As described in Example 14 herein, in an animal model of fibrotic kidney disease (unilateral ureteral ligation UUO), it was confirmed that mice lacking the MASP-2 gene (MASP-2- / -) exhibited significantly less kidney disease compared to wild-type control animals, as indicated by inflammatory cell infiltrates (75% reduction) and histological markers of fibrosis, such as collagen deposition (1 / 3 reduction). Furthermore, as shown in Example 15, wild-type mice systemically treated with anti-MASP-2 monoclonal antibodies that selectively block the lectin pathway while leaving the classical pathway intact were protected from kidney fibrosis compared to wild-type mice treated with an isotype control antibody. These results demonstrate that the lectin pathway is an important contributor to kidney disease, and further demonstrate that MASP-2 inhibitors that block the lectin pathway, such as MASP-2 antibodies, are effective as anti-fibrotic agents. Furthermore, as shown in Example 16, in a protein overload model, wild-type mice treated with bovine serum albumin (BSA) developed proteinuric nephropathy, whereas MASP-2- / - mice treated with the same levels of BSA had less renal damage. As shown in Example 17, in a protein overload model, wild-type mice systemically treated with an anti-MASP-2 monoclonal antibody that selectively blocks the lectin pathway while leaving the classical pathway intact were protected from renal damage. As described in Example 18, in an adriamycin-induced nephropathy model of renal fibrosis, MASP-2- / - mice had less renal inflammation and tubulointerstitial damage compared to wild-type mice. As described in Example 19, in an ongoing Phase 2 open-label renal study, IgA nephropathy patients treated with anti-MASP-2 antibodies demonstrated clinically meaningful and statistically significant reductions in urinary albumin / creatinine ratio (uACR) throughout the study and reduced 24-hour urinary protein levels from baseline to the end of treatment. Additionally, as described in Example 19, in the same Phase 2 renal study, membranous nephropathy patients treated with anti-MASP-2 antibodies also demonstrated reduced uACR during treatment.

[0063] Based on the above, the present invention relates to the use of MASP-2 inhibitors, e.g., MASP-2 inhibitory antibodies, as anti-fibrotic agents, the use of MASP-2 inhibitors for the manufacture of pharmaceutical agents for treating fibrotic conditions, and a method for preventing, treating, alleviating or reversing a fibrotic condition in a human subject in need thereof, comprising administering to the patient an effective amount of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody).

[0064] As described in Examples 20, 21, and 22, clinical improvement was observed in patients suffering from COVID-19-associated respiratory failure after treatment with narsoplimab, which inhibits MASP-2 and lectin pathway activation. As described in Example 21, all six COVID-19 patients treated with narsoplimab showed clinical improvement. In all cases, COVID-19 lung injury had progressed to ARDS prior to narsoplimab treatment, and all patients were on noninvasive mechanical ventilation at the start of treatment. No clinical or laboratory evidence of long-term sequelae was observed in narsoplimab-treated COVID-19 patients for whom follow-up (5-6 months) data were available. As further described in Example 22, additional COVID-19 patients treated with narsoplimab also showed clinical improvement. As further demonstrated in Example 22, narsoplimab-treated patients developed appropriately high titers of anti-SARS-Cov-2 antibodies. This indicates that narsoplimab treatment does not interfere with the effector functions of the adaptive immune response.

[0065] As further described in Examples 24-30 herein, the inventors observed that the concentration of MASP-2 / C1-INH in the blood (e.g., serum and / or plasma) is abnormally high in severe COVID-19 patients, and also in subjects previously infected with COVID-19 and suffering from long-term sequelae. The inventors also observed that after recovery, the concentration of MASP-2 / C1-INH complexes decreases to normal levels in most cases. The inventors believe that monitoring patients infected with SARS-CoV-2 for increased concentrations of MASP-2 / C1-INH complexes is useful for diagnosing patients as having or at risk of developing acute COVID-19, and also as having or at risk of developing post-acute COVID-19 (also called long COVID-19), and optionally treating subjects identified as at risk with a complement inhibitor, e.g., a MASP-2 inhibitor. As further described herein, the use of MASP-2 inhibitors is also useful for treating, inhibiting, alleviating, or preventing acute respiratory distress syndrome in subjects infected with a coronavirus, e.g., COVID-19, and for treating, inhibiting, alleviating, or preventing acute respiratory distress in subjects infected with influenza virus. Thus, monitoring the status of the MASP-2 / C1-INH complex may also be useful for determining whether a COVID-19 patient is responding to therapy with a complement inhibitor, e.g., a MASP-2 inhibitor, and optionally, for adjusting the dosage of the MASP-2 inhibitor as needed to bring the level of MASP-2 / C1-INH into the normal range.

[0066] The present disclosure also provides assay methods for measuring fluid-phase MASP-2 / C1-INH complexes in biological samples. Compositions, kits, and methods are also provided for determining the concentration of fluid-phase MASP-2 / C1-INH complexes in biological samples, such as biological fluids obtained from subjects infected with SARS-CoV-2.

[0067] Thus, the methods of the invention can be used to treat, inhibit, reduce, prevent, or reverse coronavirus-induced pneumonia or acute respiratory distress syndrome in a human subject suffering from a coronavirus, such as COVID-19, SARS, or MERS, as further described herein. The methods of the invention can also be used to treat, inhibit, reduce, prevent, or reverse influenza virus-induced pneumonia or acute respiratory distress syndrome in a human subject suffering from an influenza virus, e.g., influenza A virus serotypes (H1N1 (caused the "Spanish Flu" in 1918 and "Swine Flu" in 2009); H2N2 (caused the "Asian Flu" in 1957), H3N2 (caused the Hong Kong Flu in 1968), H5N1 (caused the "Bird Flu" in 2004), H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and H6N1); or an influenza B virus, or an influenza C virus.

[0068] III. Role of MASP-2 in diseases and conditions caused or exacerbated by fibrosis Fibrosis is the formation or presence of excess connective tissue in an organ or tissue, generally in response to injury or insult. The hallmark of fibrosis is the production of excess extracellular matrix after injury. In the kidney, fibrosis is characterized as a progressive, deleterious deposition of connective tissue on the renal parenchyma, which inevitably leads to reduced renal function, regardless of the primary renal disease that caused the original kidney injury. The so-called epithelial-mesenchymal transition (EMT), a cellular characteristic change in which tubular epithelial cells turn into mesenchymal fibroblasts, constitutes the main mechanism of renal fibrosis. Fibrosis can affect almost all tissues and organ systems and can occur as a repair or replacement response to stimuli such as tissue injury or inflammation. The normal physiological response to injury results in the deposition of connective tissue, but if this process is pathological, tissue structure and function are altered as highly differentiated cells are replaced by scarred connective tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased stiffness, microvascular compression, and hypoxia.Currently, there are no effective treatments or cures for fibrosis, but both animal studies and anecdotal reports in humans suggest that fibrotic tissue damage may be reversed (Tampe and Zeisberg, Nat Rev Nephrol, Vol 10:226-237, 2014).

[0069] Diseases of the kidney (e.g., chronic kidney disease, IgA nephropathy, C3 glomerulopathy, and other glomerulonephritides), lung (e.g., idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis), liver (e.g., cirrhosis, nonalcoholic fatty liver disease), heart (e.g., myocardial infarction, atrial fibrosis, valvular fibrosis, endomyocardial fibrosis), brain (e.g., stroke), skin (e.g., excessive wound healing, scleroderma, systemic sclerosis, keloids), vasculature (e.g., atrial fibrosis, pulmonary ... Many diseases cause fibrosis that leads to progressive organ failure, including atherosclerotic vascular disease), intestine (e.g., Crohn's disease), eye (e.g., anterior subcapsular cataract, posterior capsular opacification), musculoskeletal soft tissue structures (e.g., adhesive capsulitis, Dupuytren's contracture, myelofibrosis), reproductive organs (e.g., endometriosis, Peyronie's disease), and some infections (e.g., coronaviruses, alphaviruses, hepatitis C, hepatitis B, etc.).

[0070] Fibrosis occurs in many tissues and diseases, but its pathology has common molecular and cellular mechanisms. The deposition of extracellular matrix by fibroblasts is accompanied by immune cell infiltrates, primarily mononuclear cells (see Wynn T., Nat Rev Immunol 4(8):583-594, 2004, incorporated herein by reference). A strong inflammatory response results in the expression of growth factors (TGF-β, VEGF, hepatocyte growth factor, connective tissue growth factor), cytokines and hormones (endothelin, IL-4, IL-6, IL-13, chemokines), digestive enzymes (elastase, matrix metalloproteinase, cathepsin), and extracellular matrix proteins (collagen, fibronectin, integrin).

[0071] Furthermore, the complement system is activated in numerous fibrotic diseases. Complement components, including the membrane attack complex, have been identified in numerous fibrotic tissue specimens. For example, components of the lectin pathway have been found in fibrotic lesions of kidney disease (Satomura et al., Nephron. 92(3):702-4 (2002); Sato et al., Lupus 20(13):1378-86 (2011); Liu et al., Clin Exp Immunol, 174(1):152-60 (2013)); liver disease (Rensen et al., Hepatology 50(6): 1809-17 (2009)); and lung disease (Olesen et al., Clin Immunol 121(3):324-31 (2006)).

[0072] Excessive complement activation has been identified as an important contributor to immune complex-mediated as well as antibody-independent glomerulonephritis. However, there is strong evidence that uncontrolled in situ complement activation is intrinsically involved in the pathophysiological progression of TI fibrosis in nonrenal glomerular diseases (Quigg RJ, J Immunol 171:3319-3324, 2003; Naik A. et al., Semin Nephrol 33:575-585, 2013; Mathern DR et al., Clin J Am Soc Nephrol 10:P1636-1650, 2015). The potent proinflammatory signals elicited by local complement activation may be caused by complement components that enter the proximal tubules by filtration and subsequently enter the interstitial space, or by aberrant synthesis of complement components by tubular cells or other resident and infiltrating cells, or by altered expression of complement regulatory proteins on renal cells, or by absence or loss or gain of function mutations of complement regulatory components (Mathern DR et al., Clin J Am Soc Nephrol 10:P1636-1650, 2015, Sheerin NS, et al., FASEB J 22: 1065-1072, 2008). For example, in mice, deficiency of the complement regulatory protein CR1-related gene / protein y (Crry) leads to tubulointerstitial (TI) complement activation and subsequent inflammation and fibrosis that are typical of the injury seen in human TI disease (Naik A. et al., Semin Nephrol 33:575-585, 2013, Bao L. et al., J Am Soc Nephrol 18:811-822, 2007). Exposure of renal tubular epithelial cells to the anaphylatoxin C3a leads to an epithelial-mesenchymal transition (Tsang Z. et al., J Am Soc Nephrol 20:593-603, 2009).Recently, blocking C3a signaling exclusively at the C3a receptor has been shown to attenuate renal TI fibrosis in proteinuric and non-proteinuric animals (Tsang Z. et al., J Am Soc Nephrol 20:593-603, 2009; Bao L. et al., Kidney Int. 80: 524-534, 2011).

[0073] As described herein, the inventors have identified a central role for the lectin pathway in the initiation and disease progression of tubular kidney pathologies. This has been shown to be a key factor in the development of various kidney diseases, including IgA nephropathy, C3 glomerulopathy, and other glomerulonephritides (Endo M. et al., Nephrol Dialysis Transplant 13: 1984-1990, 1998; Hisano S. et al., Am J Kidney Dis 45:295-302, 2005; Roos A. et al., J Am Soc Nephrol 17: 1724-1734, 2006; Liu LL et al., Clin Exp. Immunol 174:152-160, 2013; Lhotta K. et al., Nephrol Dialysis Transplant 14:881-886, 1999; Pickering et al., Kidney International 84:1079-1089, 2013), diabetic nephropathy (Hovind P. et al. This implies an important role for lectin pathway activation in the pathophysiology of inflammatory bowel disease (Asgari E. et al., Diabetes 54:1523-1527, 2005), ischemia-reperfusion injury (Asgari E. et al., FASEB J 28:3996-4003, 2014), and transplant rejection (Berger SP et al., Am J Transplant 5:1361-1366, 2005).

[0074] As further described herein, the inventors have demonstrated that MASP-2 inhibition reduces inflammation and fibrosis in mouse models of tubulointerstitial disease. Thus, MASP-2 inhibitors are expected to be useful in treating renal fibrosis, including tubulointerstitial inflammation and fibrosis, proteinuria, IgA nephropathy, C3 glomerulopathy, and other glomerulonephritides and renal ischemia-reperfusion injury.

[0075] Pulmonary disease Pulmonary fibrosis is the formation or development of excess fibrous connective tissue in the lungs, where normal lung tissue is replaced by fibrotic tissue. This scarring leads to stiffening of the lungs and impaired lung structure and function. In humans, pulmonary fibrosis is thought to result from repeated injury to the tissue within and between the tiny air sacs (alveoli) of the lungs. In the experimental setting, various animal models have reproduced aspects of the human disease. For example, foreign agents such as bleomycin, fluorescein isothiocyanate, silica, or asbestos may be instilled into the trachea of ​​animals (Gharaee-Kermani et al., Animal Models of Pulmonary Fibrosis. Methods Mol. Med., 2005, 117:251-259).

[0076] Thus, in certain embodiments, the present disclosure provides a method for inhibiting pulmonary fibrosis in a subject suffering from a pulmonary disease or disorder caused or exacerbated by fibrosis and / or inflammation, such as coronavirus-induced ARDS, comprising administering a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody, to a subject in need of inhibiting pulmonary fibrosis. The method comprises administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit, reduce, and / or improve pulmonary function. Improved symptoms of pulmonary function include improved pulmonary function and / or lung capacity, reduced fatigue, and improved oxygen saturation.

[0077] The MASP-2 inhibitory composition may be administered locally to the area of ​​fibrosis, for example by local application of the composition directly or remotely, for example by catheter, during surgery or local injection. Alternatively, the MASP-2 inhibitor may be administered systemically to the subject, for example by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, potentially by oral administration in the case of non-peptidic substances. Administration may be repeated as determined by the physician until the condition is resolved or controlled.

[0078] In certain embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered in combination with one or more drugs or treatment modalities appropriate to the underlying pulmonary disease or condition.

[0079] infectious disease Infections such as coronaviruses and chronic infections such as hepatitis C and B cause tissue inflammation and fibrosis, where high lectin pathway activity can be detrimental. In such diseases, MASP-2 inhibitors may be beneficial. For example, MBL and MASP-1 levels have been found to be important predictors of liver fibrosis severity in hepatitis C virus (HCV) infection (Brown et al., Clin Exp Immunol. 147(1):90-8, 2007; Saadanay et al., Arab J Gastroenterol. 12(2):68-73, 2011; Saeed et al., Clin Exp Immunol. 174(2):265-73, 2013). MASP-1 has previously been shown to be a potent activator of MASP-2 and the lectin pathway (Megyeri et al., J Biol Chem. 29: 288(13)8922-34, 2013). Alphaviruses such as Chikungunya and Ross River viruses induce a strong host inflammatory response that leads to arthritis and myositis, a pathology mediated by MBL and the lectin pathway (Gunn et al., PLoS Pathog. 8(3):e1002586, 2012).

[0080] Thus, in certain aspects, the present disclosure provides a method for preventing, treating, reversing, inhibiting, and / or reducing fibrosis and / or inflammation in a subject suffering from or who has previously suffered from an infectious disease, such as a coronavirus or influenza virus, that causes inflammation and / or fibrosis, comprising administering a MASP-2 inhibitor, e.g., a MASP-2 inhibitory antibody, to a subject in need of prevention, treatment, reversal, inhibition, and / or reduction of fibrosis and / or inflammation.

[0081] The MASP-2 inhibitory composition may be administered locally to the area of ​​fibrosis, for example by local application of the composition directly or remotely, for example by catheter, during surgery or local injection. Alternatively, the MASP-2 inhibitor may be administered systemically to the subject, for example by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, potentially by oral administration in the case of non-peptidic substances. Administration may be repeated as determined by the physician until the condition is resolved or controlled.

[0082] In certain embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered in combination with one or more drugs or treatment modalities appropriate to the underlying infection.

[0083] In some embodiments, the infectious disease that causes inflammation and / or fibrosis is selected from the group consisting of coronavirus, alphavirus, hepatitis A, hepatitis B, hepatitis C, tuberculosis, HIV, and influenza.

[0084] In certain embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody or a MASP-2 inhibitory small molecule compound) is administered in combination with one or more drugs or therapeutic modalities appropriate to the underlying disease or disorder.

[0085] In certain embodiments of any of the various methods and pharmaceutical compositions described herein, the MASP-2 inhibitory antibody or small molecule compound selectively blocks the lectin pathway while leaving the classical pathway intact.

[0086] IV. MASP-2 inhibitors In various aspects, the present invention provides a method for inhibiting the side effects of fibrosis and / or inflammation, comprising administering a MASP-2 inhibitor to a subject in need of inhibition of the side effects of fibrosis and / or inflammation. The MASP-2 inhibitor is administered in an amount effective to inhibit MASP-2-dependent complement activation in a living subject. In carrying out this aspect of the present invention, representative MASP-2 inhibitors include molecules that inhibit the biological activity of MASP-2 (e.g., small molecule inhibitors, anti-MASP-2 antibodies (e.g., MASP-2 inhibitory antibodies), or blocking peptides that interact with MASP-2 or interfere with protein-protein interactions), as well as molecules that reduce MASP-2 expression, thereby preventing MASP-2 from activating the lectin complement pathway (e.g., MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes). MASP-2 inhibitors may be used alone as a primary therapy or in combination with other therapeutic agents as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments.

[0087] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in complement system components that occur as a result of administration of a MASP-2 inhibitor according to the methods of the present invention: inhibition of generation or production of MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (measured, for example, as described in Example 2), reduced C4 cleavage and C4b deposition (measured, for example, as described in Example 2), or reduced C3 cleavage and C3b deposition (measured, for example, as described in Example 2).

[0088] In accordance with the present invention, MASP-2 inhibitors are utilized that are effective in inhibiting respiratory distress (or, in other words, improving respiratory function) in subjects infected with coronavirus.

[0089] Respiratory function assessment may be performed periodically, for example, hourly, daily, weekly, or monthly. This assessment is preferably performed at several time points for a particular subject, or at one or several time points for a particular subject and healthy control. Assessment may be performed at regular time intervals, for example, hourly, daily, weekly, or monthly. When an assessment leads to the discovery of a reduction in respiratory distress (i.e., an increase in respiratory function), the MASP-2 inhibitor, for example, the MASP-2 inhibitor antibody, is said to be effective in treating subjects suffering from coronavirus-induced acute respiratory distress syndrome.

[0090] MASP-2 inhibitors useful in the practice of this aspect of the invention include, for example, MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors, and expression inhibitors. MASP-2 inhibitors may inhibit the MASP-2-dependent complement activation system by blocking the biological function of MASP-2. For example, inhibitors may effectively block MASP-2 protein-protein interactions, may interfere with MASP-2 dimerization or assembly, and may inhibit Ca 2+ It may block binding, it may interfere with the MASP-2 serine protease active site, or it may reduce MASP-2 protein expression.

[0091] In some embodiments, the MASP-2 inhibitor selectively inhibits MASP-2 complement activation without impairing the function of the C1q-dependent complement activation system.

[0092] In one embodiment, the MASP-2 inhibitor useful in the method of the present invention is a specific MASP-2 inhibitor that specifically binds to a polypeptide comprising SEQ ID NO:6 with at least 10-fold higher affinity than other antigens of the complement system. In another embodiment, the MASP-2 inhibitor specifically binds to a polypeptide comprising SEQ ID NO:6 with at least 100-fold higher affinity than other antigens of the complement system. In one embodiment, the MASP-2 inhibitor specifically binds to at least one of (i) the CCP1-CCP2 domain (aa 300-431 of SEQ ID NO:6) or the serine protease domain (aa 445-682 of SEQ ID NO:6) of MASP-2 and inhibits MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof that specifically binds to MASP-2. The binding affinity of the MASP-2 inhibitor can be determined using a suitable binding assay.

[0093] MASP-2 polypeptides show a molecular structure similar to MASP-1, MASP-3, and C1r and C1s, which are proteases of the C1 complement system. The cDNA molecule shown in SEQ ID NO:4 encodes a representative example of MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:5), providing a human MASP-2 polypeptide with a leader sequence (aa1-15) that is cleaved after secretion to yield the mature human MASP-2 (SEQ ID NO:6). As shown in FIG. 2, the human MASP2 gene contains 12 exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. As shown in FIG. 2, an alternative splice results in a 20 kDa protein called MBL-associated protein 19 (also called "MAp19", "sMAP") (SEQ ID NO:2), which is encoded by exons B, C, D, and E (SEQ ID NO:1). The cDNA molecule shown in SEQ ID NO:50 encodes mouse MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:51) and provides a mouse MASP-2 polypeptide having a leader sequence that is cleaved after secretion to yield mature mouse MASP-2 (SEQ ID NO:52). The cDNA molecule shown in SEQ ID NO:53 encodes rat MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:54) and provides a rat MASP-2 polypeptide having a leader sequence that is cleaved after secretion to yield mature rat MASP-2 (SEQ ID NO:55).

[0094] Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO:4, SEQ ID NO:50, and SEQ ID NO:53 are single alleles of human MASP-2, mouse MASP-2, and rat MASP-2, respectively, and that allelic variations and alternative splicing are expected to occur. Allelic variants of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:50, and SEQ ID NO:53 are within the scope of the present invention, including allelic variants containing silent mutations and allelic variants in which the mutation changes the amino acid sequence. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals, following standard procedures.

[0095] The domains of the human MASP-2 protein (SEQ ID NO:6) are shown in Figure 1 and Figure 2A and include an N-terminal C1r / C1s / sea urchin Vegf / bone morphogenetic protein (CUBI) domain (aa1-121 of SEQ ID NO:6), an epidermal growth factor-like domain (aa122-166), another CUBI domain (aa167-293), as well as a tandem arrangement of complement control protein domains and a serine protease domain. Alternative splicing of the MASP2 gene results in MAp19, shown in Figure 1. MAp19 is a non-enzymatic protein that contains the N-terminal CUBI-EGF region of MASP-2 and four additional residues (EQSL) derived from exon E, as shown in Figure 1.

[0096] Several proteins have been shown to bind to or interact with MASP-2 through protein-protein interactions. For example, MASP-2 binds to the lectin proteins MBL, H-ficolin, and L-ficolin, and interacts with them in Ca2+ binding. 2+It is known that MASP-2 / lectin complexes form a complement-dependent complex. Each MASP-2 / lectin complex has been shown to activate complement through MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med 176:197-2284, 2000; Matsushita, M., et al., J. Immunol.168:3502-3506, 2002). Studies have shown that the CUBI-EGF domain of MASP-2 is essential for the binding of MASP-2 to MBL (Thielens, NM, et al., J. Immunol. 166:5068, 2001). The CUBIEGFCUBII domain has also been shown to mediate MASP-2 dimerization, which is necessary for the formation of an active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000). Thus, it is possible to identify MASP-2 inhibitors that bind to or interfere with MASP-2 target regions known to be important for MASP-2-dependent complement activation.

[0097] Anti-MASP-2 inhibitor antibody In some embodiments of this aspect of the invention, the MASP-2 inhibitor comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complement activation system. Anti-MASP-2 antibodies useful in this aspect of the invention include polyclonal, monoclonal, or recombinant antibodies derived from any antibody-producing mammal and may be multispecific, chimeric, humanized, anti-idiotypic, and antibody fragments. Antibody fragments include Fab, Fab', F(ab) and / or Fab' antibodies, as further described herein. 2 , F(ab') 2 , Fv fragments, scFv fragments, and single chain antibodies.

[0098] The ability of MASP-2 antibodies to inhibit the MASP-2-dependent complement activation system and anti-fibrogenic activity and / or the ability to inhibit kidney damage associated with proteinuria or adriamycin-induced nephropathy can be screened using the assays described herein. Some MASP-2 antibodies have been described in the literature, some newly created, some of which are listed in Table 1 below. For example, as described in Example 10 and Example 11 herein, anti-MASP-2 Fab2 antibodies that block MASP-2-dependent complement activation have been identified. As described in Example 12 and also in WO2012 / 151481, which is incorporated herein by reference, fully human MASP-2 scFv antibodies (e.g., OMS646) that block MASP-2-dependent complement activation have been identified. As described in Example 13, and also as described in WO2014 / 144542, which is incorporated by reference herein, SGMI-2 peptide amino acid sequences (SEQ ID NO:72, 73, or 74) were fused to the amino or carboxy terminus of the heavy and / or light chains of human MASP-2 antibodies to generate MASP-2 antibodies and fragments thereof containing the SGMI-2 peptide and having MASP-2 inhibitory activity (e.g., OMS646-SGMI-2).

[0099] Thus, in one aspect, a MASP-2 inhibitor for use in the methods of the present invention comprises a human antibody, such as, for example, OMS646. Thus, in one aspect, a MASP-2 inhibitor for use in the compositions and methods of the present invention comprises a human antibody that binds to a polypeptide consisting of human MASP-2 (SEQ ID NO:6). The antibody may comprise: (I) (a) a heavy chain variable region comprising i) a heavy chain CDR-H1 comprising an amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising an amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising an amino acid sequence from 95-107 of SEQ ID NO:67; and b) a light chain variable region comprising i) a light chain CDR-L1 comprising an amino acid sequence from 24-34 of SEQ ID NO:69; and ii) a light chain CDR-L2 comprising an amino acid sequence from 50-56 of SEQ ID NO:69; and iii) a light chain CDR-L3 comprising an amino acid sequence from 89-97 of SEQ ID NO:69; or (II) a light chain variable region comprising at least 90% identity (e.g., SEQ ID NO:69) to SEQ ID NO:67 (e.g., SEQ ID NO:69). and a light chain variable region having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO:69 and variants thereof.

[0100] In some aspects, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, the composition comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:67 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:69.

[0101] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or antigen-binding fragment thereof which specifically recognizes at least a portion of the epitope on human MASP-2 recognized by reference antibody OMS646, which comprises a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:69. In one embodiment, the MASP-2 inhibitor for use in the methods of the present invention comprises human antibody OMS646.

[0102] Table 1. Exemplary MASP-2-specific antibodies TIFF2024509702000008.tif202155

[0103] Anti-MASP-2 antibodies with reduced effector function In some embodiments of this aspect of the present invention, the anti-MASP-2 antibody has reduced effector function to alleviate inflammation that may result from activation of the classical complement pathway. It has been shown that the ability of IgG molecules to trigger the classical complement pathway resides within the Fc portion of the molecule (Duncan, AR. et al., Nature 332:738-740 1988). IgG molecules in which the Fc portion of the molecule has been removed by enzymatic cleavage do not have this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Thus, by having a genetically engineered Fc sequence that minimizes effector function, or by using human IgG 2 or IgG 4 By isotyping, antibodies can be generated that have reduced effector functions as a result of lacking the Fc portion of the molecule.

[0104] Antibodies with reduced effector functions can be generated by standard molecular biology manipulations of the Fc portion of an IgG heavy chain, as described herein, and also in Jolliffe et al., Int'l Rev. Immunol. 10:241-250, 1993 and Rodrigues et al., J. Immunol. 151:6954-6961, 1998. Antibodies with reduced effector functions also include human IgG2 and IgG4 isotypes with reduced ability to activate complement and / or interact with Fc receptors (Ravetch, JV, et al., Annu. Rev. Immunol. 9:457-492, 1991; Isaacs, JD, et al., J. Immunol. 148:3062-3071, 1992; van de Winkel, JG, et al., Immunol Today 14:215-221, 1993). Humanized or fully human antibodies specific for human MASP-2, consisting of IgG2 or IgG4 isotypes, can be produced by one of several methods known to those skilled in the art, as described in Vaughan, TJ, et al., Nature Biotechnical 16:535-539, 1998.

[0105] Preparation of anti-MASP-2 antibody Anti-MASP-2 antibodies can be generated using MASP-2 polypeptides (e.g., full-length MASP-2) or antigenic MASP-2 epitope-containing peptides (e.g., portions of MASP-2 polypeptides). Immunogenic peptides can be as small as 5 amino acid residues. For example, a MASP-2 polypeptide comprising the entire amino acid sequence of SEQ ID NO:6 may be used to induce anti-MASP-2 antibodies useful in the methods of the present invention. Certain MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, and the region containing the serine-protease active site, may be expressed as recombinant polypeptides as described in Example 3 and used as antigens. In addition, peptides comprising at least 6 amino acid portions of the MASP-2 polypeptide (SEQ ID NO:6) are also useful for inducing MASP-2 antibodies. Further examples of MASP-2-derived antigens useful for inducing MASP-2 antibodies are shown in Table 2 below. The MASP-2 peptides and polypeptides used to generate antibodies may be isolated as natural or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides, e.g., MASP-2A, as further described herein. In some embodiments of this aspect of the invention, anti-MASP-2 antibodies are obtained using transgenic mouse strains, as described herein.

[0106] Antigens useful in producing anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2 or a portion thereof with an immunoglobulin polypeptide or maltose binding protein. Polypeptide immunogens may be full-length molecules or portions thereof. If the polypeptide portion is haptenic, such portions may be conveniently conjugated or linked to macromolecular carriers (e.g., keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

[0107] Table 2. MASP-2-derived antigens TIFF2024509702000009.tif186153

[0108] Polyclonal antibodies Polyclonal antibodies against MASP-2 can be prepared by immunizing animals with MASP-2 polypeptides or immunogenic portions thereof using methods well known to those skilled in the art. See, for example, Green et al., "Production of Polyclonal Antisera", Immunochemical Protocols (Manson, ed.), p. 105. The immunogenicity of MASP-2 polypeptides can be enhanced using adjuvants including mineral gels, such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surfactants, such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Polyclonal antibodies are typically produced in animals, such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep. Alternatively, anti-MASP-2 antibodies useful in the present invention may also be derived from subhuman primates. General techniques for producing diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO91 / 11465, and Losman, MJ, et al., Int. J. Cancer 46:310, 1990. Serum containing immunologically active antibodies is then generated from the blood of such immunized animals using standard procedures well known in the art.

[0109] Monoclonal antibodies In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly specific because they are produced against a single MASP-2 epitope. The modifier "monoclonal" as used herein indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring the antibody to be produced by a particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molecules by continuous cell lines, for example, the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975. Alternatively, monoclonal antibodies can be produced by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352:624-628, 1991, and Marks, JD, et al., J. Mol Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

[0110] For example, monoclonal antibodies can be obtained by injecting a composition containing a MASP-2 polypeptide or a portion thereof into a suitable mammal (e.g., a BALB / c mouse). After a predetermined period of time, spleen cells are removed from the mouse and suspended in a cell culture medium. The spleen cells are then fused with an immortal cell line to form hybridomas. The formed hybridomas are grown in cell culture and screened for their ability to produce monoclonals against MASP-2. Examples further describing the production of anti-MASP-2 monoclonal antibodies are provided herein (see also Current Protocols in Immunology, Vol.1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991).

[0111] Human monoclonal antibodies can be obtained using transgenic mice engineered to produce specific human antibodies in response to antigen challenge. In this technique, elements of the human immunoglobulin heavy and light chain loci are introduced into a line of mice derived from an embryonic stem cell line that contains targeted disruptions of the endogenous immunoglobulin heavy and light chain loci. The transgenic mice are capable of synthesizing human antibodies specific to human antigens, such as the MASP-2 antigens described herein, and can be used to generate human MASP-2 antibody-secreting hybridomas by fusing B cells from such animals with a suitable myeloma cell line using conventional Kohler-Millstein technology as further described herein. Transgenic mice with human immunoglobulin genomes are commercially available (e.g., from Abgenix, Inc., Fremont, CA. and Medarex, Inc., Annandale, NJ). Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, LL, et al., Nature Genet. 7:13, 1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, LD, et al., Int. Immun. 6:579, 3994.

[0112] Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques, including affinity chromatography with Protein A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Coligan, pp. 2.7.1-2.7.12 and 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)", Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pp. 79-104, 1992).

[0113] Once a polyclonal, monoclonal or phage-derived antibody is produced, it is first tested for specific MASP-2 binding. To detect an antibody that specifically binds to MASP-2, various assays known to those skilled in the art can be used. Exemplary assays include Western blot or immunoprecipitation analysis by standard methods (e.g., as described in Ausubel et al.,), immunoelectrophoresis, enzyme-linked immunosorbent assay, dot blot, inhibition or competitive assay, and sandwich assay (as described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once an antibody that specifically binds to MASP-2 is identified, the anti-MASP-2 antibody is tested for its ability to function as a MASP-2 inhibitor in one of several assays, for example, a lectin-specific C4 cleavage assay (described in Example 2), a C3b deposition assay (described in Example 2), or a C4b deposition assay (described in Example 2).

[0114] The affinity of an anti-MASP-2 monoclonal antibody can be easily determined by one skilled in the art (see, for example, Scatchard, A., NY Acad. Sci. 51:660-672, 1949). In one embodiment, an anti-MASP-2 monoclonal antibody useful in the method of the present invention binds to MASP-2 with a binding affinity of <100 nM, preferably <10 nM, and most preferably <2 nM.

[0115] Chimeric / Humanized Antibodies Monoclonal antibodies useful in the methods of the invention include chimeric antibodies in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (U.S. Pat. No. 4,816,567 to Cabilly; and Morrison, SL, et al., Proc. Nat'l Acad. Sci. USA 81:6851-6855, 1984).

[0116] One form of chimeric antibody useful in the present invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g., mouse) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulin. Humanized monoclonal antibodies are made by introducing non-human (e.g., mouse) complementarity determining regions (CDRs) derived from the variable heavy and variable light chains of mouse immunoglobulin into a human variable domain. Human antibody residues are then typically substituted in the framework regions of the non-human counterpart. In addition, humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine the performance of the antibody. Generally, humanized antibodies contain substantially all of at least one, and typically two, variable domains. All or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the Fv framework regions correspond to the Fv framework regions of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, PT, et al., Nature 321:522-525, 1986; Reichmann, L., et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992.

[0117] The humanized antibodies useful in the present invention include human monoclonal antibodies that contain at least the MASP-2 binding CDRH3 region. In addition, the Fc portion may be exchanged to generate IgA or IgM antibodies as well as human IgG antibodies. Such humanized antibodies are considered to be particularly useful in clinical applications because they specifically recognize human MASP-2 but do not induce an immune response against the antibody itself in humans. As a result, such humanized antibodies are more suitable for in vivo administration in humans, especially when repeated or long-term administration is required.

[0118] An example of producing a humanized anti-MASP-2 antibody from a mouse anti-MASP-2 monoclonal antibody is shown in Example 6 of the present specification. Techniques for producing humanized monoclonal antibodies are described, for example, in Jones, PT, et al., Nature 321:522, 1986; Carter, P., et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu, JS, Crit. Rev. Biotech. 12:437, 1992; Singer, II, et al., J. Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, "Engineering Therapeutic Antibodies", Protein Engineering: Principles and Practice, Cleland et al.(eds.), John Wiley & Sons, Inc., pp. 399-434, 1996; and Queen, No. 5,693,762 to the United States Patent No. 5,693,762 issued to Robert J. Schneider, 1997. Additionally, there are commercial entities that synthesize humanized antibodies from specific murine antibody regions, such as Protein Design Labs (Mountain View, Calif.).

[0119] Recombinant antibodies Anti-MASP-2 antibodies can also be produced using recombinant techniques. For example, human antibody fragments (V H , V L , Fv, Fd, Fab, or F(ab') 2 Human antibodies can be made by using a human immunoglobulin expression library (available, for example, from Stratagene, Corp., La Jolla, Calif.) to generate fragments of human antibodies. These fragments are then used to construct whole human antibodies using techniques similar to those used to make chimeric antibodies.

[0120] Anti-idiotype antibodies Once anti-MASP-2 antibodies with the desired inhibitory activity have been identified, these antibodies can be used to generate anti-idiotypic antibodies that resemble portions of MASP-2 using techniques well known in the art. See, for example, Greenspan, NS, et al., FASEB J. 7:437, 1993. For example, an antibody that binds to MASP-2 and completely inhibits the MASP-2 protein interaction required for complement activation can be used to generate anti-idiotypic antibodies that resemble the MBL binding site on the MASP-2 protein and thus bind to and neutralize the binding ligand of MASP-2, such as MBL.

[0121] Immunoglobulin fragments MASP-2 inhibitors useful in the methods of the present invention include intact immunoglobulin molecules as well as antibody fragments formed therefrom, such as Fab, Fab', F(ab) and / or Fab' antibodies. 2 , F(ab') 2 , and well-known fragments including Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies.

[0122] It is well known in the art that the binding of an antibody to its epitope involves only a small portion of the antibody molecule, the paratope (see, e.g., Clark, WR, The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of an antibody are effectors of the classical complement pathway but are not involved in antigen binding. Antibodies from which the pFc' region has been enzymatically cleaved or which have been produced without the pFc' region are called F(ab') 2 These fragments, called F(ab') fragments, retain both of the antigen-binding sites of an intact antibody. 2 The fragment is referred to as a bivalent monoclonal fragment because of its two antigen-binding sites. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, is called a Fab fragment, and retains one of the antigen-binding sites of the intact antibody molecule.

[0123] Antibody fragments can be obtained by proteolytic hydrolysis of whole antibodies by conventional methods, such as pepsin or papain digestion. For example, antibody fragments can be obtained by enzymatic cleavage of antibodies with pepsin to give F(ab') fragments. 2 Fab' can be produced by obtaining a 5S fragment, called 3.5S Fab'. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be performed using a sulfhydryl blocking group that cleaves disulfide bonds. Alternatively, two monovalent Fab fragments and one Fc fragment are generated directly by enzymatic cleavage using pepsin. These methods are described, for example, in U.S. Patent No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, RR, Biochem, J. 73:119, 1959; Edelman, et al., Methods in Enzymology 1:422, Academic Press, 1967; and in Coligan, pages 2.8.1-2.8.10 and 2.10.-2.10.4.

[0124] In some embodiments, it is preferable to use antibody fragments without Fc regions to avoid activation of the classical complement pathway, which is initiated when Fc binds to Fcγ receptors. There are several ways that MoAbs can be made that avoid Fcγ receptor interactions. For example, the Fc region of a monoclonal antibody can be chemically removed using partial digestion with a proteolytic enzyme (e.g., ficin digestion), thereby producing, for example, an antigen-binding antibody fragment, such as a Fab fragment or F(ab) fragment. 2Fragments can be generated (Mariani, M., et al., Mol. Immunol. 28:69-71, 1991). Alternatively, human γ4 IgG isotypes, which do not bind to Fcγ receptors, can be used in constructing humanized antibodies as described herein. Antibodies without Fc domains, single chain antibodies, and antigen binding domains can also be engineered using recombinant methods described herein.

[0125] single chain antibody fragment Alternatively, peptide single-chain binding molecules specific for MASP-2 can be made in which the heavy chain Fv region and the light chain Fv region are connected. The Fv fragments may be connected with a peptide linker to form a single-chain antigen-binding protein (scFv). These single-chain antigen-binding proteins are composed of VFv fragments connected with an oligonucleotide. H and V L scFvs are prepared by constructing a structural gene containing a DNA sequence encoding the V domains. The structural gene is inserted into an expression vector, which is then introduced into a host cell, such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for making scFvs are described, for example, in Whitlow, et al., "Methods: A Companion to Methods in Enzymology" 2:97, 1991; Bird, et al., Science 242:423, 1988; U.S. Patent No. 4,946,778 to Ladner; Pack, P., et al., Bio / Technology 11:1271, 1993.

[0126] As an illustrative example, MASP-2-specific scFvs can be obtained by exposing lymphocytes to MASP-2 polypeptides in vitro and selecting antibody display libraries in phage vectors or similar vectors (e.g., by using immobilized or labeled MASP-2 proteins or peptides). Genes encoding polypeptides with potential MASP-2 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phages or bacteria, such as E. coli. These random peptide display libraries can be used to screen peptides that interact with MASP-2. Techniques for generating and screening such random peptide display libraries are well known in the art (U.S. Patent No. 5,223,409 to Lardner; U.S. Patent No. 4,946,778 to Ladner; U.S. Patent No. 5,403,484 to Ladner; U.S. Patent No. 5,571,698 to Ladner; and Kay et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996). Random peptide display libraries and kits for screening such libraries are commercially available from, for example, CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Ipswich, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ).

[0127] Another form of anti-MASP-2 antibody fragment useful in this aspect of the present invention is a peptide encoding a single complementarity determining region (CDR) that binds to an epitope on the MASP-2 antigen and inhibits MASP-2-dependent complement activation. CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDRs of the antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, e.g., Larrick et al., Methods; A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies", Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., (eds.), p. 166, Cambridge University Press, 1995; and Ward et al., "Genetic Manipulation and Expression of Antibodies", Monoclonal Antibodies: Principles and Applications, Birch et al., (eds,), p. 137, Wiley-Liss, Inc., 1995).

[0128] To inhibit MASP-2-dependent complement activation, the MASP-2 antibody described herein is administered to a subject in need thereof. In some embodiments, the MASP-2 inhibitor is a high-affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.

[0129] Peptide inhibitors In some embodiments of this aspect of the present invention, the MASP-2 inhibitor comprises an isolated MASP-2 peptide inhibitor, including isolated natural and synthetic peptide inhibitors, that inhibit the MASP-2-dependent complement activation system. As used herein, the term "isolated MASP-2 peptide inhibitor" refers to a peptide that inhibits MASP-2-dependent complement activation by binding to MASP-2, by competing with MASP-2 for binding to another recognition molecule of the lectin pathway (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin), and / or by directly interacting with MASP-2 to inhibit MASP-2-dependent complement activation, which peptide is substantially pure and essentially free of other substances with which it may be found in nature to an extent practical and appropriate for the intended use.

[0130] Peptide inhibitors have been successfully used in vivo to disrupt protein-protein interactions and catalytic sites. For example, a peptide inhibitor against an adhesion molecule structurally related to LFA-1 was recently approved for clinical use in coagulation disorders (Ohman, EM, et al., European Heart J. 16:50-55, 1995). Short linear peptides (<30 amino acids) that block or interfere with integrin-dependent adhesion have been described (Murayama, O., et al., J. Biochem. 120:445-51, 1996). Longer peptides ranging from 25 to 200 amino acid residues in length have also been successfully used to block integrin-dependent adhesion (Zhang, L., et al., J. Biol. Chem. 271(47):29953-57, 1996). Generally, long peptide inhibitors have higher affinity and / or slower off-rate than short peptides, and therefore can be potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective integrin inhibitors in vivo for the treatment of human inflammatory diseases (Jackson, DY, et al., J. Med. Chem. 40:3359-68, 1997). One method for making cyclic peptides involves peptide synthesis in which the terminal amino acid of the peptide is cysteine, thereby allowing the peptide to exist in a cyclic form via disulfide bonds between the terminal amino acids. This cyclic form has been shown to improve affinity and half-life in vivo for the treatment of hematopoietic neoplasms (e.g., U.S. Patent No. 6,649,592 to Larson).

[0131] Synthetic MASP-2 peptide inhibitors MASP-2 inhibitory peptides useful in the methods of this aspect of the invention are exemplified by amino acid sequences that mimic target regions important for MASP-2 function. Inhibitory peptides useful in the practice of the methods of the invention range in size from about 5 amino acids to about 300 amino acids. Table 3 provides a list of exemplary inhibitory peptides that may be useful in the practice of this aspect of the invention. For example, the ability of candidate MASP-2 inhibitory peptides to function as MASP-2 inhibitors can be tested in one of several assays, including a lectin-specific C4 cleavage assay (described in Example 2) and a C3b deposition assay (described in Example 2).

[0132] In some embodiments, the MASP-2 inhibitory peptide is derived from a MASP-2 polypeptide and is selected from the full-length mature MASP-2 protein (SEQ ID NO:6), or specific domains of the MASP-2 protein, such as the CUBI domain (SEQ ID NO:8), the CUBIEGF domain (SEQ ID NO:9), the EGF domain (SEQ ID NO:11), and the serine protease domain (SEQ ID NO:12). As mentioned above, the CUBEGFCUBII region has been shown to be required for dimerization and binding to MBL (Thielens et al., supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO:16) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in a study identifying humans carrying a homozygous Asp105 to Gly105 mutation, resulting in loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et al., New England J. Med. 349:554-560, 2003).

[0133] In some embodiments, MASP-2 inhibitory peptide is derived from lectin protein that binds to MASP-2 and participates in lectin complement pathway.Several different lectins have been identified that participate in this pathway, including mannan-binding lectin (MBL), L-ficolin, M-ficolin, and H-ficolin (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. 176:1497-2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002).These lectins exist in serum as oligomers of homotrimeric subunits, each of which has N-terminal collagen-like fibers with carbohydrate recognition domains. These different lectins have been shown to bind to MASP-2, and the lectin / MASP-2 complex activates complement by cleaving proteins C4 and C2. H-ficolin has a 24 amino acid amino-terminal region, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a 12 amino acid neck domain, and a 207 amino acid fibrinogen-like domain (Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). H-ficolin binds to GlcNAc and agglutinates human erythrocytes coated with LPS from S. typhimurium, S. minnesota, and E. coli. H-ficolin has been shown to bind to MASP-2 and MAp19 and activate the lectin pathway. L-ficolin / P35 also binds to GlcNAc and has been shown to bind to MASP-2 and MAp19 in human serum, and this complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol. 164:2281, 2000).Thus, a MASP-2 inhibitory peptide useful in the present invention may comprise a region of at least five amino acids selected from MBL protein (SEQ ID NO:21), H-ficolin protein (GenBank accession number NM_173452), M-ficolin protein (GenBank accession number O00602), and L-ficolin protein (GenBank accession number NM_015838).

[0134] More specifically, the scientists found that the MASP-2 binding site on MBL is located at 12 Gly-XY triplets between the hinge and neck in the C-terminal part of the collagen-like domain of MBP. TIFF2024509702000010.tif12128 (Wallis, R. et al., J. Biol Chem. 279:14065, 2004). This MASP-2 binding site region is also highly conserved in human H-ficolin and human L-ficolin. A consensus binding site has been described that is present in all three lectin proteins and contains the amino acid sequence "OGK-X-GP" (SEQ ID NO:22), where the letter "O" represents hydroxyproline and the letter "X" is a hydrophobic residue (Wallis et al., 2004, supra). Thus, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least six amino acids in length and contain SEQ ID NO:22. Amino acid sequence Peptides derived from MBL, including TIFF2024509702000011.tif4128, have been shown to bind to MASP-2 in vitro (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides can be synthesized that are flanked by two GPO triplets at each end, which enhances triple helix formation as seen in the native MBL protein. TIFF2024509702000012.tif5132 (further described in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).

[0135] The MASP-2 inhibitor peptide also has the sequence derived from the consensus MASP-2 binding domain of H-ficolin. A sequence derived from the consensus MASP-2 binding region of L-ficolin, which may be derived from human H-ficolin, including TIFF2024509702000013.tif4145. Also included are peptides derived from human L-ficolin, including TIFF2024509702000014.tif12153.

[0136] The MASP-2 inhibitor peptide also contains a C4 cleavage site linked to the C-terminal portion of antithrombin III. It may be derived from a C4 cleavage site such as TIFF2024509702000015.tif4128 (Glover, GI, et al., Mol. Immunol. 25:1261(1988)).

[0137] Table 3: Exemplary MASP-2 inhibitory peptides TIFF2024509702000016.tif98155TIFF2024509702000017.tif255155Note: The letter "O" represents hydroxyproline. The letter "X" is a hydrophobic residue.

[0138] Peptides derived from the C4 cleavage site as well as other peptides that inhibit the MASP-2 serine protease site can be chemically modified to become irreversible protease inhibitors. For example, suitable modifications may include, but are not necessarily limited to, halomethylketones (Br, Cl, I, F) at the C-terminus, Asp, or Glu, or added to functional side chains; haloacetyl (or other alpha-haloacetyl) groups at amino groups or other functional side chains; epoxide- or imine-containing groups at the amino or carboxy terminus or functional side chains; or imidate esters at the amino or carboxy terminus or functional side chains. Such modifications would have the advantage of permanently inhibiting the enzyme through covalent binding of the peptide. This may result in lower effective doses and / or require less frequent administration of peptide inhibitors.

[0139] In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the methods of the present invention include peptides containing the MASP-2-binding CDRH3 region of an anti-MASP-2 MoAb obtained as described herein. The sequences of the CDR regions for use in peptide synthesis can be determined by methods known in the art. The heavy chain variable region is generally a peptide of 100-150 amino acids in length. The light chain variable region is generally a peptide of 80-130 amino acids in length. The CDR sequences in the heavy and light chain variable regions contain sequences of only about 3-25 amino acids that can be readily sequenced by one of skill in the art.

[0140] Those skilled in the art will recognize that substantially homologous variations of MASP-2 inhibitory peptides also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptides with insertions, deletions, replacements, and / or additional amino acids at the carboxy- or amino-terminal portions of the peptides, and mixtures thereof. Thus, homologous peptides with MASP-2 inhibitory activity are considered useful in the methods of the present invention. The peptides described may also include overlapping motifs and other modifications by conservative substitutions. Conservative variants are described elsewhere herein and include replacing an amino acid with an amino acid of similar charge, size, or hydrophobicity, etc.

[0141] To more closely resemble segments in the intact protein, the MASP-2 inhibitory peptides may be modified to increase solubility and / or to maximize positive or negative charge. Derivatives may or may not have the exact primary amino acid structure of the peptides disclosed herein, so long as they functionally retain the desired MASP-2 inhibitory properties. The modifications may include amino acid substitutions with one of the 20 commonly known amino acids or with another amino acid, amino acid substitutions with derivatized or substituted amino acids or D-amino acids that have additional desirable characteristics, such as resistance to enzymatic degradation, or substitutions with other molecules or compounds that mimic the natural conformation and function of an amino acid, a number of amino acids, or a peptide, such as carbohydrates; amino acid deletions; amino acid insertions with one of the 20 commonly known amino acids or with another amino acid, amino acid insertions with derivatized or substituted amino acids or D-amino acids that have additional desirable characteristics, such as resistance to enzymatic degradation, or substitutions with other molecules or compounds that mimic the natural conformation and function of an amino acid, a number of amino acids, or a peptide, such as carbohydrates; or substitutions with other molecules or compounds that mimic the natural conformation, charge distribution, and function of the parent peptide, such as carbohydrates or nucleic acid monomers. The peptides may also be modified by acetylation or amidation.

[0142] The synthesis of derivative inhibitory peptides may rely on known techniques such as peptide biosynthesis, carbohydrate biosynthesis, etc. As a starting point, a person skilled in the art may rely on a suitable computer program to determine the conformation of the peptide of interest. Once the conformation of the peptide disclosed herein is known, a person skilled in the art can determine what kind of substitutions can be made at one or more sites in a rational design manner to create a derivative that retains the basic conformation and charge distribution of the parent peptide, but may have features that are not present in the parent peptide or that are enhanced beyond those found in the parent peptide. Once a candidate derivative molecule is identified, the derivative can be tested using the assays described herein to determine whether it functions as a MASP-2 inhibitor.

[0143] Screening of MASP-2 inhibitor peptides Molecular modeling and rational molecular design can also be used to generate and screen peptides that mimic the molecular structure of the important binding region of MASP-2 and inhibit the complement activity of MASP-2. As previously mentioned, the molecular structures used in modeling include the CDR regions of anti-MASP-2 monoclonal antibodies, as well as target regions known to be important for MASP-2 function, including the region required for dimerization, the region involved in binding with MBL, and the serine protease active site. Methods for identifying peptides that bind to a specific target are well known in the art. For example, molecular imprinting can be used to de novo construct macromolecular structures, such as peptides, that bind to a specific molecule. See, for example, Shea, KJ, "Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sties", TRIP 2(5) 1994.

[0144] As an illustrative example, one method for preparing mimetics of MASP-2 binding peptides is as follows: A functional monomer (template) of a known MASP-2 binding peptide or a binding region of an anti-MASP-2 antibody that exhibits MASP-2 inhibition is polymerized. The template is then removed, and another class of monomer is subsequently polymerized into the void left by the template to obtain a new molecule that exhibits one or more desirable properties similar to the template. In addition to preparing peptides in this way, other MASP-2 binding molecules that are MASP-2 inhibitors can also be prepared, such as polysaccharides, nucleosides, drugs, nuclear proteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials. This method is useful for the design of a wide variety of biomimetics that are more stable than their natural counterparts. Because these biomimetics are typically prepared by free radical polymerization of functional monomers, resulting in compounds with non-biodegradable backbones.

[0145] Peptide synthesis MASP-2 inhibitory peptides can be prepared using techniques well known in the art, for example, the solid phase synthesis technique first described by Merrifield in J. Amer. Chem. Soc. 85:2149-2154, 1963. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Foster City, Calif.), following the instructions provided by the manufacturer. Other techniques can be found, for example, in Bodanszky, M., et al., Peptide Synthesis, 2nd Edition, John Wiley & Sons, 1976, as well as other references known to those skilled in the art.

[0146] Peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, peptides can be made enzymatically by inserting a nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the required amino acids. The peptide is then purified using chromatographic or electrophoretic methods, or by a carrier protein that can be fused to the peptide by inserting the peptide-encoding sequence in phase with a nucleic acid sequence encoding the carrier protein into an expression vector and subsequently cleaved. The fusion protein peptide may be isolated using chromatographic, electrophoretic, or immunological techniques (e.g., antibody-mediated binding of the resin to the carrier protein). The peptide can be cleaved using chemical methodology or enzymatically, e.g., by hydrolases.

[0147] The MASP-2 inhibitory peptide useful in the method of the present invention can also be produced in recombinant host cells according to conventional techniques.In order to express the sequence encoding the MASP-2 inhibitory peptide, the nucleic acid molecule encoding the peptide must be functionally linked to the regulatory sequence that controls transcriptional expression in an expression vector, and then introduced into host cells.In addition to the transcriptional regulatory sequence such as promoter and enhancer, the expression vector may also include a translational regulatory sequence and a marker gene that is suitable for selecting cells that carry the expression vector.

[0148] Nucleic acid molecules encoding MASP-2 inhibitory peptides can be synthesized by a "gene machine" using protocols such as the phosphoramidite method. When chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or gene fragment, each complementary strand is made separately. The creation of short genes (60-80 base pairs) is technically straightforward and can be achieved by synthesizing the complementary strands and then annealing them. To create larger genes, synthetic genes (double stranded) are assembled in a modular fashion from single-stranded fragments of 20-100 nucleotides in length. For reviews of polynucleotide synthesis, see, e.g., Glick and Pasternak, "Molecular Biotechnology, Principles and Applications of Recombinant DNA", ASM Press, 1994; Itakura, K., et al., Annu. Rev, Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nal'l Acad. Sci. USA 87:633, 1990.

[0149] Small molecule inhibitors of MASP-2 In some embodiments, MASP-2 inhibitors are small molecule inhibitors, including natural substances, semi-synthetic substances, and synthetic substances having a low molecular weight (e.g., 50 to 1000 Da), such as peptides, peptidomimetics, and non-peptide inhibitors (e.g., oligonucleotides and organic compounds). MASP-2 small molecule inhibitors can be prepared based on the molecular structure of the variable region of an anti-MASP-2 antibody.

[0150] Small molecule inhibitors may also be designed and generated based on the MASP-2 crystal structure using computational drug design (Kuntz ID, et al., Science 257:1078, 1992). The crystal structure of rat MASP-2 has been described (Feinberg, H., et al., EMBO J. 22:2348-2359, 2003). The MASP-2 crystal structure coordinates are used as input for a computer program, such as DOCK, which outputs a list of small molecule structures predicted to bind to MASP-2 using the method described in Kuntz et al. The use of such computer programs is well known to those skilled in the art. For example, crystal structures of HIV-1 protease inhibitors have been used to identify unique non-peptide ligands that are HIV-1 protease inhibitors by evaluating the fit of compounds found in the Cambridge Crystallographic database with the enzyme binding site using the program DOCK (Kuntz, ID, et al., J. Mol. Biol. 161:269-288, 1982; DesJarlais, RL, et al., PNAS 87:6644-6648, 1990).

[0151] Exemplary MASP-2 inhibitors include, but are not limited to, compounds disclosed in U.S. Patent Application Nos. 62 / 943,629, 62 / 943,622, 62 / 943,611, 62 / 943,599, 16 / 425,791, and PCT Application No. PCT / US19 / 34220, each of which is incorporated by reference in its entirety into this specification.

[0152] In some embodiments, the small molecule has formula (I-1), (IIA), (IIB), (III), or (IV): TIFF2024509702000018.tif157128, or a salt thereof; During the ceremony, Cy 1A is unsubstituted or substituted C 6~10aryl or unsubstituted or substituted 5-10 membered heteroaryl; Cy 1A The ring atoms of the 5- to 10-membered heteroaryl that forms the ring are carbon atoms and one, two, or three heteroatoms selected from O, N, and S, and Cy 1A Substitution C to form 6~10 The aryl or substituted 5- to 10-membered heteroaryl is each independently R Cy1A , halogen, C 1~6 Haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , C(=NOR a11 )NR c11 R d11 , C(=NOC(O)R b11 )NR c11 R d11 , C(=NR e11 )NR c11 C(O)OR a11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11and oxo; Each R Cy1A is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy1A The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy1A Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O)2 NR c11 R d11 and oxo; R Cy1A Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; R 11 is H or C 1~6 Alkyl, C 6~10 Aryl-C 1~6Alkyl, or 5-10 membered heteroaryl-C 1~6 is alkyl, R 11 Forming C 1~6 Alkyl is unsubstituted or independently selected from halogen, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; R 11 Forming C 6~10 Aryl-C 1~6 Alkyl or 5-10 membered heteroaryl-C 1~6 Alkyl is unsubstituted or independently C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 Rd11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; R 12 is H or C 1~6 is alkyl, or R 11 and R 12 together with the group to which they are attached form a 4- to 6-membered heterocycloalkyl ring; A 11 CR 13 R 15 or N, Each R 13 is independently 1B , (CR 13A R 13B ) n3 Cy 1B , (C 1~6 Alkylene)Cy 1B , (C 2~6 Alkenylene)Cy 1B , (C 2~6 Alkynylene)Cy 1B, or O.Cy 1B and R 13 C 1~6 Alkylene, C 2~6 Alkenylene, or C 2~6 The alkynylene moieties are unsubstituted or each independently selected from halogen, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; Each R 14 are independently H and C 1~6 alkyl; R 15 , H, R 13 , C 1~6 alkyl, and OH; R 14 Independently of other occurrences of R, a pair of R attached to adjacent carbon atoms 14group or R attached to adjacent carbon atoms 14 Groups and R 15 The pair of groups is arranged such that adjacent carbon atoms are connected by a double bond. 14 Group pair or R 14 Groups and R 15 may be replaced together by a bond connecting adjacent carbon atoms to which the pair of groups are attached, or A pair of R attached to the same carbon atom 14 group, or R attached to the same carbon atom 13 Groups and R 15 The pair of groups is R 14 Independently of other occurrences of and R 14 Group pair or R 13 Groups and R 15 Together with the carbon atom to which the pair of radicals is attached, they form a spiro-fused C 3~10 A cycloalkyl or 4-10 membered heterocycloalkyl ring may be formed, the ring atoms of the formed 4-10 membered heterocycloalkyl ring consisting of carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S, and the formed spiro-fused C 3~10 The cycloalkyl or 4-10 membered heterocycloalkyl ring is optionally independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R.c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; or R attached to adjacent carbon atoms 14 Pairs of groups or R attached to adjacent carbon atoms 14 Groups and R 15 The pair of groups is R 14 Independently of other occurrences of R 14 Group pair or R 14 Groups and R 15 Together with the adjacent carbon atoms to which the pair of groups are attached, they form a fused C 3~10 A cycloalkyl or 4-10 membered heterocycloalkyl ring may be formed, the ring atoms of the formed 4-10 membered heterocycloalkyl ring consisting of carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S, and the formed fused C 3~10 The cycloalkyl or 4-10 membered heterocycloalkyl ring is optionally independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 Rd11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; or Four R's attached to two adjacent carbon atoms 14 A group of groups or two R attached to two adjacent carbon atoms 14 Group, 1 R 13 group, and one R 15 The group of groups is R 14 Independently of other occurrences of 14 A group of groups or two R 14 Group, 1 R 13 group, and one R 15 Together with the two adjacent carbon atoms to which the group is attached, they form a fused C 6~10 Aryl or 5-10 membered heteroaryl, C 3~10 The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl ring formed may consist of carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S, and the formed fused C 6~10 Aryl or 5-10 membered heteroaryl, C 3~10 The cycloalkyl or 4-10 membered heterocycloalkyl ring is optionally independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6Alkynyl, haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; n1 is 1 or 2; n2 is 0, 1, or 2; However, the sum of n1 and n2 is 1, 2, or 3, However, if n1 is 1 or n2 is 0, then A 11 is CR 13 R 15 and n3 is 0, 1, or 2; Each R 13A are independently H or C 1~6 is alkyl, Each R 13B are independently H or C 1~6 is alkyl, or Alternatively, R attached to the same carbon atom 13A and R 13B is any other R 13A Groups and R 13B Independently and together with the -(CH 2 ) 2-5- may be formed to form a 3- to 6-membered cycloalkyl ring, Cy 1B is unsubstituted or substituted C 6~10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3~10 cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; Cy 1B The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming the ring are carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S; Cy 1B Substitution C to form 6~10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3~10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl each independently represent R Cy1B , halogen, C 1~6 Haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , C(=NOR a11 )NR c11 R d11, C(=NOC(O)R b11 )NR c11 R d11 , C(=NR e11 )NR c11 C(O)OR a11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; Each R Cy1B is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy1B The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy1B Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)Rb11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; R Cy1B Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; R 16 H, Cy 1C , C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 alkynyl, R 16 Forming, C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is unsubstituted or 1C , halogen, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R.c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo, with the proviso that R 16 One or less of the substituents of 1C and Cy 1C is unsubstituted or substituted C 6~10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3~10 cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; Cy 1C The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming the ring are carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S; Cy 1C Substitution C to form 6~10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3~10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl each independently represent R Cy1C , halogen, C 1~6 Haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , C(=NOR a11 )NR c11 R d11, C(=NOC(O)R b11 )NR c11 R d11 , C(=NR e11 )NR c11 C(O)OR a11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; Each R Cy1C is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy1C The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy1C Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)Rb11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11 C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; R Cy1C Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a11 , S.R. a11 , C(O)R b11 , C(O)NR c11 R d11 , C(O)OR a11 , O.C.(O)R b11 , O-C(O)NR c11 R d11 , N.R. c11 R d11 , N.R. c11 C(O)R b11 , N.R. c11 C(O)NR c11 R d11 , N.R. c11 C(O)OR a11 , C(=NR e11 )NR c11 R d11 , N.R. c11C(=NR e11 )NR c11 R d11 , S(O)R b11 , S(O)NR c11 R d11 , S(O) 2 R b11 , N.R. c11 S(O) 2 R b11 , S(O) 2 NR c11 R d11 and oxo; R a11 , R b11 , R c11 , and R d11 are independently H, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 6~10 Aryl-C 1~3 Alkyl, 5-10 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-10 membered heterocycloalkyl-C 1~3 alkyl; R a11 , R b11 , R c11 and R d11 forming C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 6~10 Aryl-C 1~3 Alkyl, 5-10 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-10 membered heterocycloalkyl-C 1~3 Each alkyl is independently C1~6 Alkyl, halo, CN, OR a12 , S.R. a12 , C(O)R b12 , C(O)NR c12 R d12 , C(O)OR a12 , O.C.(O)R b12 , O-C(O)NR c12 R d12 , N.R. c12 R d12 , N.R. c12 C(O)R b12 , N.R. c12 C(O)NR c12 R d12 , N.R. c12 C(O)OR a12 , C(=NR e12 )NR c12 R d12 , N.R. c12 C(=NR e12 )NR c12 R d12 , S(O)R b12 , S(O)NR c12 R d12 , S(O) 2 R b12 , N.R. c12 S(O) 2 R b12 , S(O) 2 NR c12 R d12 and oxo; Alternatively, R bonded to the same N atom c11 and R d11 together with the N atom to which they are both attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl group or a 5-membered heteroaryl group, each of which independently represents C 1~6 Alkyl, halo, CN, OR a12 , S.R. a12 , C(O)R b12 , C(O)NR c12 R d12 , C(O)OR a12 , O.C.(O)R b12 , O-C(O)NR c12 R d12 , N.R.c12 R d12 , N.R. c12 C(O)R b12 , N.R. c12 C(O)NR c12 R d12 , N.R. c12 C(O)OR a12 , C(=NR e12 )NR c12 R d12 , N.R. c12 C(=NR e12 )NR c12 R d12 , S(O)R b12 , S(O)NR c12 R d12 , S(O) 2 R b12 , N.R. c12 S(O) 2 R b12 , S(O) 2 NR c12 R d12 and oxo, R a12 , R b12 , R c12 and R d12 are independently H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Phenyl, C 3~7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1~3 Alkyl, 5-6 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-7 membered heterocycloalkyl-C 1~3 alkyl; R a12 , R b12 , R c12 , and R d12 forming C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Phenyl, C3~7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1~3 Alkyl, 5-6 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-7 membered heterocycloalkyl-C 1~3 Each alkyl is independently OH, CN, amino, NH(C 1~6 alkyl), N(C 1~6 Alkyl) 2 , Haro, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 1~6 optionally substituted with 1, 2, or 3 substituents selected from haloalkoxy, and oxo; Alternatively, R bonded to the same N atom c12 and R d12 together with the N atom to which they are both attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl group or a 5-membered heteroaryl group, each of which is unsubstituted or independently selected from OH, CN, amino, NH(C 1~6 alkyl), N(C 1~6 Alkyl) 2 , Haro, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 1~6 substituted with 1, 2, or 3 substituents selected from haloalkoxy, and oxo; R e11 and R e12 are each independently H, CN, or NO 2 and Cy 2A is unsubstituted or substituted C 6~10 aryl or unsubstituted or substituted 5-10 membered heteroaryl; Cy 2A The ring atoms of the 5- to 10-membered heteroaryl that forms the ring are carbon atoms and one, two, or three heteroatoms selected from O, N, and S, and Cy 2ASubstitution C to form 6~10 The aryl or substituted 5- to 10-membered heteroaryl is each independently R Cy2A , halogen, C 1~6 Haloalkyl, CN, OR a21 , S.R. a21 , C(O)R b21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21 , O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)R b21 , N.R. c21 C(O)NR c21 R d21 , N.R. c21 C(O)OR a21 , C(=NR e21 )NR c21 R d21 , C(=NOR a21 )NR c21 R d21 , C(=NOC(O)R b21 )NR c21 R d21 , C(=NR e21 )NR c21 C(O)OR a21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 R d21 and oxo; Each R Cy2A is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy2A The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy2A Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a21 , S.R. a21 , C(O)R b21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21 , O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)R b21 , N.R. c21 C(O)NR c21 R d21 , N.R. c21 C(O)OR a21 , C(=NR e21 )NR c21 R d21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 R d21 and oxo; R Cy2A Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a21 , S.R. a21 , C(O)R b21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21 , O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)R b21 , N.R. c21 C(O)NR c21 R d21 , N.R. c21 C(O)OR a21 , C(=NR e21 )NR c21 R d21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 R d21 and oxo; R 21 is H or C 1~6 Alkyl, C 6~10 Aryl-C 1~6 Alkyl or 5-10 membered heteroaryl-C 1~6 is alkyl, R 21 Forming, C 1~6 Alkyl is unsubstituted or independently selected from halogen, CN, OR a21 , S.R. a21 , C(O)Rb21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21 , O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)R b21 , N.R. c21 C(O)NR c21 R d21 , N.R. c21 C(O)OR a21 , C(=NR e21 )NR c21 R d21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 R d21 and oxo; R 21 Forming, C 6~10 Aryl-C 1~6 Alkyl or 5-10 membered heteroaryl-C 1~6 Alkyl is unsubstituted or independently C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a21 , S.R. a21 , C(O)R b21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21 , O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)Rb21 , N.R. c21 C(O)NR c21 R d21 , N.R. c21 C(O)OR a21 , C(=NR e21 )NR c21 R d21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 R d21 and oxo; R 22 is H or C 1~6 is alkyl, or R 21 and R 22 together with the group to which they are attached form a 4- to 6-membered heterocycloalkyl ring; A 23 is N or NR 23 and A 24 CR 24 ;N or NR 24 and A 26 CR 26 or S, however, A in formula (IIA) 23 , A 24 , and A 26 A 23 , A 24 and A 26 is selected to be a heteroaryl ring, TIFF2024509702000019.tif2128 represents an aromatic ring (standardized) bond, R 23 is H or C1~6 is alkyl, R 24 , H;C 1~6 alkyl or phenyl; R 25 Cy 2B , (CR 25A R 25B ) n25 Cy 2B , (C 1~6 Alkylene)Cy 2B , (C 2~6 Alkenylene)Cy 2B , or (C 2~6 Alkynylene)Cy 2B and R 25 C 1~6 Alkylene, C 2~6 Alkenylene, or C 2~6 The alkynylene moieties are unsubstituted or each independently represents a halogen, CN, OR a21 , S.R. a21 , C(O)R b21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21 , O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)R b21 , N.R. c21 C(O)NR c21 R d21 , N.R. c21 C(O)OR a21 , C(=NR e21 )NR c21 R d21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 Rd21 and oxo; R 26 is H or C 1~6 is alkyl, Each R 25A is H or C 1~6 is alkyl, Each R 25B is H or C 1~6 is alkyl, n25 is 0, 1, or 2; Cy 2B is unsubstituted or substituted C 6~10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3~10 cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; Cy 2B The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming the ring are carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S; Cy 2B Forming the substitution C 6~10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3~10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl each independently represents R Cy2B , halogen, C 1~6 Haloalkyl, CN, OR a21 , S.R. a21 , C(O)R b21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21 , O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)R b21 , N.R. c21 C(O)NR c21 R d21 , N.R. c21 C(O)OR a21 , C(=NRe21 )NR c21 R d21 , C(=NOR a21 )NR c21 R d21 , C(=NOC(O)R b21 )NR c21 R d21 , C(=NR e21 )NR c21 C(O)OR a21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 R d21 and oxo; Each R Cy2B is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy2B The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy2B Forming C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a21 , S.R. a21 , C(O)R b21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21, O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)R b21 , N.R. c21 C(O)NR c21 R d21 , N.R. c21 C(O)OR a21 , C(=NR e21 )NR c21 R d21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 R d21 and oxo; R Cy2B Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a21 , S.R. a21 , C(O)R b21 , C(O)NR c21 R d21 , C(O)OR a21 , O.C.(O)R b21 , O-C(O)NR c21 R d21 , N.R. c21 R d21 , N.R. c21 C(O)R b21 , N.R. c21 C(O)NR c21 R d21 , N.R.c21 C(O)OR a21 , C(=NR e21 )NR c21 R d21 , N.R. c21 C(=NR e21 )NR c21 R d21 , S(O)R b21 , S(O)NR c21 R d21 , S(O) 2 R b21 , N.R. c21 S(O) 2 R b21 , S(O) 2 NR c21 R d21 and oxo; Each is independent, H, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 6~10 Aryl-C 1~3 Alkyl, 5-10 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-10 membered heterocycloalkyl-C 1~3 alkyl; R a21 , R b21 , R c21 , and R d21 forming C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 6~10 Aryl-C 1~3 Alkyl, 5-10 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3Alkyl and 4-10 membered heterocycloalkyl-C 1~3 Each alkyl is independently C 1~6 Alkyl, halo, CN, OR a22 , S.R. a22 , C(O)R b22 , C(O)NR c22 R d22 , C(O)OR a22 , O.C.(O)R b22 , O-C(O)NR c22 R d22 , N.R. c22 R d22 , N.R. c22 C(O)R b22 , N.R. c22 C(O)NR c22 R d22 , N.R. c22 C(O)OR a22 , C(=NR e22 )NR c22 R d22 , N.R. c22 C(=NR e22 )NR c22 R d22 , S(O)R b22 , S(O)NR c22 R d22 , S(O) 2 R b22 , N.R. c22 S(O) 2 R b22 , S(O) 2 NR c22 R d22 and oxo; Alternatively, R bonded to the same N atom c21 and R d21 together with the N atom to which they are both attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl group or a 5-membered heteroaryl group, each of which independently represents C 1~6 Alkyl, halo, CN, OR a22 , S.R. a22 , C(O)R b22 , C(O)NR c22 R d22 , C(O)OR a22, O.C.(O)R b22 , O-C(O)NR c22 R d22 , N.R. c22 R d22 , N.R. c22 C(O)R b22 , N.R. c22 C(O)NR c22 R d22 , N.R. c22 C(O)OR a22 , C(=NR e22 )NR c22 R d22 , N.R. c22 C(=NR e22 )NR c22 R d22 , S(O)R b22 , S(O)NR c22 R d22 , S(O) 2 R b22 , N.R. c22 S(O) 2 R b22 , S(O) 2 NR c22 R d22 and oxo, R a22 , R b22 , R c22 , and R d22 are independently H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Phenyl, C 3~7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1~3 Alkyl, 5-6 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-7 membered heterocycloalkyl-C 1~3 alkyl; R a22 , R b22 , R c22 , and R d22 forming C 1~6 Alkyl, C1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Phenyl, C 3~7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1~3 Alkyl, 5-6 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-7 membered heterocycloalkyl-C 1~3 Each alkyl is independently OH, CN, amino, NH(C 1~6 alkyl), N(C 1~6 Alkyl) 2 , Haro, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 1~6 optionally substituted with 1, 2, or 3 substituents selected from haloalkoxy, and oxo; Alternatively, R bonded to the same N atom c22 and R d22 together with the N atom to which they are both attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl group or a 5-membered heteroaryl group, each of which is unsubstituted or substituted with OH, CN, amino, NH(C 1~6 alkyl), N(C 1~6 Alkyl) 2 , Haro, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 1~6 substituted with 1, 2, or 3 substituents selected from haloalkoxy, and oxo; R e21 and R e22 are each independently H, CN, or NO 2 and Cy 3A is unsubstituted or substituted C 6~10 aryl or unsubstituted or substituted 5-10 membered heteroaryl; Cy 3AThe ring atoms of the 5- to 10-membered heteroaryl that forms the ring are carbon atoms and one, two, or three heteroatoms selected from O, N, and S, and Cy 3A Substitution C to form 6~10 The aryl or substituted 5- to 10-membered heteroaryl is independently R Cy3A , halogen, C 1~6 Haloalkyl, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , C(=NOR a31 )NR c31 R d31 , C(=NOC(O)R b31 )NR c31 R d31 , C(=NR e31 )NR c31 C(O)OR a31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; Each RCy3A is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy3A The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy3A Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31and oxo; R Cy3A Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; R 31 is H or C 1~6 Alkyl, C 6~10 Aryl-C 1~6 Alkyl or 5-10 membered heteroaryl-C 1~6is alkyl, R 31 Forming C 1~6 Alkyl is unsubstituted or independently selected from halogen, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; R 31 Forming, C 6~10 Aryl-C 1~6 Alkyl or 5-10 membered heteroaryl-C 1~6 Alkyl is unsubstituted or independently C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31, O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; R 32 is H or C 1~6 is alkyl, or R 31 and R 32 together with the group to which they are attached form a 4- to 6-membered heterocycloalkyl ring; R 33 Cy 3B , (CR 33A R 33B ) n33 Cy 3B , (C 1~6 Alkylene)Cy 3B , (C 2~6 Alkenylene)Cy 3B , or (C 2~6 Alkynylene)Cy 3B and R 35 C 1~6 Alkylene, C 2~6 Alkenylene, or C 2~6The alkynylene moieties are unsubstituted or each independently selected from halogen, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; Each R 33A are independently H or C 1~6 is alkyl, Each R 33B are independently H or C 1~6 is alkyl, or Alternatively, R attached to the same carbon atom 33A and R 33B is any other R 33A and R 33B Independently and together with the group -(CH 2 ) 2-5- thereby forming a 3- to 6-membered cycloalkyl ring, n33 is 0, 1, 2, or 3; Cy 3B is unsubstituted or substituted C 6~10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3~10 cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; Cy 3B The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming the ring are carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S; Cy 3B Forming the substitution C 6~10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3~10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl each independently represent R Cy3B , halogen, C 1~6 Haloalkyl, CN, OR a31 , S Ra31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , C(=NOR a31 )NR c31 R d31 , C(=NOC(O)R b31 )NR c31 R d31 , C(=NR e31 )NR c31 C(O)OR a31 , N.R. c31 C(=NR e31 )NRc31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; Each R Cy3B is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy3B The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy3B Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R.c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; R Cy3B Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; R 34 H and C 1~6 alkyl; R 35 is H, unsubstituted or substituted C 1~6 Alkyl, and Cy 3C Selected from R 35 Substitution C to form 1~6 Alkyl is Cy 3C , halogen, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo, with the proviso that R 35 One or less of the substituents of3C and Cy 3C is unsubstituted or substituted C 6~10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3~10 cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; Cy 3C The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming the ring are carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S; Cy 3C Substitution C to form 6~10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3~10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl each independently represents R Cy3C , halogen, C 1~6 Haloalkyl, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , C(=NOR a31 )NR c31 R d31 , C(=NOC(O)R b31 )NR c31 R d31 , C(=NR e31 )NR c31 C(O)OR a31 , N.R. c31 C(=NR e31 )NR c31 Rd31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; Each R Cy3C is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy3C The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy3C Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NRe31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2 R b31 , S(O) 2 NR c31 R d31 and oxo; R Cy3C Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a31 , S.R. a31 , C(O)R b31 , C(O)NR c31 R d31 , C(O)OR a31 , O.C.(O)R b31 , O-C(O)NR c31 R d31 , N.R. c31 R d31 , N.R. c31 C(O)R b31 , N.R. c31 C(O)NR c31 R d31 , N.R. c31 C(O)OR a31 , C(=NR e31 )NR c31 R d31 , N.R. c31 C(=NR e31 )NR c31 R d31 , S(O)R b31 , S(O)NR c31 R d31 , S(O) 2 R b31 , N.R. c31 S(O) 2R b31 , S(O) 2 NR c31 R d31 and oxo; R 36 H and C 1~6 alkyl; R a31 , R b31 , R c31 and R d31 are independently H, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 6~10 Aryl-C 1~3 Alkyl, 5-10 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-10 membered heterocycloalkyl-C 1~3 alkyl; R a31 , R b31 , R c31 , and R d31 forming C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 6~10 Aryl-C 1~3 Alkyl, 5-10 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-10 membered heterocycloalkyl-C 1~3 Each alkyl is independently C 1~6 Alkyl, halo, CN, OR a32 , S.R. a32 , C(O)R b32 , C(O)NR c32 R d32, C(O)OR a32 , O.C.(O)R b32 , O-C(O)NR c32 R d32 , N.R. c32 R d32 , N.R. c32 C(O)R b32 , N.R. c32 C(O)NR c32 R d32 , N.R. c32 C(O)OR a32 , C(=NR e32 )NR c32 R d32 , N.R. c32 C(=NR e32 )NR c32 R d32 , S(O)R b32 , S(O)NR c32 R d32 , S(O) 2 R b32 , N.R. c32 S(O) 2 R b32 , S(O) 2 NR c32 R d32 and oxo; Alternatively, R bonded to the same N atom c31 and R d31 together with the N atom to which they are both attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl group or a 5-membered heteroaryl group, each of which independently represents C 1~6 Alkyl, halo, CN, OR a32 , S.R. a32 , C(O)R b32 , C(O)NR c32 R d32 , C(O)OR a32 , O.C.(O)R b32 , O-C(O)NR c32 R d32 , N.R. c32 R d32 , N.R. c32 C(O)R b32 , N.R. c32 C(O)NR c32 R d32 , N.R.c32 C(O)OR a32 , C(=NR e32 )NR c32 R d32 , N.R. c32 C(=NR e32 )NR c32 R d32 , S(O)R b32 , S(O)NR c32 R d32 , S(O) 2 R b32 , N.R. c32 S(O) 2 R b32 , S(O) 2 NR c32 R d32 and oxo, R a32 , R b32 , R c32 , and R d32 are independent of each other, H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Phenyl, C 3~7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1~3 Alkyl, 5-6 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-7 membered heterocycloalkyl-C 1~3 alkyl; R a32 , R b32 , R c32 , and R d32 forming C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Phenyl, C 3~7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1~3 Alkyl, 5-6 membered heteroaryl-C 1~3 Alkyl, C3~7 Cycloalkyl-C 1~3 Alkyl and 4-7 membered heterocycloalkyl-C 1~3 Each alkyl is independently OH, CN, amino, NH(C 1~6 alkyl), N(C 1~6 Alkyl) 2 , Haro, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 1~6 substituted with 1, 2, or 3 substituents selected from haloalkoxy, and oxo; Alternatively, R bonded to the same N atom c32 and R d32 together with the N atom to which they are both attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl group or a 5-membered heteroaryl group, each of which is unsubstituted or independently selected from OH, CN, amino, NH(C 1~6 alkyl), N(C 1~6 Alkyl) 2 , Haro, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 1~6 substituted with 1, 2, or 3 substituents selected from haloalkoxy, and oxo; R e31 and R e32 are each independently H, CN, or NO 2 and Cy 4A is unsubstituted or substituted C 6~10 aryl or unsubstituted or substituted 5-10 membered heteroaryl; Cy 4A The ring atoms of the 5- to 10-membered heteroaryl that forms the ring are carbon atoms and one, two, or three heteroatoms selected from O, N, and S, and Cy 4A Substitution C to form 6~10 The aryl or substituted 5- to 10-membered heteroaryl is each independently R Cy4A , halogen, C 1~6 Haloalkyl, CN, OR a41 , S.R.a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , C(=NOR a41 )NR c41 R d41 , C(=NOC(O)R b41 )NR c41 R d41 , C(=NR e41 )NR c41 C(O)OR a41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; Each R Cy4A is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy4AThe ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy4A Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; R Cy4A Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; R 41 is H or C 1~6 Alkyl, C 6~10 Aryl-C 1~6 Alkyl or 5-10 membered heteroaryl-C 1~6 is alkyl, R 41 Forming C 1~6 Alkyl is unsubstituted or independently selected from halogen, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41, O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; R 41 Forming C 6~10 Aryl-C 1~6 Alkyl or 5-10 membered heteroaryl-C 1~6 Alkyl is unsubstituted or independently C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)ORa41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; R 42 , H, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, or Cy 4B and R 42 Forming, C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Each alkynyl is unsubstituted or 4B , halogen, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)Rb41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo, provided that not more than one of the substituents is Cy. 4B and Cy 4B is unsubstituted or substituted C 6~10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3~10 cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; Cy 4B The ring atoms of the 5- to 10-membered heteroaryl or unsubstituted or substituted 4- to 10-membered heterocycloalkyl forming the ring are carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S, and Cy 4B Forming the substitution C 6~10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3~10 Cycloalkyl or 4-10 membered heterocycloalkyl each independently represents R Cy4B , halogen, C 1~6 Haloalkyl, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 Rd41 , C(=NOR a41 )NR c41 R d41 , C(=NOC(O)R b41 )NR c41 R d41 , C(=NR e41 )NR c41 C(O)OR a41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; Each R Cy4B is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy4B The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy4B Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 Rd41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo, each R Cy4B Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)ORa41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; Or, R 41 and R 42 are the atoms to which they are attached and R 41 and R 42 together with the nitrogen atom linked to the atom to which R is bonded, form a 4- to 7-membered heterocycloalkyl ring, Cy4B , halogen, C 1~6 Haloalkyl, CN, OR a41 , S Ra41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , C(=NOR a41 )NR c41 R d41 , C(=NOC(O)R b41 )NR c41 R d41 , C(=NRe41 )NR c41 C(O)OR a41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo, R 43 , H, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, or Cy 4C and R 43 Forming, C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Each alkynyl is unsubstituted or each independently is Cy. 4C , halogen, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41, S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo, with the proviso that R 43 Forming, C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Up to one substituent on an alkynyl group may be Cy. 4C and Cy 4C is unsubstituted or substituted C 6~10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3~10 cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; Cy 4B The ring atoms of the 5- to 10-membered heteroaryl or unsubstituted or substituted 4- to 10-membered heterocycloalkyl forming the ring are carbon atoms and 1, 2, or 3 heteroatoms selected from O, N, and S, and Cy 4C Substitution C to form 6~10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3~10 Cycloalkyl or 4-10 membered heterocycloalkyl each independently represent R Cy4C , halogen, C 1~6 Haloalkyl, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)Rb41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , C(=NOR a41 )NR c41 R d41 , C(=NOC(O)R b41 )NR c41 R d41 , C(=NR e41 )NR c41 C(O)OR a41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; Each R Cy4C is independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 cycloalkyl, and 4- to 10-membered heterocycloalkyl; R Cy4C The ring atoms of the 5- to 10-membered heteroaryl or 4- to 10-membered heterocycloalkyl forming R Cy4C Each C 1~6 Alkyl, C 2~6 Alkenyl, or C 2~6 Alkynyl is independently unsubstituted or independently selected from halogen, CN, OR a41 , S.R.a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41 R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo, each R Cy4A Each C 6~10 Aryl, 5-10 membered heteroaryl, C 3~10 Cycloalkyl, and 4-10 membered heterocycloalkyl are independently unsubstituted or independently selected from halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, CN, OR a41 , S.R. a41 , C(O)R b41 , C(O)NR c41 R d41 , C(O)OR a41 , O.C.(O)R b41 , O-C(O)NR c41 R d41 , N.R. c41R d41 , N.R. c41 C(O)R b41 , N.R. c41 C(O)NR c41 R d41 , N.R. c41 C(O)OR a41 , C(=NR e41 )NR c41 R d41 , N.R. c41 C(=NR e41 )NR c41 R d41 , S(O)R b41 , S(O)NR c41 R d41 , S(O) 2 R b41 , N.R. c41 S(O) 2 R b41 , S(O) 2 NR c41 R d41 and oxo; R a41 , R b41 , R c41 , and R d41 are independently H, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 6~10 Aryl-C 1~3 Alkyl, 5-10 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-10 membered heterocycloalkyl-C 1~3 alkyl; R a41 , R b41 , R c41 , and R d41 Forming C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~7Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 6~10 Aryl-C 1~3 Alkyl, 5-10 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-10 membered heterocycloalkyl-C 1~3 Each alkyl is independently C 1~6 Alkyl, halo, CN, OR a42 , S.R. a42 , C(O)R b42 , C(O)NR c42 R d42 , C(O)OR a42 , O.C.(O)R b42 , O-C(O)NR c42 R d42 , N.R. c42 R d42 , N.R. c42 C(O)R b42 , N.R. c42 C(O)NR c42 R d42 , N.R. c42 C(O)OR a42 , C(=NR e42 )NR c42 R d42 , N.R. c42 C(=NR e42 )NR c42 R d42 , S(O)R b42 , S(O)NR c42 R d42 , S(O) 2 R b42 , N.R. c42 S(O) 2 R b42 , S(O) 2 NR c42 R d42 and oxo; Alternatively, R bonded to the same N atom c41 and R d41together with the N atom to which they are both attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl group or a 5-membered heteroaryl group, each of which independently represents C 1~6 Alkyl, halo, CN, OR a42 , S.R. a42 , C(O)R b42 , C(O)NR c42 R d42 , C(O)OR a42 , O.C.(O)R b42 , O-C(O)NR c42 R d42 , N.R. c42 R d42 , N.R. c42 C(O)R b42 , N.R. c42 C(O)NR c42 R d42 , N.R. c42 C(O)OR a42 , C(=NR e42 )NR c42 R d42 , N.R. c42 C(=NR e42 )NR c42 R d42 , S(O)R b42 , S(O)NR c42 R d42 , S(O) 2 R b42 , N.R. c42 S(O) 2 R b42 , S(O) 2 NR c42 R d42 and oxo, R a42 , R b42 , R c42 , and R d42 are independently H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Phenyl, C 3~7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1~3Alkyl, 5-6 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-7 membered heterocycloalkyl-C 1~3 alkyl; R a42 , R b42 , R c42 , and R d42 forming C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Phenyl, C 3~7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1~3 Alkyl, 5-6 membered heteroaryl-C 1~3 Alkyl, C 3~7 Cycloalkyl-C 1~3 Alkyl and 4-7 membered heterocycloalkyl-C 1~3 Each alkyl is independently OH, CN, amino, NH(C 1~6 alkyl), N(C 1~6 Alkyl) 2 , Haro, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 1~6 optionally substituted with 1, 2, or 3 substituents selected from haloalkoxy, and oxo; Alternatively, R bonded to the same N atom c42 and R d42 together with the N atom to which they are both attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl group or a 5-membered heteroaryl group, each of which is unsubstituted or substituted with OH, CN, amino, NH(C 1~6 alkyl), N(C 1~6 Alkyl) 2 , Haro, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 1~6substituted with 1, 2, or 3 substituents selected from haloalkoxy, and oxo; R e41 and R e42 are each independently H, CN, or NO 2 It is.

[0153] In some embodiments, the small molecule has the formula (VA) or (VB): TIFF2024509702000020.tif49128 or a salt thereof, During the ceremony, A 1 -(C=NH)-, -(C=NOR a )-, -[C=NO(C=O)R a ]-, -[C=N[O(C=O)ZR b ]}-, fused 5- or 6-membered heterocyclyl, and fused 5- or 6-membered heteroaryl; A 1 When is -(C=NH)-, Y 1 is -NH 2 , -NH(C=O)R a , and -NH(C=O)ZR b is selected from the group consisting of A 1 -(C=NOR a )-, -[C=NO(C=O)R a ]-, or -{C=N[O(C=O)ZR b ]}-, then Y 1 Ha-NH 2 and A 1 When Y is a fused heterocyclyl or heteroaryl, 1 Ha-NH 2 or halo, A 1 m further R 1 is substituted with a group, Each R a and R b is independently 1 ~C 6 Alkyl, C 3 ~C 10Cycloalkyl, C 6 ~C 10 Aryl, and C 7 ~C 12 arylalkyl; R a is C 1 ~C 6 Alkyl, Hydroxyl, Hydroxyl (C 1 ~C 6 Alkyl), C 1 ~C 6 Alkoxy, C 2 ~C 9 Alkoxyalkyl, amino, C 1 ~C 6 or alternatively, R a and R b are linked to form a heterocyclyl ring, and m substituents are C 1 ~C 6 Alkyl, Hydroxyl, C 1 ~C 6 alkoxy, and halo; Each Z is independently selected from the group consisting of O and S; A 2 is C 3 ~C 6 Heteroaryl, C 6 Aryl, and C 2 ~C 6 is a member selected from the group consisting of alkyl, A 2 C 3 ~C 6 When Y is heteroaryl, 2 is -NH 2 , -CH 2 NH 2 , chloro, -(C=NH)NH 2 , -(C=NH)NH(C=O)R a , -(C=NH)NH(C=O)ZR b , -(C=NOR a )NH 2 , -[C=NO(C=O)R a ]NH 2 , and -{C=N[O(C=O)ZRb ]}NH 2 A is selected from the group consisting of 2 m further R 1 is substituted with a group, A 2 C 6 When Y is aryl, 2 is selected from the group consisting of aminomethyl, hydroxy, and halo; A 2 m further R 1 is substituted with a group, A 2 C 2 ~C 6 When it is alkyl, Y 2 is -NH(C=NH)NH 2 , -NH(C=NH)NH(C=O)R a , and -NH(C=NH)NH(C=O)ZR b is selected from the group consisting of Each R 1 is independently 1 ~C 6 Alkyl, Hydroxyl, C 1 ~C 6 Alkoxy, Amino, C 1 ~C 6 is a member selected from the group consisting of alkylamino, alkylamino, and halo; each m and n is an integer independently selected from 0 to 3; L is -(O) p -(C(R 2a )(R 2b )) q - and Each R 2a or R 2b is independently a member selected from the group consisting of hydrogen and fluoro; p is an integer from 0 to 1; q is an integer from 1 to 2, R 3 is hydrogen, C 1 ~C 6 Alkyl, C 1 ~C 6 Fluoroalkyl, and carboxy (C 1 ~C 6or alternatively, R 3 and R 4 are linked to form an azetidine, pyrrolidine, or piperidine ring, R 4 is hydrogen and C 1 ~C 6 or alternatively, R 4 and R 3 are linked to form an azetidine, pyrrolidine, or piperidine ring, R 5 is C 3 ~C 7 Cycloalkyl, C 4 ~C 8 Cycloalkyl, alkyl, heteroaryl, and C 7 ~C 12 Arylalkyl or 0 to 3 R 13 or alternatively, R 5 and R 6 are combined to form 0 to 3 R 13 Forming a substituted heterocyclic ring, R 6 is hydrogen, C 1 ~C 6 Alkyl, C 3 ~C 7 Cycloalkyl, carboxy (C 1 ~C 6 Alkyl), C 7 ~C 12 Arylalkyl or 0 to 3 R 13 Substituted heteroaryl alkyl, amino (C 1 ~C 8 alkyl); and amide (C 1 ~C 8 or alternatively, R 6 and R 5 are combined to form 0 to 3 R 13 Forming a substituted heterocyclic ring, Each R 13is independently 1 ~C 6 Alkyl, C 6 ~C 10 Aryl, (C 6 ~C 10 Aryl)C 1 ~C 6 Alkyl, carboxy (C 1 ~C 6 alkyloxy), heteroaryl, (C 6 ~C 10 Heteroaryl)C 1 ~C 6 Alkyl, heterocyclyl, hydroxyl, hydroxyl (C 1 ~C 6 Alkyl), C 1 ~C 6 Alkoxy, C 2 ~C 9 Alkoxyalkyl, amino, C 1 ~C 6 Amide, C 1 ~C 6 or alternatively, two R 13 The groups are bonded to form a condensed C 6 ~C 10 Aryl, C 6 ~C 10 Heteroaryl, or C 5 ~C 7 It forms a cycloalkyl ring.

[0154] In some embodiments, the small molecule has formula (VIA) or (VIB): TIFF2024509702000021.tif42128, or a salt thereof; During the ceremony, A 1 -(C=NH)-, -(C=NOR a )-, -[C=NO(C=O)R a ]-, -[C=N[O(C=O)ZR b ]-, fused 5- or 6-membered heterocyclyl, and fused 5- or 6-membered heteroaryl; A1 When is -(C=NH)-, Y 1 is -NH 2 , -NH(C=O)R a , and -NH(C=O)ZR b is selected from the group consisting of A 1 -(C=NOR a )-, -[C=NO(C=O)R a ]-, or -{C=N[O(C=O)ZR b ]}-, then Y 1 Ha-NH 2 and A 1 When Y is a fused heterocyclyl or heteroaryl, 1 Ha-NH 2 or halo, A 1 is m further R 1 is substituted with a group, Each R a and R b is independently 1 ~C 6 Alkyl, C 3 ~C 10 Cycloalkyl, C 6 ~C 10 Aryl, and C 7 ~C 12 arylalkyl; R a is C 1 ~C 6 Alkyl, Hydroxyl, Hydroxyl (C 1 ~C 6 Alkyl), C 1 ~C 6 Alkoxy, C 2 ~C 9 Alkoxyalkyl, amino, C 1 ~C 6 or alternatively, R a and R b are linked to form a heterocyclyl ring, and m substituents are C 1 ~C 6 Alkyl, Hydroxyl, C 1 ~C6 alkoxy, and halo; Each Z is independently selected from the group consisting of O and S; A 2 is C 3 ~C 6 Heteroaryl and C 2 ~C 6 is a member selected from the group consisting of alkyl, A 2 C 3 ~C 6 When Y is heteroaryl, 2 is -NH 2 , -CH 2 NH 2 , chloro, -(C=NH)NH 2 , -(C=NH)NH(C=O)R a , -(C=NH)NH(C=O)ZR b , -(C=NOR a )NH 2 , -[C=NO(C=O)R a ]NH 2 , and -{C=N[O(C=O)ZR b ]}NH 2 A is selected from the group consisting of 2 is m further R 1 is substituted with a group, A 2 C 2 ~C 6 When it is alkyl, Y 2 is -NH(C=NH)NH 2 , -NH(C=NH)NH(C=O)R a , and -NH(C=NH)NH(C=O)ZR b is selected from the group consisting of Each R 1 is independently 1 ~C 6 Alkyl, Hydroxyl, C 1 ~C 6 Alkoxy, Amino, C 1 ~C 6 is a member selected from the group consisting of alkylamino, alkylamino, and halo; Each m and n is independently an integer selected from 0 to 3; X and X 2 Each is NR 8 , C.H., and C.R. 10 is a member selected from the group consisting of Each R 8 are independently hydrogen and C 1 ~C 6 is a member selected from the group consisting of alkyl, Each R 10 is independently 1 ~C 6 Alkyl, 0 to 3 R 13 Substituted Heteroaryl or C 6 ~C 10 Aryl, Hydroxyl, Hydroxyl (C 1 ~C 6 Alkyl), C 1 ~C 6 Alkoxy, C 2 ~C 9 Alkoxyalkyl, amino, C 1 ~C 6 or alternatively, two R 10 The groups are bonded to form a condensed C 6 Aryl, heteroaryl, or 0 to 3 R 13 Substituted C 5 ~C 7 forming a cycloalkyl ring, r is an integer from 0 to 4; Each R 13 is independently 1 ~C 6 Alkyl, C 6 ~C 10 Aryl, Carboxy (C 1 ~C 6 alkyloxy), heteroaryl, heterocyclyl, hydroxyl, hydroxyl (C 1 ~C 6 Alkyl), C 1 ~C 6 Alkoxy, C 2 ~C 9Alkoxyalkyl, amino, C 1 ~C 6 Amide, C 1 ~C 6 or alternatively, two R 13 The groups are bonded to form a condensed C 6 ~C 10 Aryl, C 6 ~C 10 Heteroaryl, or C 5 ~C 7 It forms a cycloalkyl ring.

[0155] In certain embodiments, the small molecule has formula (VIIA) or (VIIB): TIFF2024509702000022.tif73128, or a salt thereof; During the ceremony, A 1 -(C=NH)-, -(C=NOR a )-, -[C=NO(C=O)R a ]-, -[C=N[O(C=O)ZR b ]}-, fused 5- or 6-membered heterocyclyl, and fused 5- or 6-membered heteroaryl; A 1 When is -(C=NH)-, Y 1 is -NH 2 , -NH(C=O)R a , and -NH(C=O)ZR b is selected from the group consisting of A 1 -(C=NOR a )-, -[C=NO(C=O)R a ]-, or -{C=N[O(C=O)ZR b ]}-, then Y 1 Ha-NH 2 and A 1 When Y is a fused heterocyclyl or heteroaryl, 1 Ha-NH 2 or halo, A 1is m further R 1 is substituted with a group, Each R a and R b is independently 1 ~C 6 Alkyl, C 3 ~C 10 Cycloalkyl, C 6 ~C 10 Aryl, and C 7 ~C 12 arylalkyl; R a is C 1 ~C 6 Alkyl, Hydroxyl, Hydroxyl (C 1 ~C 6 Alkyl), C 1 ~C 6 Alkoxy, C 2 ~C 9 Alkoxyalkyl, amino, C 1 ~C 6 or alternatively, R a and R b are linked to form a heterocyclyl ring, and m substituents are C 1 ~C 6 Alkyl, Hydroxyl, C 1 ~C 6 alkoxy, and halo; Each Z is independently selected from the group consisting of O and S; A 2 is C 3 ~C 6 Heteroaryl and C 2 ~C 6 is a member selected from the group consisting of alkyl, A 2 C 3 ~C 6 When Y is heteroaryl, 2 is -NH 2 , -CH 2 NH 2 , chloro, -(C=NH)NH 2 , -(C=NH)NH(C=O)R a, -(C=NH)NH(C=O)ZR b , -(C=NOR a )NH 2 , -[C=NO(C=O)R a ]NH 2 , and -{C=N[O(C=O)ZR b ]}NH 2 A is selected from the group consisting of 2 is m further R 1 is substituted with a group, A 2 C 2 ~C 6 When it is alkyl, Y 2 is -NH(C=NH)NH 2 , -NH(C=NH)NH(C=O)R a , and -NH(C=NH)NH(C=O)ZR b is selected from the group consisting of Each R 1 is independently 1 ~C 6 Alkyl, Hydroxyl, C 1 ~C 6 Alkoxy, Amino, C 1 ~C 6 is a member selected from the group consisting of alkylamino, alkylamino, and halo; Each m and n is independently an integer selected from 0 to 3; L is -(O)p-(C(R 2a )(R 2b ))q-, Each R 2a or R 2b is independently a member selected from the group consisting of hydrogen and fluoro; p is an integer from 0 to 1; q is an integer from 1 to 2, R 3 is hydrogen, C 1 ~C 6 Alkyl, and carboxy (C 1 ~C 6 alkyl), Each R 11 is independently1 ~C 6 Alkyl, Hydroxyl, C 1 ~C 6 Alkoxy, Amino, C 1 ~C 6 Alkylamino, halo, and (R 14 )(R 14 )N(CO)—, or alternatively, two R 11 The groups are bonded to form a condensed C 6 Aryl, heteroaryl, or 0 to 3 R 13 Substituted C 5 ~C 7 forming a cycloalkyl ring, r is an integer from 0 to 4; Each Z is independently O and NR 8 is a member selected from the group consisting of Each R 8 are independently hydrogen and C 1 ~C 6 is a member selected from the group consisting of alkyl, Each R 12 are independently hydrogen, C 1 ~C 6 Alkyl and 0 to 3 R 13 Substituted C 7 ~C 14 arylalkyl; Each R 13 is independently 1 ~C 6 Alkyl, Hydroxyl, Hydroxyl (C 1 ~C 6 Alkyl), C 1 ~C 6 Alkoxy, C 2 ~C 9 Alkoxyalkyl, amino, C 1 ~C 6 or alternatively, two R 13 The groups are bonded to form a condensed C 6 Aryl, heteroaryl, or C5 ~C 7 forming a cycloalkyl ring, Each R 14 are independently hydrogen, C 1 ~C 6 Alkyl, C 3 ~C 7 Cycloalkyl, C 4 ~C 8 Cycloalkylalkyl, C 7 ~C 14 Arylalkyl, and heteroaryl (C 1 ~C 6 or alternatively, two R 13 The groups are joined to form a fused heterocyclyl ring.

[0156] In some embodiments, the small molecule has the following structure: TIFF2024509702000023.tif37128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i , or R 2j are independently hydrogen, halo, C(=O)OR 5 ,OC(=O)R 5 , hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminylalkyl, carboxyalkyl, NR 5 R 6 , C(=O)NR 5 R 6 , N(R 5 )C(=O)R 6 , N.R.5 C(=O)NR 6 , S(O) t , S.R. 5 , Nitro, N(R 5 )C(O)OR 6 , C(=NR 5 )NR 6 R 7 , N(R 5 )C(=NR 6 )NR 7 R 8 , S(O)R 5 , S(O)NR 5 R 6 , S(O) 2 R 5 , N(R 5 )S(O) 2 R 6 , S(O) 2 NR 5 R 6 , aryl, heteroaryl, heterocyclyl, cycloalkyl, and oxo, where R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i , or R 2j At least one occurrence of is not hydrogen, R 3 is NR 3a R 3b and R 3a and R 3b are each independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH 2 ) n C(=O)OR 6 , or (CH 2 ) n P(=O)(OR 6 ) 2 and Or, R 3a and R 3btogether with the nitrogen to which they are attached form an optionally substituted 4- to 7-membered heteroaryl or an optionally substituted 4- to 7-membered heterocyclyl; Or, R 3a and R 4 each taken together with the nitrogen and carbon to which they are attached forms an optionally substituted 4- to 7-membered heterocyclyl; R 4 is, when n is 2, 3, 4, 5, or 6, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocyclyl; or R 4 is a substituted or unsubstituted monocyclic heteroaryl or substituted or unsubstituted heterocyclyl when n is 0 or 1; R 5 , R 6 , R 7 , and R 8 is, independently at each occurrence, hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl; X is a direct bond, -CR 2e R 2f - or -CR 2e R 2f -CR 2g R 2h - and Y is a direct bond or -CR 2i R 2j - and n is an integer from 0 to 6, t is 1 to 3.

[0157] In some embodiments, the small molecule has the following structure: TIFF2024509702000024.tif37128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 1is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i , or R 2j are independently hydrogen, halo, OR 5 , C(=O)OR 5 ,OC(=O)R 5 , hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminylalkyl, carboxyalkyl, NR 5 R 6 , C(=O)NR 5 R 6 , N(R 5 )C(=O)R 6 , N.R. 5 C(=O)NR 6 , S(O) t , S.R. 5 , Nitro, N(R 5 )C(O)OR 6 , C(=NR 5 )NR 6 R 7 , N(R 5 )C(=NR 6 )NR 7 R 8 , S(O)R 5 , S(O)NR 5 R 6 , S(O) 2 R 5 , N(R 5 )S(O) 2 R 6 , S(O) 2 NR 5 R 6 , aryl, heteroaryl, heterocyclyl, cycloalkyl, and oxo, where R 2a , R 2b , R 2c , R 2d , R 2e , R2f , R 2g , R 2h , R 2i , or R 2j At least one occurrence of is not hydrogen, R 3 is NR 3a R 3b and R 3a and R 3b are each independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, -CH 2 C(C=O)OH, -CH 2 C(═O)Oalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, or cycloalkyl; Or, R 3a and R 3b together with the nitrogen to which they are attached form an optionally substituted 4- to 7-membered heteroaryl or an optionally substituted 4- to 7-membered heterocyclyl; R 4 is, when n is 2, 3, 4, 5, or 6, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 4 is a substituted or unsubstituted monocyclic heteroaryl or substituted or unsubstituted heterocyclyl when n is 0 or 1; R 5 , R 6 , R 7 , and R 8 is, independently at each occurrence, hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl; X is a direct bond, -[C(R 2e )R 2f ]-, or -[C(R 2e )R 2f ]-[C(R 2g )R 2h ]- and Y is a direct bond or -[C(R 2i )R 2j ]- and n is an integer from 0 to 6, t is 1 to 3; however, a)R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i , or R 2j If one occurrence of is OH, then R 1 has the following structure: Does not have TIFF2024509702000025.tif17128, b)R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , or R 2h is -OH, then n is an integer from 2 to 6; c)R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i , or R 2j When one occurrence of is unsubstituted phenyl, R 3a Also 3b Also, the following structure: Does not have TIFF2024509702000026.tif12128.

[0158] In some embodiments, the small molecule has the following structure: TIFF2024509702000027.tif29128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 17is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 18a , R 18b , R 18c , R 18d , R 18e , R 18f , R 18g , R 18h , R 18i , or R 18j are independently hydrogen, halo, -OR 21 , C(=O)OR 21 ,OC(=O)R 21 , hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminylalkyl, carboxyalkyl, NR 21 R 22 , C(=O)NR 21 R 22 , N(R 21 )C(=O)R 22 , N.R. 21 C(=O)NR 22 , S(O) t , S.R. 21 , Nitro, N(R 21 )C(O)OR 22 , C(=NR 21 )NR 22 R 23 , N(R 21 )C(=NR 22 )NR 23 R 24 , S(O)R 21 , S(O)NR 21 R 22 , S(O) 2 R 21 , N(R 21 )S(O) 2 R 22 , S(O) 2 NR 21 R 22 , aryl, heteroaryl, heterocyclyl, cycloalkyl, and oxo, where R 18a , R 18b , R 18c , R 18d , R 18e, R 18f , R 18g , R 18h , R 18i , or R 18j At least one occurrence of is not hydrogen, R 19 is NR 19a R 19b and R 19a and R 19b each independently represents hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH 2 ) n C(=O)OR 5 , or (CH 2 ) n P(=O)(OR 5 ) 2 or Or, R 19a and R 19b together with the nitrogen to which they are attached form an optionally substituted 4- to 7-membered heteroaryl or an optionally substituted 4- to 7-membered heterocyclyl; R 20 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 21 , R 22 , R 23 , and R 24 is, independently at each occurrence, hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl; X is a direct bond, -CR 2e R 2f - or -CR 2e R 2f -CR 2g R 2h - and Y is a direct bond or -CR 2i R 2j - and Z is O or S; m is an integer from 0 to 6; t is 1 to 3.

[0159] In certain specific embodiments, the small molecule has the following structure: TIFF2024509702000028.tif35128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 25 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 26a , R 26b , R 26c , or R 26d are independently hydrogen, halo, -OR 29 , C(=O)OR 29 ,OC(=O)R 29 , hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminylalkyl, carboxyalkyl, NR 29 R 30 , C(=O)NR 29 R 30 , N(R 29 )C(=O)R 30 , N.R. 29 C(=O)NR 30 , S(O) t , S.R. 29 , Nitro, N(R 29 )C(O)OR 30 , C(=NR 29 )NR 30 R 31 , N(R 29 )C(=NR 30 )NR 31 R 32 , S(O)R 29 , S(O)NR 29 R 30 , S(O) 2 R 30 , N(R 29 )S(O) 2 R 30 , S(O)2 NR 29 R 30 , aryl, heteroaryl, heterocyclyl, cycloalkyl, and oxo, where R 26a , R 26b , R 26c , or R 26d At least one occurrence of is not hydrogen, R 27 is NR 27a R 27b and R 27a and R 27b are each independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH 2 ) n C(=O)OR 29 , or (CH 2 ) n P(=O)(OR 29 ) 2 or Or, R 27a and R 27b together with the nitrogen to which they are attached form an optionally substituted 4- to 7-membered heteroaryl or an optionally substituted 4- to 7-membered heterocyclyl; R 28 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 29 , R 30 , R 31 , and R 32 is, independently at each occurrence, hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl; X is a direct bond or -CR 26c R 26d - and p is an integer from 0 to 6; t is 1 to 3.

[0160] In some embodiments, the small molecule has the following structure: TIFF2024509702000029.tif29128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 2 is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, or cycloalkyl; R 3 is hydrogen, alkyl, haloalkyl, or cycloalkyl; Or, R 2 and R 3 each taken together with the carbon and nitrogen to which they are attached forms an optionally substituted 4- to 7-membered heterocyclyl; R 4 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 5a is hydrogen, alkyl, haloalkyl, cycloalkyl, phosphonalkyl, (CH 2 ) n C(=O)OR 6 , C(=O)R 6 , C(=O)OR 6 , or C(=O)NR 6 R 7 and R 5b is an electron pair or an alkyl, R 6 and R 7 is independently, at each occurrence, hydrogen, alkyl, haloalkyl, cycloalkyl, or arylalkyl; R 8is alkyl, haloalkyl, aminylalkyl, substituted or unsubstituted arylalkyl, n is 1, 2, 3, 4, 5, 6, 7, or 8; however, A)R 5a is an alkyl, haloalkyl, cycloalkyl, phosphonalkyl, (CH 2 ) n C(=O)OR 6 , C(=O)R 6 , C(=O)OR 6 or C(=O)NR 6 R 7 or R 1 is a substituted heteroaryl, C(=NH)NHC(=O)OR 8 , C(=NOC(=O)R 8 )NH 2 , C(=NOC(=O)OR 8 )NH 2 , and C(=NOH)NH 2 and is substituted with one or more substituents selected from the group consisting of B)R 2 and R 3 do not form an optionally substituted 4- to 7-membered heterocyclyl together with the carbon and nitrogen to which they are attached, R 5a is alkyl or (CH 2 ) n C(=O)OR 6 If R 1 has the following structure: Does not have TIFF2024509702000030.tif14128.

[0161] In some embodiments, the small molecule has the following structure: TIFF2024509702000031.tif29128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 1is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 2 is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, or cycloalkyl; R 3 is hydrogen, alkyl, haloalkyl, or cycloalkyl; Or, R 2 and R 3 each taken together with the carbon and nitrogen to which they are attached forms an optionally substituted 4- to 7-membered heterocyclyl; R 4 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 5a is hydrogen, alkyl, haloalkyl, cycloalkyl, phosphonalkyl, (CH 2 ) n C(=O)OR 6 , C(=O)R 6 , C(=O)OR 6 or C(=O)NR 6 R 7 and R 5b is an electron pair or an alkyl, R 6 and R 7 is independently, at each occurrence, hydrogen, alkyl, haloalkyl, cycloalkyl, or arylalkyl; R 8 is alkyl, haloalkyl, aminylalkyl, substituted or unsubstituted arylalkyl, n is 1, 2, 3, 4, 5, 6, 7, or 8; however, A)R 5a is an alkyl, haloalkyl, cycloalkyl, phosphonalkyl, (CH 2 ) n C(=O)OR 6 , C(=O)R 6, C(=O)OR 6 , or C(=O)NR 6 R 7 or R 1 is a substituted heteroaryl, C(=NH)NHC(=O)OR 8 , C(=NOC(=O)R 8 )NH 2 , C(=NOC(=O)OR 8 )NH 2 , and C(=NOH)NH 2 and is substituted with one or more substituents selected from the group consisting of B) The compound of structure (I) has the following structure: It is not possible to have one of TIFF2024509702000032.tif175152TIFF2024509702000033.tif99152.

[0162] In some embodiments, the small molecule has the following structure: TIFF2024509702000034.tif39128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 2 is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, or cycloalkyl; R 3 is hydrogen, alkyl, haloalkyl, or cycloalkyl; Or, R 2 and R 3 each taken together with the carbon and nitrogen to which they are attached forms an optionally substituted 4- to 7-membered heterocyclyl; R 4 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R5a and R 5b has, independently in each occurrence, the structure: TIFF2024509702000035.tif20141 or Or, R 5a and R 5b together with the phosphorus atom to which they are attached form an optionally substituted 4- to 7-membered heterocyclyl; R 6a is alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; R 6b is, independently at each occurrence, hydrogen or alkyl; R 7 is, independently at each occurrence, alkyl, haloalkyl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, or heterocyclylalkyl; R 8 is an amino acid side chain, n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0163] In some embodiments, the small molecule has the following structure: TIFF2024509702000036.tif34128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, TIFF2024509702000037.tif2128 represents a double bond or a single bond, R 1 is a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; R 2 is hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, haloalkoxy, or cycloalkyl; R 3 is hydrogen, alkyl, haloalkyl, or cycloalkyl, or R 2 and R3 each taken together with the carbon and nitrogen to which they are attached forms an optionally substituted 4- to 7-membered heterocyclyl; R 4 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; R 5 is hydrogen, alkyl, haloalkyl, cycloalkyl, phosphonalkyl, (CH 2 ) m C(=O)OR 6 , C(=O)R 6 , C(=O)OR 6 , (CH 2 ) m NR 6 S(O) 2 R 7 or C(=O)NR 6 R 7 and R 6 and R 7 is independently, at each occurrence, hydrogen, alkyl, haloalkyl, cycloalkyl, or arylalkyl; L 1 is a direct bond, -CR 8a R 8b -, -S(O) t -, NR 8c or -O-, R 8a and R 8b are each independently hydrogen, alkyl, or R 8a and R 8b which, taken together with the carbon to which they are attached, form an optionally substituted 3- to 6-membered cycloalkyl ring; R 8cis hydrogen, alkyl, haloalkyl, (C=O)alkyl, (C=O)Oalkyl, (C=O)cycloalkyl, (C=O)Ocycloalkyl, (C=O)aryl, (C=O)Oaryl, (C=O)heteroaryl, (C=O)Oheteroaryl, (C=O)heterocyclyl, (C=O)Oheterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl, or substituted or unsubstituted heterocyclylalkyl; n is 1 or 2; m is 1, 2, 3, 4, 5, or 6; t is 0, 1, or 2.

[0164] In some embodiments, the small molecule has the following structure: TIFF2024509702000038.tif34128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is a substituted or unsubstituted heteroaryl; R 2 is a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; R 3 is hydrogen or alkyl, R 4 is heterocyclyl substituted with a substituent selected from the group consisting of alkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted pyridinyl; or R 3 and R 4 each taken together with the nitrogen and carbon to which they are attached forms an optionally substituted 4- to 10-membered heterocyclyl; R 5a is hydrogen or halo, R 5bis hydrogen, alkyl, haloalkyl, (C=O)alkyl, (C=O)Oalkyl, (C=O)cycloalkyl, (C=O)Ocycloalkyl, (C=O)aryl, (C=O)Oaryl, (C=O)heteroaryl, (C=O)Oheteroaryl, (C=O)heterocyclyl, (C=O)Oheterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl, or substituted or unsubstituted heterocyclylalkyl; L 1 is a direct bond, -CH 2 -, -S(O) t -, NR 5b , -O-, -C=C-, or -C≡C-; t is 0, 1, or 2; however, A)R 2 has the following structure: It never has one of TIFF2024509702000039.tif23128, B)R 1 has the following structure: TIFF2024509702000040.tif23142, C)R 2 is unsubstituted phenyl, R 1 has the following structure: You will never have one of TIFF2024509702000041.tif40131.

[0165] In certain more specific embodiments, the small molecule has the following structure: TIFF2024509702000042.tif26128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 6 is a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; R 7 is alkyl, -SR 10 , substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 8 is hydrogen, alkyl, haloalkyl, or cycloalkyl; R 9 is a substituted or unsubstituted arylalkyl, a substituted or unsubstituted heteroarylalkyl, or R 8 and R 9 together with the nitrogen to which they are attached form an optionally substituted 4- to 10-membered heterocyclyl; R 10 is hydrogen, alkyl, haloalkyl, or cycloalkyl; however, A)R 7 is unsubstituted phenyl, 3-((methylsulfonyl)amino)phenyl, 2-methylphenyl, 3-(dimethylamino)phenyl, 3-(methylamino)phenyl, 3-methylphenyl, 3-aminomethylphenyl, 3-aminophenyl, unsubstituted pyridinyl, 3-(methylamino)-2-thienyl, 3,4-diamino-2-thienyl, 3-((methylsulfonyl)amino)-2-thienyl, 3-amino-2-thienyl, 3-amino-5-5(aminocarbonyl)phenyl, or the structure: If you have one of the following files: TIFF2024509702000043.tif139150 R 6 has the following structure: does not have TIFF2024509702000044.tif22128, B)R 7 is unsubstituted phenyl, R 6 has the following structure: Does not have TIFF2024509702000045.tif21128.

[0166] In some embodiments, the small molecule has the following structure: TIFF2024509702000046.tif22128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 11 has the following structure: TIFF2024509702000047.tif40133 R 12 is methyl or halo, R 13 is a substituted or unsubstituted aryl; n is 1 or 2; however, Compounds of structure (III) have the following structure: Does not have TIFF2024509702000048.tif32128.

[0167] In some more specific embodiments, the small molecule has the following structure: TIFF2024509702000049.tif27128, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, During the ceremony, R 14 is a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; R 15 is a substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl; L 2 is a direct bond, -C(=O), or -S(=O)t-; t is 0, 1, or 2.

[0168] Inhibitor of MASP-2 expression In another embodiment of this aspect of the present invention, the MASP-2 inhibitor is a MASP-2 expression inhibitor capable of inhibiting MASP-2-dependent complement activation. In implementing this aspect of the present invention, representative MASP-2 expression inhibitors include MASP-2 antisense nucleic acid molecules (e.g., antisense mRNA, antisense DNA, or antisense oligonucleotides), MASP-2 ribozymes, and MASP-2 RNAi molecules.

[0169] Antisense RNA and DNA molecules act to directly block the translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing the translation of MASP-2 protein. Antisense nucleic acid molecules may be constructed in a number of different ways, provided that they are capable of interfering with the expression of MASP-2. For example, antisense nucleic acid molecules may be constructed by inverting the coding region (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) relative to the normal transcription direction, so that the complementary strand of the coding region (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) can be transcribed.

[0170] Antisense nucleic acid molecules are usually substantially identical to at least a portion of a target gene or genes. However, the nucleic acid does not need to be completely identical to inhibit expression. Generally, high homology can be used to compensate for the use of short antisense nucleic acid molecules. The minimum percent identity is typically greater than about 65%, although higher percent identity may more effectively suppress the expression of endogenous sequences. A fairly high percent identity, greater than about 80%, is typically preferred, while about 95% to complete identity is typically most preferred.

[0171] Antisense nucleic acid molecules need not have the same intron or exon pattern as the target gene. Non-coding segments of a target gene may be equally effective as coding segments in achieving antisense inhibition of target gene expression. DNA sequences of at least about 8 or so nucleotides may be used as antisense nucleic acid molecules, although longer sequences are preferred. A representative example of a MASP-2 inhibitor useful in the present invention is an antisense MASP-2 nucleic acid molecule that is at least 90 percent identical to the complementary strand of a MASP-2 cDNA consisting of the nucleic acid sequence shown in SEQ ID NO:4. The nucleic acid sequence shown in SEQ ID NO:4 encodes a MASP-2 protein consisting of the amino acid sequence shown in SEQ ID NO:5.

[0172] Targeting of antisense oligonucleotides that bind to MASP-2 mRNA is another mechanism that can be used to reduce the level of MASP-2 protein synthesis. For example, synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptor is inhibited by antisense oligonucleotides against the respective mRNA sequences (U.S. Pat. No. 5,739,119 to Cheng and U.S. Pat. No. 5,759,829 to Shewmaker). Further examples of antisense inhibition include nuclear protein cyclin, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA A Receptor, and human EGF (see, e.g., U.S. Pat. No. 5,801,154 to Baracchini; U.S. Pat. No. 5,789,573 to Baker; U.S. Pat. No. 5,718,709 to Considine; and U.S. Pat. No. 5,610,288 to Reubenstein).

[0173] A system has been described that allows a person skilled in the art to determine which oligonucleotides are useful in the present invention. This system involves probing the appropriate site in the target mRNA using RnaseH cleavage as an indicator of the accessibility of sequences in the transcript. Scherr, M., et al., Nucleic Acids Res. 25:5079-5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:3665-3673, 2001. To create a site vulnerable to RNAse H, a mixture of antisense oligonucleotides complementary to a certain region of the MASP-2 transcript is added to a cell extract expressing MASP-2, e.g., hepatocytes, and hybridized. This method can be combined with computer-assisted sequence selection, which can predict the optimal sequence selection of antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to target mRNA in host cells. These secondary structure analyses and target site selection studies can be performed using OLIGO primer analysis software (Rychlik, I., 1997) and BLASTN 2.0.5 algorithm software (Altschul, SF, et al., Nucl. Acids Res. 25:3389-3402, 1997). Antisense compounds against target sequences preferably contain about 8 to about 50 nucleotides in length. Antisense oligonucleotides containing about 9 to about 35 nucleotides, etc., are particularly preferred. The inventors contemplate that all oligonucleotide compositions within the range of 9 to 35 nucleotides (i.e., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases in length) are highly preferred for the implementation of the methods of the invention based on antisense oligonucleotides.Highly preferred target regions of MASP-2 mRNA are those at or near the AUG translation initiation codon and those sequences that are substantially complementary to the 5' region of the mRNA, for example, the -10 to +10 region of the MASP-2 gene nucleotide sequence (SEQ ID NO:4). Exemplary MASP-2 expression inhibitors are provided in Table 4.

[0174] Table 4: Exemplary expression inhibitors of MASP-2 TIFF2024509702000050.tif72155

[0175] As mentioned above, the term "oligonucleotide" as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers oligonucleobases consisting of natural nucleotides, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides with non-natural modifications. These modifications allow the introduction of certain desirable properties not offered by natural oligonucleotides, such as low toxicity, high stability against nuclease degradation, and high cellular uptake. In an exemplary embodiment, the antisense compounds of the present invention differ from natural DNA by a phosphodiester backbone modification in which the phosphate substituents are replaced by phosphorothioates, which increases the life span of the antisense oligonucleotide. Similarly, one or both ends of the oligonucleotide may be replaced with one or more acridine derivatives that intercalate between adjacent base pairs in the nucleic acid strand.

[0176] Another alternative to antisense is the use of "RNA interference" (RNAi). Double-stranded RNA (dsRNA) can induce gene silencing in mammals in vivo. The natural function of RNAi and cosuppression appears to be the protection of genomes from invasion by mobile genetic elements such as retrotransposons and viruses that, when activated, produce aberrant RNA or dsRNA in the host cell (see, e.g., Jensen, J., et al., Nat. Genet. 27:209-12, 1999). Double-stranded RNA molecules can be prepared by synthesizing two RNA strands capable of forming a double-stranded RNA molecule, each of which is about 19-25 (e.g., 19-23 nucleotides) in length. For example, dsRNA molecules useful in the methods of the invention may include RNA corresponding to the sequences listed in Table 4 and their complementary strands. Preferably, at least one RNA strand has a 3' overhang of 1-5 nucleotides. The synthesized RNA strands are combined under conditions to form a double-stranded molecule. The RNA sequence may be 25 nucleotides or less in length and contain at least an 8 nucleotide portion of SEQ ID NO:4. Designing siRNA sequences for a particular target is within the ordinary skill in the art. Commercial services are available that design siRNA sequences and guarantee at least 70% knockdown of expression (Qiagen, Valencia, Calif).

[0177] dsRNA is administered as pharmaceutical composition, and can be carried out by known method that nucleic acid is introduced into desired target cell.Commonly used gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection and virus method.Such method is disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

[0178] Ribozymes, such as ribozymes targeting MASP-2 mRNA, can also be utilized to reduce the amount and / or biological activity of MASP-2. Ribozymes are catalytic RNA molecules that can cleave nucleic acid molecules with sequences that are completely or partially homologous to the ribozyme sequence. Ribozyme transgenes can be designed that code for RNA ribozymes that specifically pair with target RNA and cleave the phosphodiester backbone at a specific position, thereby functionally inactivating the target RNA. When performing this cleavage, the ribozyme itself does not change, and therefore can be reused to cleave other molecules. The inclusion of a ribozyme sequence in an antisense RNA confers RNA cleavage activity to the antisense RNA, thereby increasing the activity of the antisense construct.

[0179] Ribozymes useful in the practice of the present invention typically contain a hybridizing region of at least about nine nucleotides that is complementary in nucleotide sequence to at least a portion of the target MASP-2 mRNA, and a catalytic region adapted to cleave the target MASP-2 mRNA (see generally EPA No. 0321201; WO88 / 04300; Haseloff, J., et al., Nature 534:585-591, 1988; Fedor, MJ, et al., Proc. Natl. Acad, Sci. USA 87:1668-1672, 1990; Cech, TR, et al., Ann. Rev, Biochem. 55:599-629, 1986).

[0180] Ribozymes may be directly targeted to cells in the form of RNA oligonucleotides incorporating ribozyme sequences, or may be introduced into cells as expression vectors encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same manner as described for antisense polynucleotides.

[0181] Antisense RNA and DNA, ribozymes, and RNAi molecules useful in the method of the present invention can be prepared by any method known in the art for synthesizing DNA and RNA molecules.These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as solid-phase phosphoramidite chemical synthesis.Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding antisense RNA molecules.Such DNA sequences can be incorporated into a wide variety of vectors incorporating suitable RNA polymerase promoters, such as T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be stably introduced into cell lines.

[0182] Various well-known modifications of DNA molecules can be introduced as a means of increasing stability and extending half-life. Useful modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase linkages in the oligodeoxyribonucleotide backbone.

[0183] V. Pharmaceutical Compositions and Delivery Methods dosage In another aspect, the present invention provides a composition for inhibiting the side effects of MASP-2-dependent complement activation in a subject suffering from a disease or condition disclosed herein, comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitor and a pharma- ceutically acceptable carrier. To treat or ameliorate a condition associated with MASP-2-dependent complement activation, the MASP-2 inhibitor can be administered to a subject in need thereof at a therapeutically effective dose. A therapeutically effective dose refers to an amount of the MASP-2 inhibitor sufficient to ameliorate symptoms associated with a disease or condition.

[0184] The toxicity and therapeutic efficacy of MASP-2 inhibitors can be determined by standard pharmaceutical procedures using experimental animal models, such as the mouse MASP-2- / - mouse model expressing the human MASP-2 transgene described in Example 1. Using such animal models, NOAEL (no observed adverse effect level) and MED (minimal effective dose) can be determined using standard methods. The dose ratio between the NOAEL effect and the MED effect is the therapeutic ratio, expressed as the ratio NOAEL / MED. MASP-2 inhibitors that show a large therapeutic ratio or index are most preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of MASP-2 inhibitors is preferably within a range of circulating concentrations that include the MED with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized.

[0185] In some embodiments, the therapeutic efficacy of a MASP-2 inhibitor for treating, inhibiting, reducing or preventing fibrosis in a mammalian subject suffering from or at risk of developing a disease or disorder caused or exacerbated by fibrosis and / or inflammation is determined by one or more of the following: a reduction in one or more markers of inflammation and scarring in renal tissue (e.g., TGFβ-1, CTFF, IL-6, apoptosis, fibronectin, laminin, collagen, EMT, infiltrating macrophages); a reduction in release of soluble markers of inflammation and fibrotic kidney disease into urine and plasma (e.g., by measuring renal excretory function).

[0186] For any compound formulation, the therapeutically effective dose can be evaluated using animal model.For example, the dose that reaches the circulating plasma concentration range including MED can be formulated in animal model.The quantitative level of MASP-2 inhibitor in plasma can also be measured, for example, by high performance liquid chromatography.

[0187] In addition to toxicity studies, effective dosages may also be assessed based on the amount of MASP-2 protein present in a living subject and the binding affinity of the MASP-2 inhibitor. MASP-2 levels in normal human subjects are present in serum at low levels in the range of 500 ng / ml, and MASP-2 levels in a particular subject can be determined using a quantitative MASP-2 assay as described in Moller-Kristensen M., et al., J. Immunol Methods 282:159-167, 2003.

[0188] In general, the dosage of an administered composition comprising a MASP-2 inhibitor will vary depending on factors such as the subject's age, weight, height, sex, general medical condition, and medical history. By way of example, a MASP-2 inhibitor such as an anti-MASP-2 antibody can be administered within a dosage range of approximately 0.010-10.0 mg / kg of subject body weight, preferably 0.010-1.0 mg / kg of subject body weight, and more preferably 0.010-0.1 mg / kg of subject body weight. In some embodiments, the composition comprises a combination of an anti-MASP-2 antibody and a MASP-2 inhibitory peptide.

[0189] The therapeutic efficacy of the MASP-2 inhibitory compositions and methods of the present invention in a particular subject, as well as the appropriate dosage, can be determined according to complement assays well known to those skilled in the art. Complement generates a large number of specific products. Over the past decade, highly sensitive and specific assays have been developed and are commercially available for most of these activation products, including the small activation fragments C3a, C4a, and C5a, and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodies that react with neoantigens that are exposed on the fragments but not on the native proteins from which they are formed. For this reason, these assays are very simple and specific. Most rely on ELISA technology, although in the case of C3a and C5a, radioimmunoassays may still be used. These latter assays measure both the unprocessed fragments and their "desArg" fragments, which are the predominant forms found in the circulation. The unprocessed fragments and C5a desArg is rapidly cleaved upon binding to cell surface receptors and is therefore present in very low concentrations. desArg does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive pathway-dependent index of complement activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Detection of sC5b-9, a fluid-phase product of membrane attack pathway activation, provides evidence that complement is activated to the end. Because both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide information about which of these two pathways generated the activation product.

[0190] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in complement system components that result from administration of a MASP-2 inhibitor according to the methods of the present invention: inhibition of the generation or production of MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (measured, for example, as described in Example 2), reduced C4 cleavage and C4b deposition (measured, for example, as described in Example 10), or reduced C3 cleavage and C3b deposition (measured, for example, as described in Example 10).

[0191] Further active substances In certain embodiments, the method of preventing, treating, reversing, and / or inhibiting fibrosis and / or inflammation includes administering a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) as part of a therapeutic regimen together with one or more other drugs, biologics, or therapeutic interventions suitable for inhibiting fibrosis and / or inflammation. In certain embodiments, the additional drugs, biologics, or therapeutic interventions are suitable for the particular symptoms associated with a disease or disorder caused or exacerbated by fibrosis and / or inflammation. By way of example, a MASP-2 inhibitory antibody may be administered as part of a therapeutic regimen together with one or more immunosuppressants, such as methotrexate, cyclophosphamide, azathioprine, and mycophenolate mofetil. As a further example, a MASP-2 inhibitory antibody may be administered as part of a therapeutic regimen together with one or more agents designed to increase blood flow (e.g., nifedipine, amlodipine, diltiazem, felodipine, or nicardipine). As a further example, the MASP-2 inhibitory antibody may be administered as part of a therapeutic regimen together with one or more agents intended to reduce fibrosis, such as d-penicillamine, colchicine, PUVA, relaxin, cyclosporine, TGFβ blockers, and / or p38 MAPK blockers. As a further example, the MASP-2 inhibitory antibody may be administered as part of a therapeutic regimen together with steroids or bronchodilators.

[0192] Compositions and methods comprising a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) may optionally include one or more additional therapeutic agents that may enhance the activity of the MASP-2 inhibitor or provide an additive or synergistic associated therapeutic function. For example, in the context of treating a subject suffering from a disease or disorder caused or exacerbated by fibrosis and / or inflammation, one or more MASP-2 inhibitors may be administered in combination (including simultaneous administration) with one or more additional anti-fibrotic agents and / or one or more anti-viral agents and / or anti-inflammatory agents and / or immunosuppressants.

[0193] MASP-2 inhibitors (e.g., MASP-2 inhibitory antibodies) can be used in combination with other therapeutic agents, such as general antiviral or immunosuppressant drugs, such as corticosteroids, immunosuppressants or cytotoxic agents, and / or antifibrotic agents.

[0194] In some embodiments of the methods described herein, the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody or small molecule inhibitor of MASP-2) is used as a monotherapy to treat subjects suffering from coronavirus or influenza virus. In some embodiments of the methods described herein, the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody or small molecule inhibitor of MASP-2) is combined with other therapeutic agents, such as antiviral agents, therapeutic antibodies, corticosteroids, and / or other agents shown to be effective in treating subjects suffering from coronavirus or influenza virus. In some embodiments, the pharmaceutical composition comprises the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody or small molecule inhibitor of MASP-2) and at least one additional therapeutic agent, such as an antiviral agent (e.g., remdesivir), a therapeutic antibody against a target other than MASP-2, a corticosteroid, an anticoagulant, such as a low molecular weight herparin (e.g., enoxaparin), and an antibiotic (e.g., azithromycin).

[0195] In such combination therapy, the MASP-2 inhibitor may be formulated with, administered simultaneously with, prior to, or subsequent to one or more other desired COVID-19 therapeutic agents, such as antiviral agents (e.g., remdesivir), therapeutic antibodies against targets other than MASP-2, corticosteroids, or anticoagulants. Each component of the combination therapy may be formulated in a variety of ways known in the art. For example, the MASP-2 inhibitor and the second agent of the combination therapy may be formulated together or separately. The MASP-2 inhibitor and the additional agent may be administered to the COVID-19 patient at one time or over a course of treatment as appropriate.

[0196] Exemplary antiviral agents include, for example, darunavir (which may be used with ritonavir or cobicistat to increase darunavir levels), favipiravir, lopinavir, ritonavir, remdesivir, galidesivir, ebastine, danoprevir, ASC09, emtricitabine, tenofovir, umifunovir, baloxavir marboxil, azuvudine, and / or ISR-50. Exemplary therapeutic antibodies include, for example, vascular growth factor inhibitors (e.g., bevacizumab), PD-1 blocking antibodies (e.g., thymosin, camrelizumab), CCR5 antagonists (e.g., leronlimab), IL-6 receptor antagonists (e.g., sarilumab, tocilizumab), IL-6 targeted inhibitors (e.g., siltuximab), anti-GMCSF antibodies (e.g., dimcirumab, TJM2), GMCSF receptor alpha blocking antibodies (e.g., mavrilimumab), anti-C5 antibodies (e.g., eculizumab, ravulizumab), and / or anti-C5a antibodies (IFX-1).

[0197] In some embodiments of the methods described herein, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, e.g., OMS646, or a small molecule inhibitor of MASP-2) is combined with an antiviral agent such as remdesivir to treat a subject suffering from COVID-19.

[0198] Other agents that may be effective in treating coronaviruses and / or influenza viruses include, for example, chloroquine / hydroxychloroquine, camostat mesylate, ruxolinib, peginterferon alpha-2b, novafenone, ifenprodil, recombinant ACE2, APN01, brilacidin, BXT-25, BIO-11006, fingolimod, WP1122, interferon beta-1a, nafamostat, losartan, and / or alteplase.

[0199] Pharmaceutical Carriers and Delivery Vehicles Generally, the MASP-2 inhibitor composition of the present invention, in combination with any other selected therapeutic agent, is optionally included in a pharma- ceutically acceptable carrier. The carrier is selected to be non-toxic, biocompatible, and not adversely affect the biological activity of the MASP-2 inhibitor (and any other therapeutic agent combined therewith). Exemplary pharma- ceutically acceptable carriers for peptides are described in U.S. Patent No. 5,211,657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptides useful in the present invention may be formulated into solid, semi-solid, gel, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections, allowing oral, parenteral, or surgical administration. The present invention also contemplates local administration of the composition by coating a medical device or the like.

[0200] Carriers suitable for parenteral delivery via injection, infusion, or irrigation, and local delivery include distilled water, physiological phosphate buffered saline, normal Ringer's solution or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol. In addition, sterile non-volatile oils may be used as solvents or dispersion media. For this purpose, any biocompatible oils may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are useful in the preparation of injections. Carriers and agents may be formulated as liquids, suspensions, polymerizable or non-polymerizable gels, pastes, or ointments.

[0201] The carrier may also include a delivery vehicle to sustain (i.e., extend, delay, or modulate) the delivery of the agent or to enhance the delivery, uptake, stability, or pharmacokinetics of the therapeutic agent. Such delivery vehicles may include, by way of non-limiting example, microparticles, microspheres, nanospheres, or nanoparticles of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include PEO:PHB:PEO copolymers and copolymer / cyclodextrin complexes disclosed in WO2004 / 009664A2, and PEO and PEO / cyclodextrin complexes disclosed in US Patent Application Publication No. 2002 / 0019369A1. Such hydrogels may be injected locally at the desired site of action, or subcutaneously or intramuscularly to form a sustained release depot.

[0202] For intra-articular delivery, the MASP-2 inhibitor may be carried in an injectable liquid or gel carrier as described above, an injectable sustained release delivery vehicle as described above, or hyaluronic acid or a hyaluronic acid derivative.

[0203] For oral administration of non-peptide agents, the MASP-2 inhibitor may be delivered in an inert filler or diluent such as sucrose, corn starch, or cellulose.

[0204] For local administration, the MASP-2 inhibitor may be delivered in an ointment, lotion, cream, gel, eye drops, suppository, spray, liquid or powder, or may be delivered via a transdermal patch in a gel or microencapsulated delivery system.

[0205] A variety of nasal and pulmonary delivery systems, including aerosols, metered dose inhalers, dry powder inhalers, and nebulizers, are under development and can be suitably adapted for delivery of the present invention in aerosol, inhalation, or nebulized delivery vehicles, respectively.

[0206] For intrathecal (IT) or intracerebroventricular (ICV) delivery, the compositions of the invention can be administered using an appropriately sterile delivery system (eg, liquid; gel, suspension, etc.).

[0207] The compositions of the present invention may also contain biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, flavorings (for oral administration).

[0208] Pharmaceutical Carriers for Antibodies and Peptides More specifically, with respect to anti-MASP-2 antibodies and inhibitory peptides, exemplary formulations can be administered parenterally as a solution or suspension of an injection dose of the compound dissolved in a physiologically acceptable diluent, together with a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerol, or ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances, etc., may be present in compositions containing anti-MASP-2 antibodies and inhibitory peptides. Additional components of pharmaceutical compositions include petroleum oils (e.g., petroleum oils of animal, vegetable, or synthetic origin), such as soybean oil and mineral oil. In general, glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers for injections.

[0209] Anti-MASP-2 antibodies and inhibitory peptides can also be administered in the form of depot injection or implant preparations, which can be formulated for slow or pulsatile release of the active agent.

[0210] Pharmaceutically acceptable carriers for expression inhibitors More specifically, with respect to the expression inhibitors useful in the methods of the present invention, compositions are provided that include the expression inhibitors and a pharma- ceutically acceptable carrier or diluent, and may further include a colloidal dispersion system.

[0211] Pharmaceutical compositions containing expression inhibitors may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Preparation of such compositions typically involves combining expression inhibitors with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose, or dextrins, chelating agents such as EDTA, glutathione, and other stabilizers and excipients. Examples of suitable diluents are neutral buffered saline or saline mixed with nonspecific serum albumin.

[0212] In some embodiments, the composition can be prepared and formulated as an emulsion, which is typically a heterogeneous system in which one liquid is dispersed in the form of droplets within another liquid (see Idson, Pharmaceutical Dosage Forms, Vol.1, Rieger and Banker (eds.), Marcek Dekker, Inc., NY, 1988). Examples of natural emulsifiers used in emulsion formulations include gum arabic, beeswax, lanolin, lecithin, and phosphatides.

[0213] In one embodiment, the composition comprising nucleic acid can be formulated as microemulsion.Microemulsion as used herein refers to a system of water, oil and amphiphilic substances that is a single, optically isotropic and thermodynamically stable liquid solution (see Rosoff, Pharmaceutical Dosage Forms, Vol.1).The method of the present invention may also use liposome to introduce and deliver antisense oligonucleotide to desired site.

[0214] Pharmaceutical compositions and formulations of expression inhibitors for topical administration may include transdermal patches, ointments, lotions, creams, gels, eye drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, as well as aqueous, powder, or oily bases and thickeners, may be used.

[0215] Method of administration Pharmaceutical compositions containing MASP-2 inhibitors can be administered in a number of ways, depending on whether a local or systemic administration method is best suited for the condition being treated. Additionally, the compositions of the present invention can be delivered by coating or incorporating them onto or into an implantable medical device.

[0216] systemic delivery The terms "systemic delivery" and "systemic administration" as used herein are intended to include, but are not limited to, oral and parenteral routes, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal, and other routes of administration that effectively distribute the delivered agent to one or more desired therapeutic action sites. Preferred systemic delivery routes of the present compositions include intravenous, intramuscular, subcutaneous, and inhalation routes. It will be understood that the exact systemic administration route of a selected agent used in a particular composition of the invention will be determined, in part, to account for the agent's susceptibility to metabolic conversion pathways associated with a particular administration route. For example, peptide agents can be most suitably administered by routes other than oral.

[0217] MASP-2 inhibitory antibodies and polypeptides can be delivered to a subject in need thereof by any suitable means. Methods for delivering MASP-2 antibodies and polypeptides include oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), inhalation (e.g., inhalation via fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual administration, and can be formulated in a dosage form suitable for each administration route.

[0218] As a representative example, MASP-2 inhibitory antibodies and peptides can be introduced into the body by applying the polypeptide to the body membrane that can absorb it, such as the nasal, gastrointestinal, and rectal membranes.The polypeptide is typically applied to the absorbent membrane together with a penetration enhancer (see, for example, Lee, VHL, Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, VHL, J. Controlled Release 13:213, 1990; Lee, VHL,, Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, AG, et al., J. Controlled Release 13:241, 1990).For example, STDHF is a synthetic derivative of fusidic acid, a steroid surfactant that is structurally similar to bile salts and has been used as a penetration enhancer for nasal delivery (Lee, WA, Biopharm. 22, Nov. / Dec. 1990).

[0219] To protect the polypeptide from enzymatic degradation, MASP-2 inhibitory antibodies and polypeptides can be introduced by conjugating them with other molecules such as lipids. For example, the covalent attachment of polymers, particularly polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body, thus extending their half-life (Fuertges, P., et al., J. Controlled Release 11:139, 1990). Many polymeric systems for protein delivery have been reported (Bae, YH, et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813, 1989:Yamakawa, L, et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., Controlled Release 10:195, 1989; Asano, M., et al., J. Controlled Release 9:111, 1989; Rosenblatt, J., et al., J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990; Takakura, Y., et al., J. Pharm. Sci. 78:117, 1989; Takakura, Y., et al., J. Pharm. Sci. 78:219, 1989).

[0220] Recently, liposomes with improved serum stability and circulation half-life have been developed (see, e.g., U.S. Patent No. 5,741,516 to Webb). In addition, various methods of liposomes and liposome-like preparations as potential drug carriers have been investigated (see, e.g., U.S. Patent No. 5,567,434 to Szoka; U.S. Patent No. 5,552,157 to Yagi; U.S. Patent No. 5,565,213 to Nakamori; U.S. Patent No. 5,738,868 to Shinkarenko; and U.S. Patent No. 5,795,587 to Gao).

[0221] For transdermal application, the MASP-2 inhibitory antibodies and polypeptides may be combined with other suitable components, such as carriers and / or adjuvants. There is no limit to what such other components are, except that they must be pharma- ceutically acceptable for the intended administration and cannot degrade the activity of the active components of the composition. Examples of suitable vehicles include ointments, creams, gels, or suspensions, with or without purified collagen. The MASP-2 inhibitory antibodies and polypeptides may also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.

[0222] The compositions of the present invention may be administered systemically periodically at intervals determined to maintain a desired level of therapeutic effect. For example, the compositions may be administered, for example, by subcutaneous injection every 2-4 weeks or less frequently. The dosing schedule will be determined by the physician taking into consideration various factors that may affect the action of the combination of agents. These factors will include the degree of progression of the condition being treated, the age, sex, and weight of the patient, as well as other clinical factors. The dosage of each individual agent will vary as a function of the MASP-2 inhibitor contained in the composition, as well as the presence and content of any drug delivery vehicles (e.g., sustained release delivery vehicles). Additionally, the dosage can be adjusted to account for variations in the frequency of administration and pharmacokinetic behavior of the delivery agents.

[0223] Local delivery The term "local" as used herein encompasses application of a drug in or around the site of a desired localized effect, and may include, for example, local delivery to the skin or other affected tissue, ocular delivery, intrathecal (IT), intracerebroventricular (ICV), intraarticular, intrasinus, intracranial, or intravesicular administration, placement, or irrigation. Local administration may be preferred to allow for low dose administration to avoid systemic side effects, as well as for more precise control over the timing of delivery and concentration of active agents at the local delivery site. Local administration provides a known concentration at the target site regardless of patient-to-patient variability in metabolism, blood flow, etc. Improved dosage control is also provided by direct delivery methods.

[0224] Localized delivery of MASP-2 inhibitors can be achieved during procedures such as, for example, surgery, in the context of surgical methods to treat diseases or disorders caused or exacerbated by fibrosis and / or inflammation.

[0225] Treatment regimen In prophylactic applications, a pharmaceutical composition comprising a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody or a MASP-2 inhibitory small molecule compound) is administered to a subject susceptible to or otherwise at risk of developing coronavirus-induced acute respiratory distress syndrome or influenza virus-induced acute respiratory distress syndrome in an amount sufficient to inhibit MASP-2-dependent complement activation and thereby reduce or eliminate symptoms of the respiratory syndrome or reduce the risk of developing the symptoms. In both prophylactic and therapeutic regimens, the composition comprising a MASP-2 inhibitor may be administered in multiple doses until a satisfactory therapeutic outcome is achieved in the subject. The application of the MASP-2 inhibitory composition of the present invention may be carried out by a single administration of the composition in the case of treatment of acute conditions associated with fibrosis and / or inflammation, or by a limited series of administrations. Alternatively, the composition may be administered at regular intervals over an extended period of time in the case of treatment of chronic conditions associated with fibrosis and / or inflammation.

[0226] In both preventive and therapeutic regimens, the composition comprising the MASP-2 inhibitor may be administered in multiple doses until a sufficient therapeutic outcome is achieved in the subject. In one embodiment of the present invention, the MASP-2 inhibitor comprises a MASP-2 antibody, which may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of 0.1 mg to 10,000 mg, more suitably 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg, and even more suitably 50.0 mg to 500 mg. In the case of pediatric patients, the dosage may be adjusted in proportion to the patient's weight. The application of the MASP-2 inhibitor composition of the present invention may be performed by a single administration of the composition or by a limited series of administrations in the case of treatment of a subject suffering from or at risk of developing a disease or disorder caused or exacerbated by fibrosis and / or inflammation. Alternatively, the compositions may be administered at regular intervals over an extended period of time, for example daily, twice weekly, weekly, biweekly, monthly or bimonthly, in the case of treatment of a subject suffering from or at risk of developing a disease or disorder caused or exacerbated by fibrosis and / or inflammation.

[0227] In both preventive and therapeutic regimens, a composition containing a MASP-2 inhibitor may be administered in multiple dosages until a sufficient therapeutic outcome is achieved in the subject.

[0228] VI. Use of the MASP-2 / C1-INH complex as a biomarker for severe COVID-19 In another aspect, the present disclosure provides a biomarker of MASP-2-mediated lectin pathway activation, namely, the fluid-phase MASP-2 / C1-INH complex, the presence and / or changes in concentration of which are associated with the presence or risk of developing an acute disease associated with COVID-19 infection, the presence or risk of developing one or more long-term sequelae associated with COVID-19 infection, and / or a clinically meaningful treatment of COVID-19 infection with a complement inhibitor. Also provided are compositions, kits, and methods for determining the concentration of the fluid-phase MASP-2 / C1-INH complex in biological fluids, such as biological fluids obtained from subjects infected with COVID-19. The compositions and methods are particularly useful for assessing the risk of developing an acute disease associated with COVID-19, diagnosing COVID-19 and / or long-term sequelae induced by COVID-19, monitoring the progression or remission of COVID-19-related disease, and / or monitoring the response to treatment with a complement inhibitor, such as a MASP-2 inhibitor, or optimizing such treatment.

[0229] As described herein in Examples 25-30, a feature was exploited to determine the activation state of the lectin pathway (LP) effector enzyme human MASP-2 (shown in SEQ ID NO:6), taking advantage of the fact that human C1 inhibitor (C1-INH) (shown in SEQ ID NO:86), which acts as a pseudo-substrate upon activation of MASP-2, forms a covalent fluid-phase MASP-2 / C1-INH complex. Thus, the level of MASP-2 / C1-INH complex in plasma or serum samples is a clear measure of recent LP activation.

[0230] As described in Examples 25-30, the inventors discovered that the concentration of MASP-2 / C1-INH in the blood (e.g., serum and / or plasma) was abnormally high in severe COVID-19 patients, as well as in subjects previously infected with COVID-19 and suffering from long-term sequelae. The inventors also observed that the concentration of MASP-2 / C1-INH complexes decreased to normal levels in most cases after recovery. As further described in Examples 29 and 30, the inventors confirmed that subjects suffering from acute COVID-19 had high levels of MASP-2 / C1-INH before treatment with narsoplimab, and that this level rapidly decreased after treatment with narsoplimab. The inventors believe that monitoring patients infected with SARS-CoV-2 for increased concentrations of MASP-2 / C1-INH complexes may be useful for diagnosing patients with or at risk of developing acute COVID-19, and also for diagnosing subjects with or at risk of developing post-acute COVID-19 (also called long COVID-19), and optionally for treating subjects identified as at risk with a complement inhibitor, such as a MASP-2 inhibitor. Monitoring the status of the MASP-2 / C1-INH complex may also be useful for determining whether a COVID-19 patient is responding to therapy with a complement inhibitor, such as a MASP-2 inhibitor, and may optionally be useful for adjusting the dosage of the MASP-2 inhibitor as needed to bring the level of MASP-2 / C1-INH into the normal range.

[0231] In accordance with the foregoing, in one aspect, the present disclosure provides, inter alia, compositions, kits and methods for measuring the amount of MASP-2 / C1-INH complex as a biomarker of MASP-2-mediated lectin pathway activation, the concentration of which in biological fluids is abnormally elevated in patients suffering from acute COVID-19 disease associated with SARS-Cov2 infection and / or subjects previously infected with SARS-Cov2, as well as in subjects suffering from or at risk of developing long COVID-19 sequelae.

[0232] Thus, in one aspect, the present invention relates to a monoclonal antibody (mAb C#7 or mAb C#8) that specifically binds to MASP-2 and is capable of binding to MASP-2 in a complex with C1-INH (also referred to as MASP-2 / C1-INH complex), and the use of this antibody in a method for detecting the presence or amount of MASP-2 / C1-INH complex in a biological sample. In another aspect, the present invention relates to an immunoassay comprising the use of a MASP-2-specific monoclonal antibody and a C1-INH-specific antibody for measuring the presence or amount of MASP-2 / C1-INH complex in a mammalian subject suffering from or at risk of developing an infection with a coronavirus or influenza virus to determine the activation status of the lectin pathway, optionally before and after treatment with a complement inhibitor, such as a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody (e.g., narsoplimab), wherein the MASP-2 inhibitory antibody is capable of inhibiting the lectin pathway.

[0233] In one embodiment, the presence or amount of MASP-2 / C1-INH complex is useful as a biomarker for determining the presence or risk of developing severe COVID-19 or long COVID-19 in a subject infected with SARS-CoV-2, and a high MASP-2 / C1-INH level in a subject, when compared to a normal uninfected subject or a pool of subjects, or a threshold value, indicates that the subject is suffering from severe COVID-19 or has a high risk of developing severe COVID-19 or has a high risk of developing long COVID-19. In some embodiments, the method further comprises administering a complement inhibitor to the subject determined to have an increased level of MASP-2 / C1-INH complex. In some embodiments, the present disclosure provides a method for determining the effectiveness of a complement inhibitor, such as a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, such as narsoplimab), in a subject and / or a method for monitoring dosing in a subject undergoing treatment with a complement inhibitor. In some embodiments, the subject is suffering from a coronavirus, e.g., COVID-19, or influenza virus, or other lectin pathway disease or disorder (e.g., HSCT-TMA, IgAN, GvHD, or other lectin pathway disease or disorder).

[0234] A. Anti-MASP-2 monoclonal antibodies for use in sensitive ELISA assays and bead-based assays to detect MASP-2 / C1-INH complexes in biological samples As described in Examples 25 to 30 herein, the inventors have generated anti-MASP-2 antibody mAb clones #C7 and #C8 suitable for use in the MASP-2 / C1-INH complex detection assay and the methods described herein.

[0235] As described in Examples 25 and 26, the variable heavy and variable light chain fragments of mAb clone #C7 and mAb clone #C8 have been cloned and sequenced.

[0236] The heavy chain variable regions and light chain variable regions of anti-MASP-2 mAb clone #C7 and clone #C8 are shown below. SEQ ID NO:87: mAb clone #C7 HC variable region SEQ ID NO:88: mAb clone #C7 LC variable region SEQ ID NO:97: mAb clone #C8 HC variable region SEQ ID NO:98: mAb clone #C8 LC variable region

[0237] Thus, in one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to MASP-2 when complexed with C1-INH, wherein the antibody comprises (a) HC-CDR1, HC-CDR-2, and HC-CDR2 in the heavy chain variable region shown in SEQ ID NO:87, and LC-CDR1, LC-CDR-2, and LC-CDR-3 in the light chain variable region shown in SEQ ID NO:88, or (b) HC-CDR1, HC-CDR-2, and HC-CDR2 in the heavy chain variable region shown in SEQ ID NO:97, and LC-CDR1, LC-CDR-2, and LC-CDR-3 in the light chain variable region shown in SEQ ID NO:98. In one embodiment, the isolated antibody comprises a heavy chain variable region comprising HC-CDR-1 comprising SEQ ID NO:89, HC-CDR2 comprising SEQ ID NO:90, and HC-CDR3 comprising SEQ ID NO:91, and a light chain variable region comprising LC-CDR1 comprising SEQ ID NO:92, LC-CDR2 comprising SEQ ID NO:83, and LC-CDR3 comprising SEQ ID NO:94. In one embodiment, the anti-MASP-2 antibody is a humanized antibody, a chimeric antibody, or a fully human antibody. In one embodiment, the anti-MASP-2 antibody fragment is an Fv, Fab, Fab', F(ab) 2 and F(ab') 2In one embodiment, the anti-MASP-2 antibody is a single chain molecule. In one embodiment, the anti-MASP-2 antibody is an IgG molecule selected from the group consisting of IgG1, IgG2, and IgG4. In one embodiment, the anti-MASP-2 antibody or antigen-binding fragment thereof is labeled with a detectable moiety, for example a detectable moiety suitable for use in an immunoassay as further described herein. In one embodiment, the anti-MASP-2 antibody or fragment thereof is immobilized on a support, for example a support suitable for use in an immunoassay as further described herein.

[0238] In one embodiment, an anti-MASP-2 antibody or fragment thereof (i.e., an antibody or fragment thereof that specifically binds to human MASP-2 in complex with C1-INH) comprises a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in a heavy chain variable region comprising SEQ ID NO:87 and LC-CDR1, LC-CDR2, and LC-CDR3 in a light chain variable region comprising SEQ ID NO:88, the CDRs being numbered according to the Kabat numbering system. In one embodiment, an anti-MASP-2 antibody or fragment thereof (i.e., an antibody or fragment thereof that specifically binds to human MASP-2 in complex with C1-INH) comprises a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in a heavy chain variable region comprising SEQ ID NO:97 and LC-CDR1, LC-CDR2, and LC-CDR3 in a light chain variable region comprising SEQ ID NO:98, the CDRs being numbered according to the Kabat numbering system.

[0239] In one aspect, the anti-MASP-2 antibody or fragment thereof comprises a binding domain comprising the following six CDRs: (a) HC-CDR1 comprising the amino acid sequence SEQ ID NO:89; (b) HC-CDR2 comprising the amino acid sequence SEQ ID NO:90, (c) HC-CDR3 comprising the amino acid sequence SEQ ID NO:91; (d) LC-CDR1 comprising the amino acid sequence SEQ ID NO:92; (e) LC-CDR2 comprising the amino acid sequence SEQ ID NO:93, and (f) LC-CDR3 comprising the amino acid sequence SEQ ID NO:94.

[0240] In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a VH domain having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence of SEQ ID NO:87 or SEQ ID NO:97. In one embodiment, the MASP-2-specific antibody or fragment thereof comprises a VL domain having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence of SEQ ID NO:88. In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a VH comprising SEQ ID NO:87 and a VL comprising SEQ ID NO:88 or SEQ ID NO:98.

[0241] In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a binding domain comprising the following six CDRs: (a) HC-CDR1 comprising SEQ ID NO:89, (b) HC-CDR2 comprising SEQ ID NO:90; (c) HC-CDR3 comprising SEQ ID NO:91; (d) LC-CDR1 comprising SEQ ID NO:92, (e) LC-CDR2 comprising SEQ ID NO:93, and (f) LC-CDR3 comprising SEQ ID NO:94. In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a VH domain having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence of SEQ ID NO:87. In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a VL domain having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence of SEQ ID NO:88. In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a VH comprising SEQ ID NO:87 and a VL comprising SEQ ID NO:88.

[0242] In another aspect, the present disclosure provides a nucleic acid encoding a complementarity determining region (CDR) of a heavy chain variable region of an anti-MASP-2 antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 when complexed with C1-INH, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:95 and the CDRs are numbered according to the Kabat numbering system.In another aspect, the present disclosure provides a nucleic acid encoding a complementarity determining region (CDR) of a light chain variable region of an anti-MASP-2 antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 when complexed with C1-INH, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:96 and the CDRs are numbered according to the Kabat numbering system.

[0243] In another aspect, the present disclosure provides a cloning vector or expression vector comprising a nucleic acid encoding the complementarity determining regions (CDRs) of the heavy chain variable region and / or light chain variable region of an antibody or antigen-binding fragment thereof that specifically binds to human MASP-2, wherein (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:87 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:88, or (b) the amino acid sequence set forth in SEQ ID NO:97 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:98, and the CDRs are numbered according to the Kabat numbering system.

[0244] In another aspect, the present disclosure provides a cell containing a cloning vector or expression vector comprising a nucleic acid encoding the complementarity determining regions (CDRs) of the heavy chain variable region and / or light chain variable region of an antibody or antigen-binding fragment thereof that specifically binds to human MASP-2, wherein (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:87 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:88, or (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:97 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:98, and the CDRs are numbered according to the Kabat numbering system.

[0245] In another aspect, the present disclosure provides a method for producing an anti-MASP-2 antibody, the method comprising culturing a cell containing an expression vector containing a nucleic acid encoding one or both of the heavy and light chain polypeptides of the MASP-2 antibody or antigen-binding fragment disclosed herein. The cell or cell culture is cultured under conditions and for a time sufficient to allow expression by the cell (or cell culture) of the antibody or antigen-binding fragment encoded by the nucleic acid. The method may also include isolating the antibody or antigen-binding fragment from the cell (or cell culture) or from the medium in which the cells are cultured.

[0246] In one aspect, the present disclosure provides a composition comprising any of the anti-MASP-2 antibodies or antigen-binding fragments disclosed herein.

[0247] In one aspect, the present disclosure provides a support for use in an immunoassay comprising at least one or more of the anti-MASP-2 antibodies or antigen-binding fragments disclosed herein.

[0248] In one aspect, the disclosure provides a kit for detecting the presence or amount of MASP-2 / C1-INH complex in a test sample, such as a biological sample, comprising (a) at least one container and (b) at least one or more of any of the MASP-2 antibodies or antigen-binding fragments disclosed herein. In some aspects, the kit comprises an anti-MASP-2 antibody comprising a binding domain comprising (a) HC-CDR1, HC-CDR-2, and HC-CDR2 in the heavy chain variable region shown in SEQ ID NO:87, and LC-CDR1, LC-CDR-2, and LC-CDR-3 in the light chain variable region shown in SEQ ID NO:88, or (b) HC-CDR1, HC-CDR-2, and HC-CDR2 in the heavy chain variable region shown in SEQ ID NO:97, and LC-CDR1, LC-CDR-2, and LC-CDR-3 in the light chain variable region shown in SEQ ID NO:98. In some embodiments, the kit comprises an anti-MASP-2 antibody comprising a binding domain comprising the following six CDRs: (a) HC-CDR1 comprising SEQ ID NO:89, (b) HC-CDR2 comprising SEQ ID NO:90; (c) HC-CDR3 comprising SEQ ID NO:91; (d) LC-CDR1 comprising SEQ ID NO:92, (e) LC-CDR2 comprising SEQ ID NO:93, and (f) LC-CDR3 comprising SEQ ID NO:94. In some embodiments, the kit further comprises at least one antibody that specifically binds to C1-INH.

[0249] Anti-MASP-2 antibodies labeled with detectable moieties In another aspect, the present invention provides an anti-MASP-2 antibody (e.g., mAb clone #C7 or mAb clone #C8) labeled with a detectable moiety (i.e., a moiety that allows detection and / or quantification). In various embodiments, the antibodies described herein are conjugated to a detectable label that can be detected directly or indirectly. In this regard, an antibody "conjugate" refers to an anti-MASP-2 antibody covalently linked to a detectable label. In the present invention, monoclonal antibodies, their antigen-binding fragments, and their antibody derivatives, such as single-chain-variable fragment antibodies or epitope-tagged antibodies, may all be covalently...

Claims

1. A pharmaceutical composition comprising an MASP-2 inhibitor for treating, inhibiting, reducing or preventing acute respiratory distress syndrome, pneumonia or any other pulmonary or other acute manifestation of COVID-19, such as thrombosis, in a mammalian subject infected with SARS-CoV-2, comprising: (i) the level of MASP-2 / C1-INH complex in a biological sample obtained from the subject is determined, and an increased level of MASP-2 / C1-INH complex compared to a healthy control sample indicates an increased risk of developing one or more acute symptoms of COVID-19; and (ii) a subject having an increased level of MASP-2 / C1-INH complex is administered an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation, optionally wherein the amount of the MASP-2 inhibitor is sufficient to reduce the level of MASP-2 / C1-INH to a control level or reference standard; The pharmaceutical composition.

2. The pharmaceutical composition of claim 1, wherein the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:

6.

3. The pharmaceutical composition of claim 1, wherein the MASP-2 inhibitor is a small molecule MASP-2 inhibitor compound.

4. The pharmaceutical composition of claim 3, wherein the MASP-2 inhibitory compound is a synthetic small molecule or a semi-synthetic small molecule.

5. The pharmaceutical composition of claim 1, wherein the MASP-2 inhibitor is an inhibitor of MASP-2 expression.

6. The pharmaceutical composition of claim 2, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

69.

7. A pharmaceutical composition comprising a MASP-2 inhibitor for treating, ameliorating, preventing, or reducing the risk of developing one or more COVID-19 associated long-term sequelae in a mammalian subject that has been infected with SARS-CoV-2, comprising: (i) the level of MASP-2 / C1-INH complex in a biological sample obtained from the subject is determined, and an increased level of MASP-2 / C1-INH complex compared to a healthy control sample indicates an increased risk of developing one or more COVID-19-associated long-term sequelae; and (ii) administering to a subject having an increased level of MASP-2 / C1-INH complex an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation; The pharmaceutical composition.

8. The pharmaceutical composition described in claim 7, wherein the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:

6.

9. The pharmaceutical composition of claim 7, wherein the MASP-2 inhibitor is a small molecule MASP-2 inhibitor compound.

10. The pharmaceutical composition of claim 7, wherein the MASP-2 inhibitory compound is a synthetic small molecule or a semi-synthetic small molecule.

11. The pharmaceutical composition of claim 7, wherein the MASP-2 inhibitor is an inhibitor of MASP-2 expression.

12. The pharmaceutical composition of claim 8, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

69.

13. One or more COVID-19-related long-term sequelae include cardiovascular complications (including myocardial damage, cardiomyopathy, myocarditis, intravascular coagulation, stroke, venous and arterial complications, and pulmonary thrombosis); neurological complications (including cognitive difficulties, confusion, memory loss, also known as "brain fog", headache, stroke, dizziness, fainting, seizures, loss of appetite, insomnia, anosmia, anageusia, myoclonus, neuropathic pain, muscle pain; development of neurological diseases such as Alzheimer's disease, Guillain-Barré syndrome, Miller Fisher syndrome, Parkinson's disease, etc.), renal damage (e.g., acute kidney injury (AKI); pulmonary complications (including pulmonary fibrosis, dyspnea, pulmonary embolism), inflammatory conditions such as Kawasaki disease, Kawasaki disease-like illness, pediatric multisystem inflammatory syndrome, multisystem organ failure, extreme fatigue, muscle weakness, low-grade fever, lack of concentration, decreased memory, mood changes, difficulty sleeping, pins and needles pain in the arms and legs, etc.) 8. The pharmaceutical composition of claim 7, wherein the symptoms are selected from the group consisting of: vomiting, diarrhea, loss of taste and smell, sore throat and difficulty swallowing, new onset of diabetes and high blood pressure, skin rash, shortness of breath, chest pain, and palpitations.

14. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human MASP-2 in a complex with C1-INH, The antibody, (a) a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:87, and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:88; or (b) a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:97, and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:

98. Including, CDRs are numbered according to the Kabat numbering system. A monoclonal antibody or an antigen-binding fragment thereof.

15. 1. A method for measuring the amount of MASP-2 / C1-INH in a biological sample, comprising: The process is as follows: (b) performing an immunoassay comprising capturing and detecting MASP-2 / C1-INH complexes in a test biological sample from a human subject, wherein MASP-2 / C1-INH is captured by a monoclonal antibody that specifically binds to human MASP-2, and MASP-2 / C1-INH complexes are detected directly or indirectly by an antibody that specifically binds to C1-INH; and (c) comparing the level of MASP-2 / C1-INH complex detected according to (b) with a predetermined level or a control sample, wherein the level of MASP-2 / C1-INH complex detected in the test sample indicates the degree of lectin pathway complement activation. A method comprising:

16. 16. The method of claim 15, wherein the biological sample is a fluid sample selected from the group consisting of whole blood, serum, plasma, urine, and cerebrospinal fluid.

17. An antibody that specifically binds to MASP-2 (a) a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:87, and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:88; or (b) a binding domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region shown in SEQ ID NO:97, and LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region shown in SEQ ID NO:

98. Including, CDRs are numbered according to the Kabat numbering system.

16. The method of claim 15.

18. The method of claim 15, wherein the human subject is currently infected with or has previously been infected with SARS-CoV-2, or the subject has or is at risk for developing another lectin pathway disease or condition (e.g., HSCT-TMA, IgAN, GvHD, or other lectin pathway disease or disorder).

19. 1. A method for determining the risk of developing COVID-19 associated ARDS or other poor outcome or long-term sequelae associated with COVID-19 in a subject who is or has been infected with SARS-CoV-2, comprising: The process is as follows: (b) measuring the level of MASP-2 / C1-INH complex in a biological sample obtained from the subject; (c) comparing the measured level with a predetermined level or reference standard of MASP-2 / C1-INH complex to assess the risk of developing COVID-19-associated ARDS and / or long-term sequelae associated with COVID-19; and (d) determining the subject's risk of developing COVID-19 associated ARDS or experiencing other poor outcomes and / or developing long-term sequelae associated with COVID-19 and reporting the results to the patient, a physician, or a database; (e) optionally administering the treatment to a subject determined to be at high risk for developing acute disease and / or long-term sequelae associated with COVID-19 infection. A method comprising:

20. The method of claim 19, wherein the level of MASP-2 / C1-INH complex is measured in an immunoassay.

21. 1. A method for monitoring the effectiveness of treatment with a MASP-2 inhibitory antibody or antigen-binding fragment thereof in a mammalian subject in need thereof, comprising: The process is as follows: a first step of evaluating a first level of MASP-2 / C1-INH complex in a biological sample obtained from a mammalian subject administered a dose of a MASP-2 inhibitory antibody or antigen-binding fragment thereof at a first time point; a second step of assessing a second level of MASP-2 / C1-INH complexes in a biological sample obtained from the subject treated with the MASP-2 inhibitory antibody or antigen-binding fragment thereof at a second time point; and comparing the level of MASP-2 / C1-INH complex assessed in the first step with the level of MASP-2 / C1-INH complex assessed in the second step to determine the efficacy of the MASP-2 inhibitory antibody or antigen-binding fragment thereof in the mammalian subject. A method comprising:

22. The method of claim 21, further comprising a step of adjusting the dose of the MASP-2 inhibitory antibody or its antigen-binding fragment.

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