Method for inhibiting MASP-2 for treating and / or preventing coronavirus-induced acute respiratory distress syndrome

Abstract

In one aspect, the invention provides a method for treating, inhibiting, alleviating or preventing acute respiratory distress syndrome, pneumonia, or some other pulmonary or other manifestations of coronavirus infection, such as thrombosis, in a mammalian subject infected with a coronavirus, such as SARS-CoV-2. The method comprises the step of administering to a subject infected with a coronavirus 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 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO: 6. In one embodiment, the MASP-2 inhibitor is a small molecule MASP-2 inhibitory compound. In one embodiment, the subject is a human subject suffering from COVID-19 induced acute respiratory distress syndrome (ARDS), and supplementation of oxygen is required prior to treatment, and the MASP-2 inhibitor is administered in an amount sufficient to discontinue the need for supplementation of oxygen.

Description

[0001] This application is a divisional application of the invention patent with an application date of March 5, 2021, application number 202180019367.6, and invention name “Methods for inhibiting MASP-2 for treating and / or preventing coronavirus-induced acute respiratory distress syndrome”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 986,566, filed on March 6, 2020, and claims the benefit of U.S. Provisional Application No. 63 / 008,540, filed on April 10, 2020, and claims the benefit of U.S. Provisional Application No. 63 / 015,299, filed on April 24, 2020, and claims the benefit of U.S. Provisional Application No. 63 / 054,298, filed on July 21, 2020, and claims the benefit of U.S. Provisional Application No. 63 / 054,298, filed on August 24, 2020. No. 63 / 104,229, filed on October 22, 2020, and claims the benefit of U.S. Provisional Application No. 63 / 105,637, filed on October 26, 2020, and claims the benefit of U.S. Provisional Application No. 63 / 140,591, filed on January 22, 2021, all of which are incorporated herein by reference in their entirety. Background Art

[0004] The complement system provides an early mechanism of action in humans and other vertebrates to initiate, amplify and coordinate immune responses to microbial infections and other acute injuries (MK Liszewski and JP Atkinson, 1993, in Fundamental Immunology, 3rd edition, edited by WE Paul, Raven Press, Ltd., New York). Although complement activation provides a valuable first-line defense against potential pathogens, complement activity that promotes protective immune responses may also represent a potential threat to the host (KR Kalli et al., Springer Semin. Immunopathol. 15: 417-431, 1994; BP Organ, Eur. J. Clinical Investig. 24: 219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Although activated neutrophils are essential for host defense, they are indiscriminate in the release of their destructive enzymes and may cause organ damage. Additionally, complement activation may result in the deposition of lytic complement components on nearby host cells as well as microbial targets, leading to host cell lysis.

[0005] Currently, it is widely recognized that the complement system can be activated by three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by a complex composed of host antibodies bound to foreign particles (i.e., antigens), and therefore requires previous exposure to the antigen to generate a specific antibody response. Since the activation of the classical pathway depends on the host's previous adaptive immune response, 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.

[0006] The lectin pathway is widely believed to play 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 reduced serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572: 401-413, (2002)). Such patients display susceptibility to recurrent bacterial and fungal infections. These symptoms are usually evident early in life, during a window of vulnerability evident due to a decrease in maternally derived antibody titers, but before the full repertoire of antibody responses has developed. This syndrome often results from mutations at several sites in the collagenous portion of MBL that interfere with the proper formation of MBL oligomers. However, since MBL can act as an opsonin independently of complement, it is not known to what extent the increased susceptibility to infection is due to impaired complement activation.

[0007] All three pathways (i.e., the classical pathway, the lectin pathway, and the alternative pathway) are thought to converge at C5, which is cleaved to form products with multiple proinflammatory effects. The converging pathway has been referred to as the terminal complement pathway. C5a is the most potent anaphylatoxin, inducing changes in smooth muscle and vascular tone and vascular permeability. It is also a powerful chemotactic factor and activator for both neutrophils and monocytes. C5a-mediated cell activation can significantly amplify the inflammatory response by inducing the release of a variety of additional inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites, and reactive oxygen species. C5 cleavage leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is currently strong evidence that, in addition to its role as a lytic pore-forming complex, sublytic MAC deposition may also play an important role in inflammation.

[0008] In addition to its essential role in immune defense, the complement system also contributes to tissue damage in many clinical situations. Although there is extensive evidence suggesting that both the classical complement pathway and the alternative complement pathway are involved in the pathogenesis of non-infectious human diseases, the role of the lectin pathway is just beginning to be evaluated. Recent studies have provided evidence that activation of the lectin pathway may be responsible for complement activation and related inflammation in ischemia / reperfusion injury. Collard et al. (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and show deposition of C3 after exposure to human serum (Collard et al., Am. J. Pathol. 156: 1549-1556, (2000)). In addition, treatment of human serum with blocking anti-MBL monoclonal antibodies inhibited MBL binding and complement activation. These findings were extended to a rat model of myocardial ischemia-reperfusion, in which rats treated with a blocking antibody against rat MBL showed significantly less myocardial damage after coronary artery occlusion compared to rats treated with a control antibody (Jordan et al., Circulation 104:1413-1418, (2001)). The molecular mechanism by which MBL binds to the 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 endothelial cytokeratins (rather than glycoconjugates) (Collard et al., Am. J. Pathol. 159:1045-1054, (2001)). Other studies have implicated both the classical and alternative pathways in the pathogenesis of ischemia / reperfusion injury, while the role of the lectin pathway in this disease remains controversial (Riedermann, NC et al., Am. J. Pathol. 162:363-367, 2003).

[0009] Fibrosis is the excessive formation of connective tissue in an organ or tissue in response to damage or injury. The hallmark of fibrosis is the excessive production of extracellular matrix after local trauma. The normal physiological response to damage leads to the deposition of connective tissue, but this initially beneficial repair process may continue and become pathological, changing the architecture and function of the tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased rigidity, microvascular compression and hypoxia. The influx of inflammatory cells including macrophages and lymphocytes leads to cytokine release and expands the deposition of collagen, fibronectin and other molecular markers of fibrosis. Using corticosteroids and immunosuppressive drugs, conventional treatment methods have largely targeted the inflammatory process of fibrosis. Unfortunately, these anti-inflammatory drugs have little or even no clinical effect. There is currently no effective treatment or therapeutic agent for fibrosis, but animal studies and anecdotal reports have both suggested that fibrotic tissue damage can be reversed (Tampe and Zeisberg, Nat Rev Nephrol, Vol. 10: 226-237, 2014).

[0010] The kidney has a limited ability to recover from damage. Various renal pathological conditions lead to local inflammation, which causes scarring and fibrosis of renal tissue. The continued drive of inflammatory stimuli causes tubulointerstitial inflammation and fibrosis and progressive renal impairment in chronic kidney disease. It progresses to end-stage renal failure and is associated with significant morbidity and mortality. Since tubulointerstitial fibrosis is the common endpoint of multiple renal pathological conditions, it represents a key target for the treatment of renal failure. Risk factors (such as proteinuria) that are independent of primary renal disease contribute to the development of renal fibrosis and the loss of renal excretion function by driving local inflammation, thereby enhancing disease progression.

[0011] Given the role of fibrosis in many diseases and conditions, such as tubulointerstitial fibrosis that leads to chronic kidney disease, there is an urgent need to develop therapeutically effective agents for treating diseases and conditions caused or exacerbated by fibrosis. Further in view of the paucity of new and existing treatments that target inflammatory profibrotic pathways in kidney disease, there is a need to develop therapeutically effective agents to treat, inhibit, prevent and / or reverse kidney fibrosis and thereby prevent progressive chronic kidney disease.

[0012] Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS coronavirus 2 or SARS-CoV-2), which is a virus closely related to the SARS virus (World Health Organization (World Health Organization), 2 / 11 / 2020, Novel Coronavirus Situation Report 22). Fever, dry cough, fatigue and shortness of breath may occur in those affected by COVID-19. Cases may progress to respiratory dysfunction, including pneumonia, severe acute respiratory syndrome and the death of the most vulnerable people (see, for example, Hui DS et al., Int J Infect Dis 91: 264-266, January 14, 2020). There is no vaccine or specific antiviral treatment, and its management involves symptomatic treatment and supportive care.

[0013] Influenza (also known as "flu") is an infectious disease caused by RNA influenza viruses. Symptoms of influenza virus infection can be mild to severe and include high fever, runny nose, sore throat, muscle and joint pain, headache, cough, and feeling tired. These symptoms usually begin two days after exposure to the virus and most last less than a week, however, a cough may persist for more than two weeks. (See "Influenza Seasonal," World Health Organization 6, November 6, 2018). Complications of influenza may 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 than the common cold and last longer. Most people recover fully in about one to two weeks, but others develop life-threatening complications, such as pneumonia. Therefore, influenza can be fatal, especially for the frail, young, and elderly who have compromised immune systems or long-term illnesses. See Hilleman MR, Vaccine. 20(25–26):3068–87 (2002).

[0014] Three of the four types of influenza viruses affect humans: A, B, and C (see "Types of Influenza Viruses Seasonal Influenza (Flu)," Centers for Disease Control and Prevention (CDC). September 27, 2017). Type D is not known to infect humans but 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. The serotypes of influenza A that have been confirmed in humans are: H1N1 (causing the “Spanish flu” of 1918 and the “swine flu” of 2009); H2N2 (causing the “Asian flu” of 1957), H3N2 (causing the “flu” that appeared in Hong Kong, China in 1968), H5N1 (causing the “avian flu” of 2004), H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9 and H6N1. See World Health Organization (June 30, 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), December 7, 2018).

[0015] Common symptoms of influenza virus (also known as flu), such as fever, headache, and fatigue, are the result of large amounts of proinflammatory cytokines and chemokines (such as interferon or tumor necrosis factor) produced by cells infected with influenza. 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 massive immune response can lead to a life-threatening cytokine storm. This effect has been proposed as the cause of the extraordinary lethality of both 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 trigger programmed cell death (apoptosis), see Clinical Respiratory Medicine, Elsevier Health Sciences. p. 311 (2012).

[0016] Therefore, there is an urgent need to develop therapeutically effective agents to treat, inhibit and / or prevent coronavirus-induced pneumonia and acute respiratory distress syndrome, as well as influenza virus-induced pneumonia and acute respiratory distress syndrome.

[0017] Overview

[0018] This summary is provided to introduce selected 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.

[0019] In one aspect, the present invention provides methods for treating, inhibiting, alleviating, or preventing acute respiratory distress syndrome in a mammalian subject infected with a coronavirus, comprising administering to the subject an amount of a MASP-2 inhibitory agent 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 inhibitory agent effective to improve at least one respiratory symptom (i.e., improve respiratory function). In one embodiment, the method comprises administering the composition to a subject infected with SARS-CoV-2. In one embodiment, the method comprises administering the composition to a subject suffering from COVID-19, such as a subject suffering from acute respiratory distress syndrome (ARDS) associated with COVID-19. In one embodiment, the method comprises administering the composition to a subject infected with SARS-CoV. In one embodiment, the method comprises administering the composition to a subject infected with MERS-CoV. In one embodiment, prior to administration of the MASP-2 inhibitory agent, the subject is identified as having a coronavirus (i.e., SARS-CoV-2, SARS-CoV, or MERS-CoV). In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or an antigen-binding fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits lectin pathway complement activation while not substantially inhibiting C1q-dependent complement activation. In one embodiment, the MASP-2 inhibitory agent is a small molecule, such as a synthetic or semisynthetic small molecule, that inhibits MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory agent is an inhibitor of MASP-2 expression. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or fragment thereof is selected from 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% of human serum (i.e., normal human serum), wherein IC 5030 nM or less. In 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 set forth in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence set forth 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 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.

[0020] In another aspect, the present invention provides methods for treating, inhibiting, alleviating, or preventing acute respiratory distress syndrome in a mammalian subject infected with an influenza virus, comprising administering to the subject an amount of a MASP-2 inhibitory agent 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 inhibitory agent effective to improve at least one respiratory symptom (i.e., improve respiratory function). In one embodiment, the subject is infected with an influenza virus selected from influenza A, influenza B, and influenza C. In one embodiment, the subject is infected with influenza A. The subject is infected with an influenza A serotype selected from the group consisting of: H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and H6N1. In one embodiment, prior to administration of the MASP-2 inhibitory agent, the subject is identified as having the influenza virus. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or antigen-binding fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits lectin pathway complement activation while not substantially inhibiting C1q-dependent complement activation. In one embodiment, the MASP-2 inhibitory agent is a small molecule, such as a synthetic or semisynthetic small molecule, that inhibits MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory agent is an inhibitor of MASP-2 expression. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or fragment thereof is selected from 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% of human serum with an IC50 of 30 nM or less. In 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 set forth in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence set forth 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 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.

[0021] In another aspect, the present invention provides a method for treating, inhibiting, reducing, or preventing fibrosis in a mammalian subject having, or at risk for developing, a disease or condition caused by or exacerbated by fibrosis and / or inflammation, such as a coronavirus infection, comprising administering to the subject a MASP-2 inhibitory agent in an amount effective to inhibit fibrosis. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits lectin pathway complement activation while not substantially inhibiting C1q-dependent complement activation.

[0022] In another aspect, the present invention provides a method for treating, inhibiting, ameliorating, or preventing acute respiratory distress syndrome, pneumonia, or some other pulmonary or other manifestation of coronavirus infection, such as thrombosis, in a mammalian subject infected with a coronavirus, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent is a small molecule MASP-2 inhibitory compound.

[0023] In another aspect, the present invention provides a method for treating a human subject suffering from COVID-19-induced acute respiratory distress syndrome (ARDS) or pneumonia, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In one embodiment, the subject is on a mechanical ventilator (invasive or non-invasive) prior to treatment, and the MASP-2 inhibitory agent is administered at a dose and for a period of time sufficient to discontinue the need for mechanical ventilation. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent is a small molecule MASP-2 inhibitory compound.

[0024] In another aspect, the present invention provides a method for treating, preventing, or reducing the severity of coagulation or thrombosis in a human subject infected with SARS-CoV-2, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to treat, prevent, or reduce the severity of coagulation or thrombosis in the subject. In one embodiment, the subject has a D-dimer level above the normal range prior to treatment, and the MASP-2 inhibitory agent is administered in an amount and for a time sufficient to reduce the subject's D-dimer level to within the normal range for healthy subjects. In one embodiment, the MASP-2 inhibitory agent provides an anticoagulant and / or antithrombotic effect without affecting hemostasis. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent is a small molecule MASP-2 inhibitory compound.

[0025] In another aspect, the present invention provides a method for treating, ameliorating, preventing, or reducing the risk of developing one or more long-term sequelae associated with COVID-19 in a human subject currently infected with or previously infected with SARS-CoV-2, the method comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In one embodiment, the subject has COVID-19-induced pneumonia or ARDS, and the MASP-2 inhibitory agent is administered in an amount effective to improve respiratory function. In one embodiment, the subject has recovered from COVID-19-induced pneumonia or ARDS, and the MASP-2 inhibitory agent is administered in an amount to treat or ameliorate one or more long-term sequelae. In one embodiment, the subject has COVID-19-induced coagulation or thrombosis, and the MASP-2 inhibitory agent is administered to the subject in an amount effective to treat, prevent, or reduce the severity of coagulation or thrombosis in the subject. In one embodiment, the subject has recovered from COVID-19-induced coagulation or thrombosis, and the MASP-2 inhibitory agent is administered in an amount effective to treat or ameliorate one or more long-term sequelae. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO: 6. In one embodiment, the MASP-2 inhibitory agent is a small molecule MASP-2 inhibitory compound.

[0026] In another aspect, the present invention provides a method for treating, inhibiting, ameliorating, or preventing acute respiratory distress syndrome, pneumonia, or some other pulmonary or other manifestation of influenza virus infection in a mammalian subject infected with an influenza virus, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In one embodiment, the influenza virus is influenza A, influenza B, or influenza C. In one embodiment, the influenza A virus is selected from H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and H6N1. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent is a small molecule MASP-2 inhibitory compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The foregoing aspects of the present invention and many of the attendant advantages will become more readily understood as the same become more readily appreciated by reference to the following detailed description when considered in conjunction with the accompanying drawings, in which:

[0028] Figure 1 is a diagram showing the genomic structure of human MASP-2;

[0029] Figure 2A is a schematic diagram showing the domain structure of human MASP-2 protein;

[0030] Figure 2B is a schematic diagram showing the domain structure of human MAp19 protein;

[0031] Figure 3 is a diagram illustrating the murine MASP-2 knockout strategy;

[0032] Figure 4 is a diagram showing the human MASP-2 minigene construct;

[0033] Figure 5A The results presented demonstrate that, as described in Example 2, MASP-2 deficiency results in the loss of lectin pathway-mediated C4 activation as measured by the absence of C4b deposition on mannan;

[0034] Figure 5B The results presented demonstrate that, as described in Example 2, MASP-2 deficiency results in the loss of lectin pathway-mediated C4 activation as measured by the absence of C4b deposition on zymosan;

[0035] Figure 5CThe results presented demonstrate the relative levels of C4 activation 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;

[0036] Figure 6 The results presented demonstrate that, as described in Example 2, addition of murine 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;

[0037] Figure 7 The results presented demonstrate that, as described in Example 8, the classical pathway is functional in the MASP-2- / - strain;

[0038] Figure 8A The results presented demonstrate that, as described in Example 10, anti-MASP-2 Fab2 antibody #11 inhibits C3 convertase formation;

[0039] Figure 8B The results presented demonstrate that, as described in Example 10, anti-MASP-2 Fab2 antibody #11 binds to native rat MASP-2;

[0040] Figure 8C The results presented demonstrate that, as described in Example 10, anti-MASP-2 Fab2 antibody #41 inhibits C4 cleavage;

[0041] Figure 9 The results presented demonstrate that, as described in Example 10, all anti-MASP-2 Fab2 antibodies tested that inhibited C3 convertase formation were also found to inhibit C4 cleavage;

[0042] Figure 10 is a diagram showing recombinant polypeptides derived from rat MASP-2 used for epitope mapping of MASP-2 blocking Fab2 antibodies, as described in Example 11;

[0043] Figure 11 The results presented demonstrate binding of anti-MASP-2 Fab2 #40 and #60 to rat MASP-2 polypeptide as described in Example 11;

[0044] Figure 12A The graph shows the level of MAC deposition in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under lectin pathway specific assay conditions, demonstrating that OMS646 inhibits lectin-mediated MAC deposition with an IC 50 The value is approximately 1 nM, as described in Example 12;

[0045] Figure 12B graphically depicts the levels of MAC deposition in the presence or absence of a human MASP-2 monoclonal antibody (OMS646) under classical pathway-specific assay conditions, demonstrating that OMS646 does not inhibit classical pathway-mediated MAC deposition, as described in Example 12;

[0046] Figure 12C graphically depicts the levels of MAC deposition in the presence or absence of a human MASP-2 monoclonal antibody (OMS646) under alternative pathway-specific assay conditions, demonstrating that OMS646 does not inhibit alternative pathway-mediated MAC deposition, as described in Example 12;

[0047] Figure 13 Presented graphically is the pharmacokinetic (PK) profile of a human MASP-2 monoclonal antibody (OMS646) in mice, showing the concentration of OMS646 as a function of time following administration at the indicated doses (mean of n=3 animals / group), as described in Example 12;

[0048] Figure 14A Graphically depicted are the pharmacodynamic (PD) responses of a human MASP-2 monoclonal antibody (OMS646) measured as a decrease in systemic lectin pathway activity in mice following intravenous administration, as described in Example 12;

[0049] Figure 14B Graphically depicted are the pharmacodynamic (PD) responses of a human MASP-2 monoclonal antibody (OMS646) measured as a decrease in systemic lectin pathway activity in mice following subcutaneous administration, as described in Example 12;

[0050] Figure 15 Graphically depicted are the results of computer-based image analysis of Sirius Red-stained kidney tissue sections obtained from wild-type and MASP-2- / - mice 7 days after unilateral ureteral obstruction (UUO), and sham-operated wild-type and MASP-2- / - mice, as described in Example 14;

[0051] Figure 16 Shown graphically are the results of computer-based image analysis of kidney tissue sections stained with the F4 / 80 macrophage-specific antibody, obtained from wild-type and MASP-2- / - mice 7 days after unilateral ureteral obstruction (UUO), and sham-operated wild-type and MASP-2- / - mice, as described in Example 14.

[0052] Figure 17Shown graphically are the relative mRNA expression levels of collagen-4 in kidney tissue sections obtained from wild-type and MASP-2- / - mice 7 days after unilateral ureteral obstruction (UUO), and sham-operated wild-type and MASP-2- / - mice, as measured by quantitative PCR (qPCR), as described in Example 14.

[0053] Figure 18 Shown graphically are the relative mRNA expression levels of transforming growth factor beta-1 (TGFβ-1) in kidney tissue sections obtained from wild-type and MASP-2- / - mice 7 days after unilateral ureteral obstruction (UUO), and sham-operated wild-type and MASP-2- / - mice, as measured by qPCR, as described in Example 14.

[0054] Figure 19 Shown graphically are 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 7 days after unilateral ureteral obstruction (UUO), and sham-operated wild-type and MASP-2- / - mice, as described in Example 14.

[0055] Figure 20 Shown graphically are the relative mRNA expression levels of interferon-γ in kidney tissue sections obtained from wild-type and MASP-2- / - mice 7 days after unilateral ureteral obstruction (UUO), and sham-operated wild-type and MASP-2- / - mice, as measured by qPCR, as described in Example 14.

[0056] Figure 21 Shown graphically are the results of computer-based image analysis of Sirius Red-stained kidney tissue sections obtained from wild-type mice treated with a MASP-2 inhibitory antibody and an isotype control antibody 7 days after unilateral ureteral obstruction (UUO), as described in Example 15.

[0057] Figure 22 Shown graphically are hydroxyproline levels in kidneys harvested 7 days after unilateral ureteral obstruction (UUO) from wild-type mice treated with a MASP-2 inhibitory antibody, as described in Example 15, compared to hydroxyproline levels in tissue from obstructed kidneys from wild-type mice treated with an IgG4 isotype control.

[0058] Figure 23Shown graphically are the total amount of serum protein (mg / ml) measured on day 15 of a protein overload study in wild-type control mice that received saline only (n=2), wild-type mice that received BSA (n=6), and MASP-2- / - mice that received BSA (n=6), as described in Example 16.

[0059] Figure 24 Shown graphically is 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 receiving saline only (n=2), wild-type mice receiving BSA (n=6), and MASP-2- / - mice receiving BSA (n=6), as described in Example 16.

[0060] Figure 25 Shown are representative hematoxylin and eosin (H&E)-stained kidney tissue sections from the following groups of mice on day 15 of a protein overload study: (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), as described in Example 16.

[0061] Figure 26 Shown graphically are the results of computer-based image analysis of kidney tissue sections stained with the macrophage-specific antibody F4 / 80, obtained on day 15 of a protein overload study from wild-type control mice (n=2), wild-type mice treated with BSA (n=6), and MASP-2- / - mice treated with BSA (n=5), as described in Example 16, showing the mean staining area (%) of macrophages.

[0062] Figure 27A Graphically shown is the analysis of the presence of a macrophage-proteinuria association in each wild-type mouse (n=6) treated with BSA by plotting total excreted protein measured in urine from 24-hour samples relative to macrophage infiltration (mean % stained area), as described in Example 16.

[0063] Figure 27B Graphically shown is the analysis of the presence of a macrophage-proteinuria association in each of the MASP-2- / - mice (n=5) treated with BSA by plotting total excreted protein in the urine of a 24-hour sample relative to macrophage infiltration (mean % stained area), as described in Example 16.

[0064] Figure 28Shown graphically are the results of computer-based 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.

[0065] Figure 29 Shown graphically are the results of computer-based image analysis of tissue sections stained with anti-TNFα antibody (measured as % TNFα 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.

[0066] Figure 30 Shown graphically are the results of computer-based 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), as described in Example 16.

[0067] Figure 31 Shown graphically are the frequencies of TUNEL apoptotic cells counted in 20 consecutively selected high power fields (HPF) from tissue sections from the renal cortex in wild type control mice (n=1), MASP-2- / - control mice (n), wild type mice treated with BSA (n=7), and MASP-2- / - mice treated with BSA (n=7), as described in Example 16.

[0068] Figure 32 Shown are representative H&E-stained tissue sections from the following groups of mice at day 15 following treatment with BSA: (Panel A) wild-type control mice treated with saline, (Panel B) control mice treated with isotype antibody, and (Panel C) wild-type mice treated with a MASP-2 inhibitory antibody, as described in Example 17.

[0069] Figure 33 Shown graphically are the frequencies of TUNEL apoptotic cells counted in 20 consecutively selected high power fields (HPF) from tissue sections from the renal cortex in 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 inhibitory antibody and BSA (n=7), as described in Example 17.

[0070] Figure 34Shown graphically are the results of computer-based image analysis of tissue sections stained with anti-TGFβ antibody (measured as % TGFβ antibody stained area) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and an isotype control antibody (n=7), and wild-type mice treated with BSA and a MASP-2 inhibitory antibody (n=8), as described in Example 17.

[0071] Figure 35 Shown graphically are the results of computer-based image analysis of tissue sections stained with anti-TNFα antibody (measured as % TNFα antibody stained area) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and an isotype control antibody (n=7), and wild-type mice treated with BSA and a MASP-2 inhibitory antibody (n=8), as described in Example 17.

[0072] Figure 36 Shown graphically are the results of computer-based image analysis of tissue sections stained with anti-IL-6 antibody (measured as % IL-6 antibody stained area) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and an isotype control antibody (n=7), and wild-type mice treated with BSA and a MASP-2 inhibitory antibody (n=8), as described in Example 17.

[0073] Figure 37 Shown are representative H&E-stained tissue sections from the following groups of mice on day 14 after treatment with doxorubicin or saline alone (control): (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 doxorubicin; and (Panels C-1, C-2, C-3) MASP-2- / - mice treated with doxorubicin, as described in Example 18.

[0074] Figure 38 Graphically depicted are the results of computer-based image analysis of kidney tissue sections stained with the macrophage-specific antibody F4 / 80, showing the mean stained area (%) of macrophages from the following groups of mice on day 14 following treatment with either doxorubicin alone or saline (wild-type control): wild-type control mice treated with saline alone; wild-type mice treated with doxorubicin; MASP-2- / - mice treated with saline alone, and MASP-2- / - mice treated with doxorubicin, wherein **p=0.007, as described in Example 18;

[0075] Figure 39Presented graphically are the results of computer-based image analysis of kidney tissue sections stained with Sirius Red, showing the stained area (%) of collagen deposition at day 14 following treatment with either doxorubicin alone or saline (wild-type control) from the following groups of mice: wild-type control mice treated with saline alone; wild-type mice treated with doxorubicin; MASP-2- / - mice treated with saline alone, and MASP-2- / - mice treated with doxorubicin, wherein **p=0.005, as described in Example 18; and

[0076] Figure 40 Shown graphically are the urine albumin / creatinine ratios (uACR) in two IgA patients during the course of a twelve-week study of weekly treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 19.

[0077] Figure 41A Representative images of immunohistochemical analysis of septal vessel tissue sections from COVID-19 patient lungs (H&E, 400x) are shown, as described in Example 21.

[0078] Figure 41B Representative images of immunohistochemical analysis of septal vessel tissue sections from COVID-19 patient lungs (H&E, 400x) are shown, as described in Example 21.

[0079] Figure 41C Representative images of immunohistochemical analysis of tissue sections of medium-diameter pulmonary septal vessels from COVID-19 patients, as described in Example 21, are shown.

[0080] Figure 41D Representative images of immunohistochemical analysis of liver parenchymal tissue sections from COVID-19 patients are shown (H&E, 400x), as described in Example 21.

[0081] Figure 42A Graphically shown are circulating endothelial cell (CEC) / ml counts in the peripheral blood of normal healthy controls (n-6) compared to CEC / ml counts in COVID-19 patients (n=33) who were not part of this study, as described in Example 21.

[0082] Figure 42B Graphically shown are the CEC / ml counts in 6 patients selected for this study before (baseline) and after treatment with narsoplimab, with the boxes representing the values ​​from the first quartile to the third quartile, the horizontal line showing the median, and the whiskers indicating the minimum and maximum values, as described in Example 21.

[0083] Figure 43 The serum levels of C-reactive protein (CRP) in six COVID-19 patients at baseline before treatment (day 0) and at different time points after treatment with nasolizumab are shown graphically (median; interquartile range (IQR)), as described in Example 21.

[0084] Figure 44 Graphically shown are serum levels (median; IQR) of lactate dehydrogenase (LDH) in six COVID-19 patients at baseline before treatment (day 0) and at different time points after treatment with nasolizumab, as described in Example 21.

[0085] Figure 45 Graphically shown are serum levels (median; interquartile range (IQR)) of interleukin 6 (IL-6) in six COVID-19 patients at baseline before treatment (day 0) and at different time points after treatment with nasolizumab, as described in Example 21.

[0086] Figure 46 Graphically shown are serum levels (median; interquartile range (IQR)) of interleukin 8 (IL-8) in six COVID-19 patients at baseline before treatment (day 0) and at different time points after treatment with nasolizumab, as described in Example 21.

[0087] Figure 47A Shown is a CT scan of patient #4 on day 5 from enrollment (i.e., after treatment with nasolizumab), in which the patient was observed to have severe interstitial pneumonia with diffuse ground-glass opacities involving both peripheral and central areas, especially consolidation in the lower lobe of the left lung, and massive bilateral pulmonary emboli with filling defects in the interlobar and segmental arteries (not shown), as described in Example 21.

[0088] Figure 47B Shown is a CT scan of patient #4 on day 16 from enrollment (ie, after treatment with nasolizumab), with a significant reduction in ground-glass opacities and almost complete resolution of parenchymal consolidation, as described in Example 21.

[0089] Figure 48 The serum levels of IL-6 (pg / mL) in COVID-19 patients treated with nasolizumab at baseline and at different time points after nasolizumab treatment (after 2 doses, after 4 doses) are graphically shown, where the boxes represent the values ​​from the first quartile to the third quartile, the horizontal line shows the median, and the dots show the values ​​for all patients, as described in Example 21.

[0090] Figure 49 The serum levels of IL-8 (pg / mL) in COVID-19 patients treated with nasolizumab at baseline and at different time points after nasolizumab treatment (after 2 doses, after 4 doses) are graphically shown, where the boxes represent the values ​​from the first quartile to the third quartile, the horizontal line shows the median, and the dots show the values ​​for all patients, as described in Example 21.

[0091] Figure 50 Shown graphically are the clinical outcomes of six COVID-19 patients treated with nasolizumab, as described in Example 21.

[0092] Figure 51A The serum levels of aspartate aminotransferase (AST) before and after nasolizumab treatment are shown graphically (units / liter, U / L). The black line represents the median and interquartile range (IQR). The red line represents the normal level, and the dots show the values ​​for all patients, as described in Example 21.

[0093] Figure 51B The following table shows the serum levels of D-dimer (ng / ml) in four COVID-19 patients for whom baseline values ​​were available before the start of nasolizumab treatment. The black circles indicate when steroid treatment was initiated. The red line represents normal levels, as described in Example 21.

[0094] Figure 52A Graphically shown are serum levels of D-dimer values ​​(ng / ml) in the seventh COVID-19 patient treated with nasolizumab (patient #7) at baseline before treatment (day 0) and at different time points after treatment with nasolizumab, where administration of nasolizumab is indicated by a vertical arrow and where the horizontal line represents normal levels, as described in Example 22.

[0095] Figure 52B Graphically shown are serum levels of C-reactive protein (CRP) in patient #7 with COVID-19 at baseline before treatment (Day 0) and at different time points after treatment with nasolizumab, where administration of nasolizumab is indicated by vertical arrows and where the horizontal line represents normal levels, as described in Example 22.

[0096] Figure 52CThe serum levels of aspartate aminotransferase (AST) (units / liter, U / L) in patient #7 with COVID-19 at baseline before treatment (day 0) and at different time points after nasolizumab treatment are shown graphically, where the administration of nasolizumab is indicated by the vertical arrows and where the horizontal line represents normal levels, as described in Example 22.

[0097] Figure 52D The serum levels of alanine aminotransferase (ALT) (units / liter, U / L) in patient #7 with COVID-19 at baseline before treatment (day 0) and at different time points after nasolizumab treatment are shown graphically, where the administration of nasolizumab is indicated by the vertical arrows and where the horizontal line represents normal levels, as described in Example 22.

[0098] Figure 52E Graphically shown are serum levels of lactate dehydrogenase (LDH) in patient #7 with COVID-19 at baseline before treatment (Day 0) and at different time points after treatment with nasolizumab, where administration of nasolizumab is indicated by vertical arrows and where the horizontal line represents normal levels, as described in Example 22.

[0099] Figure 53 The anti-SARS-CoV-2 antibody titers of patient #7 over time are shown graphically, indicating that treatment with nasolizumab did not hinder the effector functions of the adaptive immune response, as described in Example 22.

[0100] Figure 54 Schematic diagram of the study design described in Example 19.

[0101] Description of Sequence Listing

[0102] SEQ ID NO: 1 Human MAp19 cDNA

[0103] SEQ ID NO: 2 Human MAp19 protein (with leader region)

[0104] SEQ ID NO: 3 Human MAp19 protein (mature)

[0105] SEQ ID NO:4 human MASP-2 cDNA

[0106] SEQ ID NO: 5 Human MASP-2 protein (with leader region)

[0107] SEQ ID NO:6 Human MASP-2 protein (mature)

[0108] SEQ ID NO:7 Human MASP-2 gDNA (exons 1-6)

[0109] Antigen: (refer to MASP-2 mature protein)

[0110] SEQ ID NO:8CUBI sequence (aa 1-121)

[0111] SEQ ID NO:9CUBEGF sequence (aa 1-166)

[0112] SEQ ID NO:10CUBEGFCUBII(aa 1-293)

[0113] SEQ ID NO:11EGF region (aa 122-166)

[0114] SEQ ID NO: 12 Serine protease domain (aa429–671)

[0115] SEQ ID NO: 13 Inactive serine protease domain (aa 610-625, with Ser618 to Ala mutation)

[0116] SEQ ID NO: 14TPLGPKWPEPVFGRL (CUBI peptide)

[0117] SEQ ID NO:15TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI peptide)

[0118] SEQ ID NO: 16TFRSDYSN (MBL binding region core)

[0119] SEQ ID NO: 17FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding region)

[0120] SEQ ID NO: 18 IDECQVAPG (EGF peptide)

[0121] SEQ ID NO: 19ANMLCAGLESGGKDSCRGDSGGALV (serine protease binding core) peptide inhibitor:

[0122] SEQ ID NO: 20MBL full-length cDNA

[0123] SEQ ID NO: 21MBL full-length protein

[0124] SEQ ID NO:22 OGK-X-GP (common region binding)

[0125] SEQ ID NO:23OGKLG

[0126] SEQ ID NO:24GLR GLQ GPO GKL GPO G

[0127] SEQ ID NO:25GPO GPO GLR GLQ GPO GKL GPO GPO GPO

[0128] SEQ ID NO:26GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG

[0129] SEQ ID NO: 27 GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-ficolin)

[0130] SEQ ID NO:28GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPNGAOGEO (human ficolin p35)

[0131] SEQ ID NO:29LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site)

[0132] Expression inhibitors:

[0133] SEQ ID NO:30 cDNA of CUBI-EGF domain (nucleotides 22-680 of SEQ ID NO:4)

[0134] SEQ ID NO:31

[0135] 5'CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC3'

[0136] Nucleotides 12-45 (sense) of SEQ ID NO: 4 including the MASP-2 translation start site

[0137] SEQ ID NO:32

[0138] 5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3'

[0139] Nucleotides 361-396 (sense) of SEQ ID NO: 4 encoding the region containing the MASP-2 MBL binding site

[0140] SEQ ID NO:33

[0141] 5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3'

[0142] Nucleotides 610-642 of SEQ ID NO: 4 encoding the region containing the CUBII domain

[0143] Cloning primers:

[0144] SEQ ID NO:34CGGGATCCATGAGGCTGCTGACCCTC (5' PCR for CUB)

[0145] SEQ ID NO:35GGAATTCCTAGGCTGCATA (3' PCR for CUB)

[0146] SEQ ID NO:36GGAATTCCTACAGGGCGCT (3' PCR for CUBIEGF)

[0147] SEQ ID NO:37GGAATTCCTAGTAGTGGAT (3' PCR for CUBIEGFCUBII)

[0148] SEQ ID NOS:38-47 are cloning primers for humanized antibodies

[0149] SEQ ID NO: 48 is a 9aa peptide

[0150] Expression vector:

[0151] SEQ ID NO:49 is the MASP-2 minigene insert

[0152] SEQ ID NO:50 is the mouse MASP-2 cDNA

[0153] SEQ ID NO:51 is murine MASP-2 protein (w / leader region)

[0154] SEQ ID NO:52 is the mature murine MASP-2 protein

[0155] SEQ ID NO: 53 Rat MASP-2 cDNA

[0156] SEQ ID NO:54 is rat MASP-2 protein (w / leader region)

[0157] SEQ ID NO: 55 is the mature rat MASP-2 protein

[0158] SEQ ID NOs: 56-59 are oligonucleotides used for site-directed mutagenesis of human MASP-2, which were used to generate human MASP-2A

[0159] SEQ ID NOs: 60-63 are oligonucleotides used for site-directed mutagenesis of murine MASP-2, which were used to generate murine MASP-2A

[0160] SEQ ID NOs: 64-65 are oligonucleotides used for site-directed mutagenesis of rat MASP-2, which were used to generate rat MASP-2A

[0161] SEQ ID NO:66 DNA encoding the heavy chain variable region (VH) of 17D20_dc35VH21N11VL (OMS646) (without the signal peptide)

[0162] SEQ ID NO:6717D20_dc35VH21N11VL(OMS646) heavy chain variable region (VH) polypeptide

[0163] SEQ ID NO:6817N16mc heavy chain variable region (VH) polypeptide

[0164] SEQ ID NO:6917D20_dc35VH21N11VL(OMS646) light chain variable region (VL) polypeptide

[0165] SEQ ID NO:70 DNA encoding the light chain variable region (VL) of 17D20_dc35VH21N11VL (OMS646)

[0166] SEQ ID NO:7117N16_dc17N9 light chain variable region (VL) polypeptide

[0167] SEQ ID NO:72: SGMI-2L (full length)

[0168] SEQ ID NO:73: SGMI-2M (medium truncated form)

[0169] SEQ ID NO:74: SGMI-2S (truncated form)

[0170] SEQ ID NO: 75: Mature polypeptide comprising VH-M2ab6-SGMI-2-N and a human IgG4 constant region with a hinge mutation

[0171] SEQ ID NO: 76: Mature polypeptide comprising VH-M2ab6-SGMI-2-C and a human IgG4 constant region with a hinge mutation

[0172] SEQ ID NO: 77: Mature polypeptide comprising VL-M2ab6-SGMI-2-N and human Igλ constant region

[0173] SEQ ID NO: 78: Mature polypeptide comprising VL-M2ab6-SGMI-2-C and human Igλ constant region

[0174] SEQ ID NO:79: Peptide linker (10aa)

[0175] SEQ ID NO:80: Peptide linker (6aa)

[0176] SEQ ID NO:81: Peptide linker (4aa)

[0177] SEQ ID NO: 82: Polynucleotide encoding a polypeptide comprising VH-M2ab6-SGMI-2-N and a human IgG4 constant region with a hinge mutation

[0178] SEQ ID NO: 83: Polynucleotide encoding a polypeptide comprising VH-M2ab 6-SGMI-2-C and a human IgG4 constant region with a hinge mutation

[0179] SEQ ID NO: 84: Polynucleotide encoding a polypeptide comprising VL-M2ab6-SGMI-2-N and a human Igλ constant region

[0180] SEQ ID NO: 85: Polynucleotide encoding a polypeptide comprising VL-M2ab6-SGMI-2-C and a human Igλ constant region Details

[0182] The present invention is based on the inventors' surprising discovery that inhibition of mannan-binding lectin-associated serine protease-2 (MASP-2), a key regulator of the complement system's lectin pathway, significantly reduces inflammation and fibrosis in various animal models of fibrotic disease, including a unilateral ureteral obstruction (UUO) model, a protein overload model, and an adriamycin-induced renal fibrosis nephropathy model. Thus, the inventors have demonstrated that inhibition of MASP-2-mediated lectin pathway activation provides an effective therapeutic approach to ameliorate, treat, or prevent renal fibrosis, such as tubulointerstitial inflammation and fibrosis, regardless of the underlying cause. As further described herein, the use of a MASP-2 inhibitory antibody (OMS646) effectively improves renal function and reduces corticosteroid requirements in human subjects with immunoglobulin A nephropathy (IgAN) and membranous nephropathy (MN). As further described herein, use of MASP-2 inhibitory agents may also be used to treat, inhibit, alleviate, or prevent acute respiratory distress syndrome in subjects infected with a coronavirus, such as SARS-CoV-2, and may also be used to treat, inhibit, alleviate, or prevent acute respiratory distress in subjects infected with an influenza virus.

[0183] I. Definition

[0184] Unless specifically defined herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art in the field of the present invention. The following definitions are provided to provide clarity regarding the terms used in the specification and claims to describe the present invention.

[0185] As used herein, the term "MASP-2-dependent complement activation" encompasses MASP-2-dependent activation of the lectin pathway, which under physiological conditions (i.e., in the presence of Ca ++ ), leading to the formation of the lectin pathway C3 convertase C4b2a, and following accumulation of the C3 cleavage product C3b, subsequently leading to the formation of the C5 convertase C4b2a(C3b)n, which has been identified as primarily responsible for opsonization.

[0186] As used herein, the term "alternative pathway" refers to complement activation triggered, for example, by zymosan from the cell walls of fungi and yeast, lipopolysaccharide (LPS) from the Gram-negative outer membrane and rabbit erythrocytes, as well as from many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites, and damaged cells, and which has traditionally been thought to arise from the spontaneous proteolytic generation of C3b from complement factor C3.

[0187] As used herein, the term "lectin pathway" refers to complement activation that occurs through 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).

[0188] As used herein, the term "classical pathway" refers to complement activation that is triggered by antibodies bound to foreign particles and requires binding of the recognition molecule CIq.

[0189] As used herein, the term "MASP-2 inhibitory agent" refers to any agent that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation, including anti-MASP-2 antibodies and MASP-2-binding fragments thereof, natural and synthetic peptides, small molecules, soluble MASP-2 receptors, expression inhibitors, and isolated natural inhibitors. It also includes peptides that compete with MASP-2 for binding to another recognition molecule in the lectin pathway (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin), but does not include antibodies that bind to such other recognition molecules. The MASP-2 inhibitory agents useful in the methods of the present invention can reduce MASP-2-dependent complement activation by greater than 20%, for example, greater than 50%, for example, greater than 90%. In one embodiment, the MASP-2 inhibitory agent reduces MASP-2-dependent complement activation by greater than 90% (i.e., results in only 10% or less MASP-2 complement activation).

[0190] As used herein, the term "fibrosis" refers to the formation or presence of excess connective tissue in an organ or tissue. Fibrosis may occur as a response to the repair or replacement of stimuli such as tissue damage or inflammation. The hallmark of fibrosis is the production of excessive extracellular matrix. Normal physiological responses to injury result in the deposition of connective tissue as part of the healing process, but this connective tissue deposition may continue and become pathological, changing the architecture and function of the tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, causing matrix contraction, increased rigidity, microvascular compression, and hypoxia.

[0191] As used herein, the term "treating fibrosis in a mammalian subject having or at risk of developing a disease or condition caused by or exacerbated by fibrosis and / or inflammation" refers to reversing, alleviating, ameliorating or inhibiting fibrosis in the mammalian subject.

[0192] As used herein, the term "proteinuria" refers to the presence of urinary protein in abnormal amounts, such as an amount exceeding 0.3 g protein in a 24-hour urine collection from a human subject, or a concentration greater than 1 g / liter in a human subject.

[0193] As used herein, the term "ameliorating proteinuria" or "reducing proteinuria" refers to reducing 24-hour urinary protein excretion in a subject with 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 subject's baseline 24-hour urinary protein excretion prior to treatment with the MASP-2 inhibitory agent. In one embodiment, treatment with a MASP-2 inhibitory agent according to the methods of the invention is effective to reduce proteinuria in a human subject so as to achieve greater than a 20 percent reduction in 24-hour urinary protein excretion, or, such as greater than a 30 percent reduction in 24-hour urinary protein excretion, or, such as greater than a 40 percent reduction in 24-hour urinary protein excretion, or, such as greater than a 50 percent reduction in 24-hour urinary protein excretion).

[0194] As used herein, the terms "small molecule," "organic small molecule," and "inorganic small molecule" refer to molecules (organic, organometallic, or inorganic), organic molecules, and inorganic molecules, respectively, that are naturally occurring or synthetic and have a molecular weight greater than about 50 Da and less than about 2500 Da. Small organic (e.g.) 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.

[0195] As used herein, the term "antibody" includes antibodies and antibody fragments thereof that are derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate, including human), or hybridoma, phage selection, recombinant expression, or transgenic animal (or other methods of producing antibodies or antibody fragments), and that specifically bind to a target polypeptide, such as MASP-2, a polypeptide, or a portion thereof. The term "antibody" is not intended to be limiting with respect to the source of the antibody or the manner in which it is produced (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; pan-specific, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotypic antibodies, and can be any intact antibody or fragment thereof. As used herein, the term "antibody" includes not only intact polyclonal antibodies or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chains (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion of an antigen-binding fragment with the desired specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site or fragment (epitope recognition site) with the desired specificity.

[0196] "Monoclonal antibody" refers to a homogeneous antibody population, wherein the monoclonal antibody comprises amino acids (naturally occurring and non-naturally occurring) that are selectively bound to an epi-position. Monoclonal antibodies are highly specific for target antigens. The term "monoclonal antibody" not only comprises complete monoclonal antibodies and full-length monoclonal antibodies, but also comprises any other modified configuration of its fragment (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), its variant, fusion protein comprising antigen-binding portion thereof, humanized monoclonal antibodies, chimeric monoclonal antibodies and immunoglobulin molecules, which comprise antigen-binding fragments (epitope recognition sites) with the ability to bind to desired specificity and epi-positions. It is not expected to be restricted in terms of the source of the antibody or the mode (such as, by hybridoma, phage selection, recombinant expression, transgenic animals, etc.) in which it is prepared. The term includes complete immunoglobulins, and the fragments described above under the definition of "antibody" etc.

[0197] 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 its antigen-binding or variable region. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0198] As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the V H and V L domains, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a V H and V L A polypeptide linker between the domains enables the scFv to form the desired structure for antigen binding.

[0199] As used herein, a "chimeric antibody" is a recombinant protein that contains the variable domains and complementarity determining regions derived from an antibody of a non-human species (eg, a rodent), while the remainder of the antibody molecule is derived from a human antibody.

[0200] As used herein, a "humanized antibody" is a chimeric antibody comprising minimal sequence identical to the specific complementary determining regions derived from a non-human immunoglobulin, which has been grafted onto a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the complementary determining regions of the antibody are of non-human origin.

[0201] As used herein, the term "mannan binding lectin" ("MBL") is equivalent to mannan binding protein ("MBP").

[0202] As used herein, "membrane attack complex" ("MAC") refers to a complex of the terminal five complement components (C5b in combination with C6, C7, C8, and C-9) (also known as C5b-9) that inserts into and destroys membranes.

[0203] As used herein, "subject" includes all mammals, including but not limited to humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents.

[0204] As used herein, amino acid residues are abbreviated 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).

[0205] In the broadest sense, naturally occurring amino acids can be divided into groups based on the chemical characteristics of the side chains of the respective amino acids. "Hydrophobic" amino acids mean Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro. "Hydrophilic" amino acids mean Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His. This grouping of amino acids can be further subclassified as follows. "Non-charged hydrophilic" amino acids mean Ser, Thr, Asn, or Gln. "Acidic" amino acids mean Glu or Asp. "Basic" amino acids mean Lys, Arg, or His.

[0206] As used herein, the term "conservative amino acid substitution" is indicated by substitution of amino acids within 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.

[0207] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or its mimetics. The term also encompasses oligonucleobases composed of naturally occurring nucleotides, sugars, and covalent internucleoside (backbone) bonds, as well as oligonucleotides with non-naturally occurring modifications.

[0208] As used herein, an "epitope" refers to a site on a protein (e.g., a human MASP-2 protein) that is bound by an antibody. An "overlapping epitope" includes at least one (e.g., two, three, four, five, or six) common amino acid residues and includes both linear and non-linear epitopes.

[0209] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modifications. The MASP-2 proteins described herein may contain or be wild-type proteins, or may be variants having no more than 50 (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions generally 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.

[0210] In some embodiments, the human MASP-2 protein can have an amino acid sequence that is equal to or greater 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 set forth in SEQ ID NO:5.

[0211] In some embodiments, the peptide fragment can be 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, 4, 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 contiguous 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 peptides in length (e.g., less than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 5 7, 8, 7, or 6) amino acid residues (e.g., less than 500 contiguous amino acid residues in any one of SEQ ID NO: 5).

[0212] The percentage (%) of amino acid sequence identity is defined as the percentage of amino acids in the candidate sequence that are identical with the amino acids in the reference sequence, after the sequences are aligned and, if necessary, introduced into spaces to achieve maximum sequence identity. Alignment for determining the purpose of sequence identity percentage can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Suitable parameters for measuring the alignment can be determined by known methods, including any algorithm required for achieving maximum alignment over the full length of the sequences to be compared.

[0213] II. Summary of the Invention

[0214] As described herein, the inventors have identified a central role for the lectin pathway in the initiation and progression of tubulorenal pathologies, thereby implicating a key role for lectin pathway activation in the pathophysiology of a diverse range of renal diseases, including IgA nephropathy, C3 glomerulopathy, and other glomerulonephritides. As further described herein, the inventors have discovered that inhibition of mannan-binding lectin-associated serine protease-2 (MASP-2), a key regulator of the lectin pathway of the complement system, significantly reduces inflammation and fibrosis in various animal models of fibrotic disease, including a unilateral ureteral obstruction (UUO) model, a protein overload model, and a doxorubicin-induced renal fibrosis model. Thus, the inventors have demonstrated that inhibition of MASP-2-mediated lectin pathway activation provides an effective therapeutic approach to ameliorate, treat, or prevent renal fibrosis, such as tubulointerstitial fibrosis, regardless of the underlying cause. As further described herein, use of MASP-2 inhibitory agents can also be used to treat, inhibit, alleviate, or prevent acute respiratory distress syndrome in subjects infected with a coronavirus, such as SARS-CoV-2.

[0215] 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-priming pathway and the associated terminal pathway amplification loop, which amplifies the activation of terminal complement effector molecules initiated by lectins. C1q is a specific recognition molecule that triggers the acquired complement system, which includes the classical initiation pathway and the associated terminal pathway amplification loop, which amplifies the activation of terminal complement effector molecules initiated by C1q. We refer to these two major complement activation systems as the lectin-dependent complement system and the C1q-dependent complement system, respectively.

[0216] In addition to its essential role in immune defense, the complement system also contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects. The recognition that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact has led to the realization that it is highly desirable to specifically inhibit only the complement activation system responsible for a particular pathological condition, without completely shutting down complement's immune defense capabilities. For example, in disease states where complement activation is predominantly mediated by the lectin-dependent complement system, it would be advantageous to specifically inhibit only that system. This would leave the C1q-dependent complement activation system intact to handle immune complex processing and aid host defense against infection.

[0217] A preferred protein component to target in the development of therapeutics to 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 both unique to the lectin-dependent complement system and essential for its function. Lectins (MBL, H-ficolin, M-ficolin, L-ficolin, and CL-11) are also unique components of the lectin-dependent complement system. However, due to lectin redundancy, loss of any one lectin component does not necessarily inhibit activation of the system. Inhibition of all five lectins is necessary to ensure inhibition of the lectin-dependent complement activation system. Furthermore, since MBL and fibrin are also known to have complement-independent opsonic activities, inhibition of lectin function would result in loss of this beneficial host defense mechanism against infection. In contrast, if MASP-2 is the target of inhibition, this complement-independent lectin opsonic activity will remain intact. An additional benefit of MASP-2 as a therapeutic target for inhibition of the lectin-dependent complement activation system is that the plasma concentration of MASP-2 is the lowest of any complement protein (≈500 ng / ml); therefore, correspondingly low concentrations of high-affinity inhibitors of MASP-2 may be sufficient to achieve complete inhibition (Moller-Kristensen, M. et al., J. Immunol Methods 282:159-167, 2003).

[0218] As described herein in Example 14, mice lacking the MASP-2 gene (MASP-2- / -) demonstrated significantly less renal disease in an animal model of fibrotic kidney disease (unilateral ureteral obstruction, UUO) compared to wild-type control animals, as demonstrated by inflammatory cell infiltration (75% reduction) and histological markers of fibrosis, such as collagen deposition (one-third reduction). As further shown in Example 15, wild-type mice treated systemically with an anti-MASP-2 monoclonal antibody that selectively blocks the lectin pathway while leaving the classical pathway intact were protected from renal fibrosis compared to wild-type mice treated with an isotype control antibody. These results demonstrate that the lectin pathway is a key contributor to kidney disease and further demonstrate that MASP-2 inhibitory agents (e.g., MASP-2 antibodies) that block the lectin pathway are effective as anti-fibrotic agents. As further shown in Example 16, wild-type mice treated with bovine serum albumin (BSA) developed proteinuric nephropathy in a protein overload model, whereas MASP-2- / - mice treated with the same levels of BSA had reduced renal damage. As shown in Example 17, 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 in a protein overload model. As described in Example 18, MASP-2- / - mice exhibited less renal inflammation and tubulointerstitial damage in a doxorubicin-induced renal fibrosis model of nephropathy compared to wild-type mice. As described in Example 19, in an ongoing Phase 2 open-label renal trial, IgA nephropathy patients treated with anti-MASP-2 antibodies demonstrated clinically meaningful and statistically significant reductions in urine albumin / creatinine ratio (uACR) throughout the trial, as well as reductions in 24-hour urine protein levels from baseline to the end of treatment. As further described in Example 19, in the same Phase 2 renal trial, patients with membranous nephropathy treated with anti-MASP-2 antibodies also demonstrated a reduction in uACR during treatment.

[0219] In light of the foregoing, the present invention relates to the use of MASP-2 inhibitory agents, such as MASP-2 inhibitory antibodies, as anti-fibrotic agents, the use of MASP-2 inhibitory agents in the manufacture of a medicament for treating a fibrotic condition, and methods for preventing, treating, alleviating, or reversing a fibrotic condition in a human subject in need thereof, the methods comprising administering an effective amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) to the patient. As described in Examples 20, 21, and 22, clinical improvement was observed in patients with COVID-19-associated respiratory failure following treatment with nasolizumab, which inhibits MASP-2 and lectin pathway activation. As described in Example 21, all six COVID-19 patients treated with nasolizumab demonstrated clinical improvement. In each case, the COVID-19 lung injury had progressed to ARDS prior to nasolizumab treatment, and all patients were receiving non-invasive mechanical ventilation at the time of treatment initiation. The COVID-19 patients treated with nasolizumab, for which follow-up (5-6 months) data were available, did not show clinical or laboratory evidence of observed long-term sequelae. As further described in Example 22, other COVID-19 patients treated with nasolizumab also demonstrated clinical improvement. Similar to the first cohort, no long-term sequelae were observed in any of these other COVID-19 patients treated with nasolizumab as described herein. As further demonstrated in Example 22, patients treated with nasolizumab developed appropriately high titers of anti-SARS-Cov-2 antibodies, indicating that treatment with nasolizumab did not hinder the effector functions of the adaptive immune response.

[0220] Accordingly, the methods of the present invention can be used to treat, inhibit, alleviate, prevent or reverse coronavirus-induced pneumonia or acute respiratory distress syndrome in a human subject suffering from a coronavirus, such as COVID-19 due to SARS-CoV-2, SARS or MERS, as further described herein. The methods of the present invention can also be used to treat, inhibit, alleviate, prevent or reverse influenza virus-induced pneumonia or acute respiratory distress syndrome in a human subject suffering from an influenza virus, such as influenza A serotype (H1N1 (causing the "Spanish flu" of 1918 and the "swine flu" of 2009); H2N2 (causing the "Asian flu" of 1957), H3N2 (causing the "flu" that appeared in Hong Kong, China in 1968), H5N1 (causing the "avian flu" of 2004), H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9 and H6N1); or influenza B virus or influenza C virus.

[0221] III. The Role of MASP-2 in Diseases and Conditions Caused or Exacerbated by Fibrosis

[0222] Fibrosis is usually response damage or injury, excessive connective tissue formation or existence in an organ or tissue.The sign of fibrosis is the generation of excessive extracellular matrix after damage.In kidney, fibrosis is characterized by the progressive harmful connective tissue deposition on renal parenchyma, which inevitably leads to decreased renal function, and is independent of the primary renal disease causing original kidney damage.So-called epithelial-mesenchymal transition (EMT), in which renal tubular epithelial cells are converted into changes in the cell characteristics of mesenchymal fibroblasts, constitutes the primary mechanism of renal fibrosis.Fibrosis affects almost all tissues and organ systems, and may occur as a repair or replacement response to stimulation (such as tissue damage or inflammation).Normal physiological responses to damage lead to the deposition of connective tissue, but if this process becomes pathological, then highly differentiated cells change the architecture and function of tissue by the replacement of scarring connective tissue.At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, causing matrix contraction, increased rigidity, microvascular compression and hypoxia. There are currently no effective treatments or therapeutics for fibrosis, but both animal studies and anecdotal human reports suggest that fibrotic tissue damage can be reversed (Tampe and Zeisberg, Nat Rev Nephrol, Vol. 10:226-237, 2014).

[0223] Many diseases lead to fibrosis that causes progressive organ failure, including 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, endocardial fibrosis), brain (e.g., stroke), skin (e.g., excessive wound healing, scleroderma, systemic sclerosis, scar tissue), and ulcers. keloids), vascular system (e.g., atherosclerotic vascular disease), intestinal tract (e.g., Crohn's disease), eye (e.g., anterior subcapsular cataract, posterior capsule opacification), musculoskeletal soft tissue structures (e.g., adhesive capsulitis, Dupuytren's contracture, myelofibrosis), reproductive organs (e.g., endometriosis, Peyronie's disease), and certain infectious diseases (e.g., coronaviruses, alphaviruses, hepatitis C, hepatitis B, etc.).

[0224] Although fibrosis occurs in many tissues and diseases, there are common molecular and cellular mechanisms underlying its pathological conditions. Extracellular matrix deposition by fibroblasts is accompanied by immune cell infiltration, predominantly monocytes (see Wynn T., Nat Rev Immunol 4(8):583-594, 2004, incorporated herein by reference). A robust 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), degradative enzymes (elastase, matrix metalloproteinases, cathepsins), and extracellular matrix proteins (collagen, fibronectin, integrin).

[0225] In addition, the complement system becomes activated in many fibrotic diseases. Complement components, including the membrane attack complex, have been identified in many fibrotic tissue samples. For example, components of the lectin pathway have been found in the following fibrotic lesions: 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)).

[0226] Overshoot complement activation has been identified as a key contributor to immune complex-mediated and antibody-independent glomerulonephritis. However, there is a strong line of evidence that uncontrolled complement activation in situ is involved in the pathophysiological progression of TI fibrosis in non-glomerular diseases in nature (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 triggered by local complement activation can be initiated by complement components filtering into the proximal tubules and subsequently entering the interstitial space, or by aberrant synthesis of complement components by the tubular or other resident and infiltrating cells, or by aberrant expression of complement regulatory proteins on renal cells, or by absence or loss or gain of function mutations in complement regulatory components (Mathern DR et al., Clin J Am Soc Nephrol 10:P1636-1650, 2015, Sheerin NS et al., FASEBJ 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, with subsequent inflammation and injury typical of fibrosis 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 epithelial-mesenchymal transition (Tsang Z. et al., J Am Soc Nephrol 20:593-603, 2009). Blockade of C3a signaling via the C3a receptor alone has recently been shown to alleviate 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).

[0227] As described herein, the inventors have identified a central role for the lectin pathway in the initiation and progression of tubular renal pathologies, implicating a key role for lectin pathway activation in the pathophysiology of a diverse range of renal 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 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 Dis 45:295-302, 2005). 84:1079-1089, 2013), diabetic nephropathy (Hovind P. 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).

[0228] As further described herein, the inventors have demonstrated that MASP-2 inhibition reduces inflammation and fibrosis in a mouse model of tubulointerstitial disease. Thus, MASP-2 inhibitory agents 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.

[0229] Lung disease

[0230] Pulmonary fibrosis is the formation or development of excessive fibrous connective tissue in the lungs, in which normal lung tissue is replaced by fibrotic tissue. This scarring causes lung stiffness and damages lung structure and function. In humans, pulmonary fibrosis is believed to be caused by repeated damage to the tissue in and between the tiny air sacs (alveoli) in the lungs. Under experimental settings, various animal models have replicated various aspects of human disease. For example, exogenous agents such as bleomycin, fluorescein isothiocyanate, silica or asbestos can be instilled into the trachea of ​​animals (Gharaee-Kermani et al., Animal Models of Pulmonary Fibrosis. Methods Mol. Med., 2005, 117: 251-259).

[0231] Accordingly, in certain embodiments, the present disclosure provides methods for inhibiting pulmonary fibrosis in a subject suffering from a pulmonary disease or condition caused by or exacerbated by fibrosis and / or inflammation, such as coronavirus-induced ARDS, comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. The method comprises administering a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit pulmonary fibrosis, reduce pulmonary fibrosis, and / or improve pulmonary function. Symptoms of improvement in pulmonary function include improvements in pulmonary function and / or capacity, reduction in fatigue, and improvements in oxygen saturation.

[0232] The MASP-2 inhibitory composition can be administered locally to the area of ​​fibrosis, for example, during surgery or local injection, by direct or remote (e.g., by catheter) local application of the composition. Alternatively, the MASP-2 inhibitory agent can be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration as with non-peptidergic agents. Administration can be repeated as determined by the physician until the condition has resolved or is under control.

[0233] In certain embodiments, a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) is administered in combination with one or more agents or treatment modalities appropriate for the underlying pulmonary disease or condition.

[0234] infectious disease

[0235] Infectious diseases such as coronaviruses and chronic infectious diseases such as hepatitis C and hepatitis B cause tissue inflammation and fibrosis, and high lectin pathway activity can be detrimental. In such diseases, inhibitors of MASP-2 may be beneficial. For example, MBL and MASP-1 levels were found to be important predictors of the severity of liver fibrosis 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 virus and Ross River virus induce a strong host inflammatory response, leading to arthritis and myositis, and this pathology is mediated by MBL and lectin pathways (Gunn et al., PLoS Pathog. 8(3):e1002586, 2012).

[0236] Accordingly, in certain embodiments, the present disclosure provides methods for preventing, treating, reversing, inhibiting and / or reducing fibrosis and / or inflammation in a subject who has or has previously had an infectious disease that causes inflammation and / or fibrosis, such as a coronavirus or influenza virus, comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to the subject in need thereof.

[0237] The MASP-2 inhibitory composition can be administered locally to the area of ​​fibrosis, for example, during surgery or local injection, by direct or remote (e.g., by catheter) local application of the composition. Alternatively, the MASP-2 inhibitory agent can be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration as with non-peptidergic agents. Administration can be repeated as determined by the physician until the condition has resolved or is under control.

[0238] In certain embodiments, a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) is administered in combination with one or more agents or treatment modalities appropriate for the underlying infectious disease. For example, in some embodiments, a patient diagnosed with COVID-19 can be treated with a combination of agents that includes one or more MASP-2 inhibitory agents, such as a combination of an antiviral agent (e.g., remdesivir) and one or more MASP-2 inhibitory agents. The agents can be administered in any suitable order, such as sequentially or simultaneously.

[0239] In some embodiments, the infectious disease causing inflammation and / or fibrosis is selected from the group consisting of coronavirus, alphavirus, hepatitis A, hepatitis B, hepatitis C, tuberculosis, HIV, and influenza.

[0240] In certain embodiments, a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody or a MASP-2 inhibitory small molecule compound) is administered in combination with one or more agents or treatment modalities appropriate for the underlying disease or condition.

[0241] 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.

[0242] IV. MASP-2 Inhibitors

[0243] In various aspects, the present invention provides methods for inhibiting the adverse effects of fibrosis and / or inflammation, comprising administering a MASP-2 inhibitory agent to a subject in need thereof. The MASP-2 inhibitory agent is administered in an amount effective to inhibit MASP-2-dependent complement activation in a living subject. In practicing this aspect of the invention, representative MASP-2 inhibitory agents 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), and molecules that reduce MASP-2 expression (e.g., MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes), thereby preventing MASP-2 from activating the lectin complement pathway. The MASP-2 inhibitory agent can be used alone as a primary therapy or in combination with other therapeutic agents as an adjunct therapy to enhance the therapeutic benefit of other medical treatments.

[0244] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in a complement system component that occur as a result of administration of a MASP-2 inhibitory agent according to the methods of the invention: inhibition of the generation or production of the MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (e.g., as measured as described in Example 2), reduction in C4 cleavage and C4b deposition (e.g., as measured as described in Example 2), or reduction in C3 cleavage and C3b deposition (e.g., as measured as described in Example 2).

[0245] According to the present invention, MASP-2 inhibitory agents are utilized that are effective in inhibiting respiratory distress (or in other words, improving respiratory function) in subjects infected with a coronavirus such as SARS-CoV-2.

[0246] Assessments of respiratory function can be performed periodically, for example, hourly, daily, weekly, or monthly. Such assessments are preferably performed at several time points for a given subject, or at one or several time points for a given subject and healthy controls. Assessments can be performed at regular intervals, for example, hourly, daily, weekly, or monthly. When an assessment has resulted in a finding of decreased respiratory distress (i.e., increased respiratory function), a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, is said to be effective in treating a subject with coronavirus-induced acute respiratory distress syndrome.

[0247] MASP-2 inhibitory agents 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 inhibitory agents can inhibit the MASP-2-dependent complement activation system by blocking the biological functions of MASP-2. For example, inhibitory agents can effectively block MASP-2 protein-protein interactions, interfere with MASP-2 dimerization or assembly, block Ca 2+ Binding to and interfering with the MASP-2 serine protease active site may reduce MASP-2 protein expression.

[0248] In some embodiments, the MASP-2 inhibitory agent selectively inhibits MASP-2 complement activation, leaving the C1q-dependent complement activation system functionally intact.

[0249] In one embodiment, a MASP-2 inhibitory agent useful in the methods of the present invention is a specific MASP-2 inhibitory agent that specifically binds to a polypeptide comprising SEQ ID NO:6 with an affinity that is at least ten times greater than that of other antigens in the complement system. In another embodiment, a MASP-2 inhibitory agent specifically binds to a polypeptide comprising SEQ ID NO:6 with an affinity that is at least 100 times greater than that of other antigens in the complement system. In one embodiment, a MASP-2 inhibitory agent 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 of MASP-2 (aa 445-682 of SEQ ID NO:6) and inhibits MASP-2-dependent complement activation. In one embodiment, a MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to MASP-2. The binding affinity of a MASP-2 inhibitory agent can be determined using a suitable binding assay. In one embodiment, a MASP-2 inhibitory agent inhibits C3b deposition in 90% of human serum with an IC of 1.5. 50 30nM or less.

[0250] MASP-2 polypeptides exhibit molecular structures similar to those of MASP-1, MASP-3, and C1r and C1s (proteases of the C1 complement system). The cDNA molecule shown in SEQ ID NO:4 encodes a representative example of MASP-2 (composed of the amino acid sequence shown in SEQ ID NO:5) and provides a human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after secretion, resulting in the mature form of human MASP-2 (SEQ ID NO:6). As shown in FIG2 , the human MASP-2 gene comprises twelve exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. Alternative splicing results in a 20 kDa protein designated MBL-associated protein 19 ("MApl9," also known as "sMAP") (SEQ ID NO:2), encoded by (SEQ ID NO:1), beginning with exons B, C, D, and E, as shown in FIG2 . The cDNA molecule set forth in SEQ ID NO:50 encodes murine MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:51) and provides a murine MASP-2 polypeptide having a leader sequence that is cleaved after secretion, resulting in the mature form of murine MASP-2 (SEQ ID NO:52). The cDNA molecule set forth in SEQ ID NO:53 encodes rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:54) and provides a rat MASP-2 polypeptide having a leader sequence that is cleaved after secretion, resulting in the mature form of rat MASP-2 (SEQ ID NO:55).

[0251] 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 represent individual alleles of human, murine, and rat MASP-2, respectively, and that allelic variation and alternative splicing are expected to occur. Allelic variants of the nucleotide sequences set forth in SEQ ID NO:4, SEQ ID NO:50, and SEQ ID NO:53, including allelic variants containing silent mutations and allelic variants in which the mutations result in changes in the amino acid sequence, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

[0252] The domain structure of the human MASP-2 protein (SEQ ID NO: 6) is shown in Figure 1 and 2Aand includes an N-terminal C1r / C1s / sea urchin Vegf / bone morphogenetic protein (CUBI) domain (aa 1-121 of SEQ ID NO: 6), an epidermal growth factor-like domain (aa 122-166), a second CUBI domain (aa 167-293), and tandem complement control protein domains and serine protease domains. Alternative splicing of the MASP 2 gene results in Figure 1 MApl9 is a non-enzymatic protein that contains the N-terminal CUBI-EGF region of MASP-2, along with four additional residues (EQSL) derived from exon E, as shown in Figure 1 As shown in .

[0253] Several proteins have been shown to bind to or interact with MASP-2 through protein-protein interactions. For example, MASP-2 is known to bind to and form Ca bonds with the lectin proteins MBL, H-ficolin, and L-ficolin. 2+ 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. 176:1497-2284, 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 MASP-2 binding to MBL (Thielens, NM et al., J. Immunol. 166:5068, 2001). The CUBIEGFCUBII domain has also been shown to mediate dimerization of MASP-2, which is required for the formation of an active MBL complex (Wallis, R. et al., J. Biol. Chem. 275:30962-30969, 2000). Thus, MASP-2 inhibitory agents can be identified that bind to or interfere with MASP-2 target regions known to be important for MASP-2-dependent complement activation.

[0254] Anti-MASP-2 antibodies

[0255] In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent 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 can be multispecific, chimeric, humanized, anti-idiotypic, and antibody fragments. Antibody fragments include Fab, Fab', F(ab)2, F(ab')2, Fv fragments, scFv fragments, and single-chain antibodies, as further described herein.

[0256] Using the assays described herein, MASP-2 antibodies can be screened for their ability to inhibit the MASP-2-dependent complement activation system and their anti-fibrotic activity, and / or their ability to inhibit renal damage associated with proteinuria or doxorubicin-induced nephropathy. Several MASP-2 antibodies have been described in the literature, and some have been recently generated, some of which are listed in Table 1 below. For example, as described in Examples 10 and 11 herein, anti-MASP-2 Fab2 antibodies that block MASP-2-dependent complement activation have been identified. As described in Example 12 and in WO 2012 / 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 in WO 2014 / 144542, incorporated herein by reference, SGMI-2 peptide-bearing MASP-2 antibodies and fragments thereof having MASP-2 inhibitory activity are generated by fusing the SGMI-2 peptide amino acid sequence (SEQ ID NO: 72, 73, or 74) to the amino or carboxyl terminus of the heavy and / or light chain of a human MASP-2 antibody (e.g., OMS646-SGMI-2).

[0257] Accordingly, in one embodiment, the MASP-2 inhibitory agent for use in the methods of the invention comprises a human antibody, such as OMS646. Accordingly, in one embodiment, the MASP-2 inhibitory agent for use in the compositions and methods of the invention comprises a human antibody that binds to a polypeptide consisting of human MASP-2 (SEQ ID NO:6), wherein the antibody comprises: (I) (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence 31-35 from SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence 50-65 from SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence 95-107 from SEQ ID NO:67, and b) a light chain variable region comprising: i) a light chain CDR-L1 comprising the amino acid sequence 24-34 from SEQ ID NO:69; and ii) a light chain CDR-L2 comprising the amino acid sequence 50-56 from SEQ ID NO:69; and iii) a heavy chain CDR-H4 comprising the amino acid sequence 95-107 from SEQ ID NO:67. NO:69, or (II) a variant thereof, comprising a heavy chain variable region that is 90% identical to SEQ ID NO:67 (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% identical to SEQ ID NO:67), and a light chain variable region that is 90% identical to SEQ ID NO:69 (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% identical).

[0258] In some embodiments, the method comprises administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, wherein the MASP-2 inhibitory antibody comprises 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.

[0259] In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by the reference antibody OMS646, comprising a heavy chain variable region as set forth in SEQ ID NO: 67 and a light chain variable region as set forth in SEQ ID NO: 69. In one embodiment, the MASP-2 inhibitory agent used in the methods of the invention comprises the human antibody OMS646.

[0260] Table 1: Exemplary MASP-2-specific antibodies

[0261]

[0262]

[0263] Anti-MASP-2 antibodies with reduced effector function

[0264] In some embodiments of this aspect of the invention, the anti-MASP-2 antibodies have reduced effector function in order to reduce inflammation that may arise from activation of the classical complement pathway. The ability of IgG molecules to trigger the classical complement pathway has been shown to be located within the Fc portion of the molecule (Duncan, AR et al., Nature 332:738-740 1988). IgG molecules from which the Fc portion of the molecule has been removed by enzymatic cleavage lack such effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function are generated by lacking the Fc portion of the molecule, either by having a genetically engineered Fc sequence that minimizes effector function, or by having a human IgG2 or IgG4 isotype.

[0265] Antibodies with reduced effector function can be generated by standard molecular biological manipulation of the Fc portion of IgG heavy chains, as described herein and 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 function also include human IgG2 and IgG4 isotypes, which have 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 composed of the IgG2 or IgG4 isotype can be generated by one of several methods known to one of ordinary skill in the art, as described in Vaughan, TJ et al., Nature Biotechnical 16:535-539, 1998.

[0266] Generation of anti-MASP-2 antibodies

[0267] Anti-MASP-2 antibodies can be generated using MASP-2 polypeptides (e.g., full-length MASP-2) or peptides bearing antigenic MASP-2 epitopes (e.g., portions of MASP-2 polypeptides). Immunogenic peptides can be as small as five amino acid residues. For example, a MASP-2 polypeptide comprising the entire amino acid sequence of SEQ ID NO:6 can be used to induce anti-MASP-2 antibodies useful in the methods of the present invention. Specific MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, and regions comprising serine protease active sites, can be expressed as recombinant polypeptides as described in Example 3 and used as antigens. In addition, peptides comprising at least six amino acids of a portion of the MASP-2 polypeptide (SEQ ID NO:6) can also be used to induce MASP-2 antibodies. Additional examples of MASP-2-derived antigens useful for inducing MASP-2 antibodies are provided in Table 2 below. MASP-2 peptides and polypeptides useful for generating antibodies can be isolated as native polypeptides, or as recombinant or synthetic peptides, as well as catalytically inactive recombinant polypeptides such as 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.

[0268] Antigens useful for raising 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. The polypeptide immunogen can be the full-length molecule or a portion thereof. If the polypeptide portion is hapten-like, such portion can be advantageously bound or linked to a macromolecular carrier (e.g., keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

[0269] Table 2: MASP-2 derived antigens

[0270]

[0271]

[0272] polyclonal antibodies

[0273] Polyclonal antibodies to MASP-2 can be prepared by immunizing animals with MASP-2 polypeptides or immunogenic portions thereof using methods well known to those of ordinary skill in the art. See, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), page 105. The immunogenicity of MASP-2 polypeptides can be increased by the use of 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 raised 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 can also be derived from humanoid monkeys. General techniques for producing diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication No. WO 91 / 11465, and Losman, MJ et al., Int. J. Cancer 46:310, 1990. Serum containing immunologically active antibodies is then produced from the blood of such immunized animals using standard procedures well known in the art.

[0274] Monoclonal antibodies

[0275] In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly specific, being directed against a single MASP-2 epitope. As used herein, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molecules by continuous cell lines in culture, such as the hybridoma method described by Kohler, G. et al., Nature 256:495, 1975, or they can be prepared by recombinant DNA methods (see, e.g., 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.

[0276] For example, monoclonal antibodies can be obtained by injecting a suitable mammal (e.g., BALB / C mouse) with a composition comprising a MASP-2 polypeptide or a portion thereof. After a predetermined period of time, spleen cells are removed from the mouse and suspended in cell culture. The spleen cells are then fused with an immortalized cell line to form hybridomas. The resulting hybridomas are grown in cell culture and screened for their ability to produce monoclonal antibodies against MASP-2. Examples further describing the generation of anti-MASP-2 monoclonal antibodies are provided herein (see also Current Protocols in Immunology, Vol. 1, John Wiley & Sons, pp. 2.5.1-2.6.7, 1991).

[0277] Human monoclonal antibodies can be obtained by using transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human immunoglobulin heavy and light chain loci are introduced into a mouse strain derived from an embryonic stem cell line that contains targeted disruptions of the endogenous immunoglobulin heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigen described herein, and can be used to generate hybridomas that secrete human MASP-2 antibodies by fusing B cells from such animals with a suitable myeloma cell line using conventional Kohler-Milstein techniques, 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, 1994.

[0278] Monoclonal antibodies can be isolated and purified from hybridoma cultures by various well-established techniques. Such separation techniques include affinity chromatography with protein A agarose, size exclusion chromatography, and ion exchange chromatography (see, e.g., Coligan at pages 2.7.1-2.7.12 and 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pp. 79-104, 1992).

[0279] Once generated, polyclonal, monoclonal, or phage-derived antibodies are first tested for specific MASP-2 binding. Antibodies that specifically bind to MASP-2 can be detected using a variety of assays known to those skilled in the art. Exemplary assays include Western blot or immunoprecipitation analysis by standard methods (e.g., as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immunosorbent assays, dot blots, inhibition or competition assays, and sandwich assays (e.g., 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 act as a MASP-2 inhibitor in one of several assays, such as 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).

[0280] The affinity of an anti-MASP-2 monoclonal antibody can be readily determined by one of ordinary skill in the art (see, e.g., Scatchard, A., NY Acad. Sci. 51:660-672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibodies useful in the methods of the invention bind to MASP-2 with an affinity of <100 nM, preferably <10 nM, and most preferably <2 nM.

[0281] Chimeric / humanized antibodies

[0282] The monoclonal antibodies useful in the methods of the present invention include chimeric antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and 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).

[0283] One form of chimeric antibody that can be used in the present invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. Humanized monoclonal antibodies are produced by transferring non-human (e.g., mouse) complementarity determining regions (CDRs) from the heavy and light chain variable regions of a mouse immunoglobulin into human variable domains. Typically, humanized monoclonal antibodies are then substituted for human antibody residues in the framework regions of the non-human counterpart. In addition, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise at least one variable domain, and typically substantially all of two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the Fv framework regions are Fv framework regions of human immunoglobulin sequences. 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.

[0284] Humanized antibodies useful in the present invention include human monoclonal antibodies that include at least the MASP-2 binding CDRH3 region. Furthermore, the Fc portion can be replaced so as to generate IgA or IgM as well as human IgG antibodies. Such humanized antibodies will have particular clinical utility because they will specifically recognize human MASP-2 but will not elicit an immune response in humans against the antibody itself. Thus, they are more suitable for in vivo administration in humans, particularly when repeated or long-term administration is desired.

[0285] An example of the generation of humanized anti-MASP-2 antibodies from murine anti-MASP-2 monoclonal antibodies is provided in Example 6 herein. Techniques for producing humanized monoclonal antibodies are also described, for example, by 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," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pp. 399-434, 1996; and U.S. Pat. No. 5,693,762 to Queen, 1997. Additionally, there are commercial entities such as Protein Design Labs (Mountain View, CA) that synthesize humanized antibodies from specific murine antibody regions.

[0286] recombinant antibodies

[0287] Anti-MASP-2 antibodies can also be produced using recombinant methods. For example, human antibodies can be produced using human immunoglobulin expression libraries (e.g., available from Stratagene, Corp., La Jolla, CA) to generate human antibody fragments (V H 、V L These fragments are then used to construct fully human antibodies using techniques similar to those used to generate chimeric antibodies.

[0288] Anti-idiotypic antibodies

[0289] Once anti-MASP-2 antibodies with the desired inhibitory activity are identified, these antibodies can be used to generate anti-idiotypic antibodies that resemble a portion of MASP-2 using techniques well known in the art. See, e.g., Greenspan, NS et al., FASEB J. 7:437, 1993. For example, antibodies that bind to MASP-2 and competitively inhibit the interaction of the MASP-2 protein required for complement activation can be used to generate anti-idiotypic antibodies that resemble the MBL binding site on the MASP-2 protein and, therefore, bind to and neutralize the binding ligand of MASP-2, such as MBL.

[0290] Immunoglobulin fragments

[0291] The MASP-2 inhibitory agents useful in the methods of the invention encompass not only intact immunoglobulin molecules, but also well-known fragments, including Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0292] It is well known in the art that only a small part of the antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (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 designated as F(ab')2 fragments, and retain the two antigen-binding sites of the intact antibody. Isolated F(ab')2 fragments are referred to as bivalent monoclonal fragments due to their two antigen-binding sites. Similarly, antibodies from which the Fc region has been enzymatically cleaved, or which have been produced without the Fc region, are designated as Fab fragments, and retain one of the antigen-binding sites of the intact antibody molecule.

[0293] Antibody fragments can be obtained by proteolytic hydrolysis, for example, by pepsin or papain digestion of intact antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted as F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab' monovalent fragment. Optionally, a protecting group for the sulfhydryl group resulting from cleavage of a disulfide bond can be used to perform the cleavage reaction. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab fragments and one Fc fragment. 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., in Methods in Enzymology 1:422, Academic Press, 1967; and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

[0294] In some embodiments, it is preferred to use an antibody fragment lacking an Fc region to avoid activation of the classical complement pathway, which is activated after Fc is bound to Fcγ receptors. There are several methods by which MoAbs that avoid Fcγ receptor interactions can be produced. For example, the Fc region of a monoclonal antibody can be chemically removed using partial digestion (e.g., ficin digestion) by proteolytic enzymes to generate, for example, antigen-binding antibody fragments, such as Fab or F(ab)2 fragments (Mariani, M. et al., Mol. Immunol. 28:69-71, 1991). Alternatively, during the construction of a humanized antibody as described herein, a human γ4 IgG isotype that does not bind to Fcγ receptors can be used. Antibodies, single-chain antibodies, and antigen-binding domains lacking an Fc domain can also be transformed using recombinant techniques as described herein.

[0295] single-chain antibody fragments

[0296] Alternatively, a single peptide chain binding molecule specific for MASP-2 can be generated in which the heavy and light chain Fv regions are linked. The Fv fragments can be linked by a peptide linker to form a single-chain antigen binding protein (scFv). These single-chain antigen binding proteins are prepared by constructing a structural gene comprising a DNA sequence encoding the V domains of the heavy and light chains linked by oligonucleotides. H and V LThe 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 having a linker peptide bridging the two V domains. Methods for producing scFv are described, for example, by 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.

[0297] 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 or similar vectors (e.g., by using immobilized or labeled MASP-2 protein or peptide). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage or bacteria (e.g., E. coli). These random peptide display libraries can be used to screen for peptides that interact with MASP-2. Techniques for generating and screening such random peptide display libraries are well known in the art (U.S. Pat. No. 5,223,409 to Lardner; U.S. Pat. No. 4,946,778 to Lardner; U.S. Pat. No. 5,403,484 to Lardner; U.S. Pat. No. 5,571,698 to Lardner; and Kay et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996), and random peptide display libraries and kits for screening such libraries are commercially available, for example, from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Ipswich, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ).

[0298] Another form of anti-MASP-2 antibody fragment useful in this aspect of the 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 an 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," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press, 1995; and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), page 137, Wiley-Liss, Inc., 1995).

[0299] The MASP-2 antibodies described herein are administered to a subject in need thereof to inhibit MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitory agent is a high-affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.

[0300] Peptide inhibitors

[0301] In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises an isolated MASP-2 peptide inhibitor, including isolated natural peptide inhibitors and synthetic peptide inhibitors, that inhibits the MASP-2-dependent complement activation system. As used herein, the term "isolated MASP-2 peptide inhibitor" refers to peptides that inhibit MASP-2-dependent complement activation by binding to MASP-2, competing with MASP-2 for binding to another recognition molecule in the lectin pathway (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin), and / or directly interacting with MASP-2 to inhibit MASP-2-dependent complement activation, said peptides being substantially pure and substantially free of other substances with which they may be found in nature, to the extent practical and appropriate for their intended use.

[0302] Peptide inhibitors have been successfully used in vivo to interfere with protein-protein interactions and catalytic sites. For example, peptide inhibitors against adhesion molecules structurally related to LFA 1 have recently been approved for clinical use in coagulopathy (Ohman, EM et al., European Heart J. 16:50-55, 1995). Short linear peptides (<30 amino acids) that prevent or interfere with integrin-dependent adhesion have been described (Murayama, O. et al., J. Biochem. 120:445-51, 1996). Longer peptides ranging in length from 25 to 200 amino acid residues have also been successfully used to block integrin-dependent adhesion (Zhang, L. et al., J. Biol. Chem. 271(47):29953-57, 1996). In general, longer peptide inhibitors have higher affinities and / or slower dissociation rates than shorter peptides and are therefore likely to be more potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective inhibitors of integrins in vivo for the treatment of human inflammatory diseases (Jackson, DY et al., J. Med. Chem. 40:3359-68, 1997). One method for producing cyclic peptides involves synthesizing peptides in which the terminal amino acid of the peptide is cysteine, thereby allowing the peptide to exist in a cyclic form through disulfide bonding between the terminal amino acids, which 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).

[0303] Synthetic MASP-2 peptide inhibitors

[0304] 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 useful in the practice of this aspect of the invention. Candidate MASP-2 inhibitory peptides can be tested for their ability to act as MASP-2 inhibitory agents in one of several assays, including, for example, the lectin-specific C4 cleavage assay (described in Example 2) and the C3b deposition assay (described in Example 2).

[0305] 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 a specific domain 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 previously described, 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 that identified humans carrying a homozygous mutation at Asp105 to Gly105, resulting in the loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K. et al., New England J. Med. 349: 554-560, 2003).

[0306] In some embodiments, the MASP-2 inhibitory peptide is derived from a lectin protein that binds to MASP-2 and is involved in the lectin complement pathway. Several different lectins involved in this pathway have been identified, 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 an N-terminal collagen-like fiber containing a carbohydrate recognition domain. These various lectins have been shown to bind to MASP-2, and the lectin / MASP-2 complex activates complement through cleavage of 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 derived from S. typhimurium, S. minnesota, and E. coli. H-ficolin has been shown to bind to MASP-2 and MApl9 and activate the lectin pathway. Ibid. 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). Accordingly, a MASP-2 inhibitory peptide useful in the present invention may comprise a region of at least 5 amino acids selected from the group consisting of MBL protein (SEQ ID NO: 21), H-ficolin (Genbank Accession No. NM_173452), M-ficolin (Genbank Accession No. 000602), and L-ficolin (Genbank Accession No. NM_015838).

[0307] More specifically, scientists have identified the MASP-2 binding site on MBL as being within 12 Gly-XY triplets: "GKD GRD GTK GEK GEP GQG LRG LQG POG KLG POG NOG PSG SOG PKG QKG DOG KS" (SEQ ID NO: 26), located between the hinge and the neck of the C-terminal portion of the collagen-like domain of MBP (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). Accordingly, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least 6 amino acids in length and comprise SEQ ID NO: 22. Peptides derived from MBL comprising the amino acid sequence "GLR GLQ GPO GKL GPO G" (SEQ ID NO: 24) 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 ("GPO GPO GLR GLQ GPO GKL GPO GGP OGP O" SEQ ID NO: 25) to enhance triple helix formation as found in native MBL protein (as further described in Wallis, R. et al., J. Biol. Chem. 279: 14065, 2004).

[0308] MASP-2 inhibitory peptides can also be derived from human H-ficolin, comprising the sequence "GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO" (SEQ ID NO: 27), which is derived from the consensus MASP-2 binding region in H-ficolin. Also included are peptides derived from human L-ficolin, comprising the sequence "GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAOGEO" (SEQ ID NO: 28), which is derived from the consensus MASP-2 binding region in L-ficolin.

[0309] MASP-2 inhibitory peptides can also be derived from a C4 cleavage site, such as "LQRALEILPNRVTIKANRPFLVFI" (SEQ ID NO:29), which is a C4 cleavage site attached to the C-terminal portion of antithrombin III (Glover, GI et al., Mol. Immunol. 25:1261 (1988)).

[0310] Table 3: Exemplary MASP-2 Inhibitory Peptides

[0311]

[0312]

[0313]

[0314] Note: The letter "O" represents hydroxyproline. The letter "X" is a hydrophobic residue.

[0315] 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 render them 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 appended to a functional side chain; haloacetyl groups (or other α-haloacetyl groups) on the amino or other functional side chain; epoxide- or imine-containing groups at the amino or carboxyl termini or on a functional side chain; or imidoesters at the amino or carboxyl termini or on a functional side chain. Such modifications offer the advantage of permanently inhibiting the enzyme through covalent attachment of the peptide. This can result in a lower effective dose and / or require less frequent administration of the peptide inhibitor.

[0316] 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 used in synthesizing the peptides can be determined by methods known in the art. The heavy chain variable region is a peptide generally ranging from 100 to 150 amino acids in length. The light chain variable region is a peptide generally ranging from 80 to 130 amino acids in length. The CDR sequences within the heavy and light chain variable regions comprise only approximately 3-25 amino acid sequences, which can be readily sequenced by one of ordinary skill in the art.

[0317] Those skilled in the art will recognize that substantially homologous variations of the above-described MASP-2 inhibitory peptides also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptides having insertions, deletions, substitutions, and / or additional amino acids at the carboxyl or amino terminal portions of the subject peptides, and mixtures thereof. Accordingly, those homologous peptides that exhibit MASP-2 inhibitory activity are contemplated as useful in the methods of the present invention. The peptides may also include repeated motifs and other modifications with conservative substitutions. Conservative variants are described elsewhere herein and include the exchange of an amino acid with another amino acid of similar charge, size, hydrophobicity, etc.

[0318] MASP-2 inhibitory peptides can be modified to increase solubility and / or maximize positive or negative charge to more closely resemble segments of the intact protein. Derivatives may or may not have the exact primary amino acid structure of the peptides disclosed herein, as long as the derivative functionally retains the desired properties of MASP-2 inhibition. Modifications can include amino acid substitutions with one of the twenty commonly known amino acids or another amino acid, amino acid substitutions with derivatives or replacement amino acids having auxiliary desired characteristics (e.g., resistance to enzymatic degradation), or amino acid substitutions with D amino acids, or substitutions with another molecule or compound, such as a carbohydrate, that mimics the natural conformation and function of one or more amino acids or peptides; amino acid deletions; amino acid insertions with one of the twenty commonly known amino acids or another amino acid, amino acid insertions with derivatives or replacement amino acids having auxiliary desired characteristics (e.g., resistance to enzymatic degradation), or amino acid insertions with D amino acids, or substitutions with another molecule or compound, such as a carbohydrate, that mimics the natural conformation and function of one or more amino acids or peptides; or substitutions with another molecule or compound, such as a carbohydrate or nucleic acid monomer, that mimics the natural conformation, charge distribution, and function of the parent peptide. The peptide can also be modified by acetylation or amidation.

[0319] The synthesis of derivative inhibitory peptides can rely on known techniques for peptide biosynthesis, carbohydrate biosynthesis, and the like. As a starting point, the skilled artisan can rely on appropriate computer programs to determine the conformation of the peptide of interest. Once the conformation of the peptides disclosed herein is known, the skilled artisan can then determine, in a rational design approach, which substitutions can be made at one or more sites to produce derivatives that retain the basic conformation and charge distribution of the parent peptide, but may possess features not present in the parent peptide or that are enhanced over those found in the parent peptide. Once candidate derivative molecules are identified, the derivatives can be tested using the assays described herein to determine whether they act as MASP-2 inhibitors.

[0320] Screening for MASP-2 inhibitory peptides

[0321] Molecular modeling and rational molecular design can also be used to generate and screen for peptides that mimic the molecular structures of the key binding regions of MASP-2 and inhibit the complement activity of MASP-2. Molecular structures used for modeling include CDR regions of anti-MASP-2 monoclonal antibodies, as well as target regions known to be important for MASP-2 function, including regions required for dimerization, regions involved in binding to MBL, and serine protease active sites, as previously described. Methods for identifying peptides that bind to specific targets are well known in the art. For example, molecular imprinting can be used to construct macromolecular structures, such as peptides that bind to specific molecules, from scratch. 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.

[0322] As an illustrative example, one method for preparing mimetics of MASP-2-binding peptides is as follows. Functional monomers of a known MASP-2-binding peptide or the binding region of an anti-MASP-2 antibody that exhibits MASP-2 inhibition (the template) are polymerized. The template is then removed, followed by polymerization of a second type of monomer in the void left by the template to provide a new molecule that exhibits one or more desired properties similar to the template. In addition to preparing peptides in this manner, other MASP-2-binding molecules that are MASP-2 inhibitory agents can also be prepared, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials. This approach can be used to design a wide variety of biomimetics that are more stable than their natural counterparts, as they are typically prepared by free radical polymerization of functional monomers, resulting in compounds with non-biodegradable backbones.

[0323] Peptide synthesis

[0324] MASP-2 inhibitory peptides can be prepared using techniques well known in the art, such as the solid phase synthesis technique originally described by Merrifield in J. Amer. Chem. Soc. 85:2149-2154, 1963. Automated synthesis can be achieved, for example, using an Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.), according to the instructions provided by the manufacturer. Other techniques can be found, for example, in Bodanszky, M. et al., Peptide Synthesis, 2nd ed., John Wiley & Sons, 1976, and other references known to those skilled in the art.

[0325] Peptide can also be prepared using standard genetic engineering techniques well known to those skilled in the art. For example, peptide can be produced by following enzymatic method: the nucleic acid encoding the peptide is inserted into the expression vector, DNA is expressed, and in the presence of the desired amino acid, DNA is translated into peptide. Then use chromatography or electrophoresis techniques or by means of carrier protein to purify the peptide, by inserting the nucleic acid sequence encoding the carrier protein into the expression vector in phase with the peptide encoding sequence, the carrier protein can be fused with the peptide and subsequently cut from the peptide. Fusion protein-peptide can be separated using chromatography, electrophoresis or immunological techniques (for example, via antibody to the carrier protein and resin binding). Chemical methods can be used or as by, for example, hydrolytic enzymes to enzymatically cut the peptide.

[0326] MASP-2 inhibitory peptides useful in the methods of the present invention can also be produced in recombinant host cells following conventional techniques. To express a MASP-2 inhibitory peptide coding sequence, a nucleic acid molecule encoding the peptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector, which is then introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can also include translational regulatory sequences and marker genes suitable for selecting cells harboring the expression vector.

[0327] Nucleic acid molecules encoding MASP-2 inhibitory peptides can be synthesized using "gene machinery" using protocols such as the phosphoramidite method. If chemically synthesized double-stranded DNA is desired for an application such as synthesis of a gene or gene fragment, each complementary strand is prepared separately. The generation of short genes (60 to 80 base pairs) is technically simple and can be accomplished by synthesizing complementary strands and then annealing them. For the generation of longer genes, synthetic genes (double-stranded) are assembled in a modular fashion from single-stranded fragments ranging from 20 to 100 nucleotides in length. For reviews of polynucleotide synthesis, see, for example, 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. Nat'l Acad. Sci. USA 87:633, 1990.

[0328] Small molecule MASP-2 inhibitors

[0329] In some embodiments, the MASP-2 inhibitory agent is a small molecule inhibitor, including natural, semisynthetic, and synthetic substances with a low molecular weight (e.g., 50 to 1000 Da), such as peptides, peptidomimetics, and non-peptide inhibitors (e.g., oligonucleotides and organic compounds). Small molecule inhibitors of MASP-2 can be generated based on the molecular structure of the variable region of an anti-MASP-2 antibody.

[0330] Computational drug design can also be used to design and generate small molecule inhibitors based on the crystal structure of MASP-2 (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). Using the methods described by Kuntz et al., the MASP-2 crystal structure coordinates are used as input to a computer program such as DOCK, which outputs a list of small molecule structures predicted to bind to MASP-2. The use of such computer programs is well known to those skilled in the art. For example, the crystal structure of an HIV-1 protease inhibitor was used to identify it as a unique non-peptide ligand for an HIV-1 protease inhibitor by evaluating the fit of compounds found in the Cambridge Crystallographic Database to 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).

[0331] Exemplary MASP-2 inhibitory agents 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 herein by reference in its entirety.

[0332] In some embodiments, the small molecule is a compound of Formula (IA), (IB), (IIA), (IIB), (III), or (IV):

[0333]

[0334]

[0335] or a salt thereof, wherein:

[0336] Cy 1A is unsubstituted or substituted C 6-10 aryl, or unsubstituted or substituted 5-10 membered heteroaryl; wherein Cy 1A The ring atoms of the 5-10 membered heteroaryl group are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein the ring atoms of the 5-10 membered heteroaryl group are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; 1A Substituted C 6-10 The aryl or substituted 5-10 membered heteroaryl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy1A , halogen, C1-6 Haloalkyl, CN, OR a11 、S Ra11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR 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 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo;

[0337] Each R Cy1A Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy1A The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, N and S, wherein R Cy1A Each C 1-6Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo, and wherein R Cy1A Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 Rd11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo;

[0338] R 11 H or C 1-6 Alkyl, C 6-10 Aryl-C 1-6 Alkyl or 5-10 membered heteroaryl-C 1-6 Alkyl, which forms R 11 C 1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo, and wherein R 11 C 6-10 Aryl-C 1-6 Alkyl or 5-10 membered heteroaryl-C 1-6 The alkyl group is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo;

[0339] R 12 H or C 1-6 alkyl; or

[0340] R 11 and R12 Together with the groups to which they are attached, they form a 4-6 membered heterocycloalkyl ring;

[0341] A 11 CR 13 R 15 or N;

[0342] Each R 13 Independently for Cy 1B 、(CR 13A R 13B ) n3 Cy 1B 、(C 1-6 Alkylene)Cy 1B 、(C 2-6 Alkenylene)Cy 1B 、(C 2-6 Alkynylidene)Cy 1B or OCy 1B , where R 13 C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 The alkynylene component is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents each independently selected from halogen, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11、S(O)2NR c11 R d11 and oxo;

[0343] Each R 14 Independently selected from H and C 1-6 alkyl;

[0344] R 15 Selected from H, R 13 、C 1-6 Alkyl and OH;

[0345] A pair of R connected to adjacent carbon atoms 14 group, or a pair of R connected to adjacent carbon atoms 14 and R 15 group, which may not depend on R 14 The other occurrences of R are replaced together by connecting the pair 14 group or the pair of R 14 and R 15 the bond to the adjacent carbon atom to which the group is attached, such that the adjacent carbon atoms are connected by a double bond; or

[0346] A pair of R attached to the same carbon atom 14 group, or a pair of R attached to the same carbon atom 13 and R 15 group, which may not depend on R 14 The other occurrences, and together with the pair of R 14 group or the pair of R 13 and R 15 The group, together with the carbon atom to which it is attached, forms a spiro-fused C 3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, wherein the ring atoms of the 4-10 membered heterocycloalkyl ring are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, wherein the spiro-fused C 3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, optionally further substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, haloalkyl, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NRc11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo; or

[0347] A pair of R connected to adjacent carbon atoms 14 group, or a pair of R connected to adjacent carbon atoms 14 and R 15 group, which may not depend on R 14 Other occurrences, together with the pair of R 14 group or the pair of R 14 and R 15 The group, together with the adjacent carbon atom to which it is attached, forms a fused C 3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, wherein the ring atoms of the 4-10 membered heterocycloalkyl ring are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, wherein the fused C 3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, optionally further substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, haloalkyl, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo; or

[0348] A group of four R atoms attached to two adjacent carbon atoms 14 A group, or a group of two R 14 , an R 13 and an R 15 group, which may not depend on R 14 Other occurrences, together with the set of four R 14 group, or a group of two R 14 , an R 13 and an R 15 The two adjacent carbon atoms to which the group is attached together form a fused C 6-10 Aryl or 5-10 membered heteroaryl, C 3-10 a cycloalkyl or 4-10 membered heterocycloalkyl ring, wherein the ring atoms of the formed 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl ring consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, and wherein the formed fused C 6-10 Aryl or 5-10 membered heteroaryl, C 3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, optionally further substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, haloalkyl, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NRc11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo;

[0349] n1 is 1 or 2;

[0350] n2 is 0, 1, or 2;

[0351] The condition is that the sum of n1 and n2 is 1, 2 or 3;

[0352] The condition is that if n1 is 1 or n2 is 0, then A 11 CR 13 R 15 ;

[0353] n3 is 0, 1, or 2;

[0354] Each R 13A are independently H or C 1-6 alkyl;

[0355] Each R 13B are independently H or C 1-6 alkyl; or

[0356] or R attached to the same carbon atom 13A and R 13B , does not depend on any other R 13A and R 13B groups, which together can form –(CH2) 2-5 -, thereby forming a 3-6 membered cycloalkyl ring; or

[0357] Cy 1B is unsubstituted or substituted C6-10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; wherein Cy 1B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

[0358] The formation of Cy 1B Substituted C 6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy1B , halogen, C 1-6 Haloalkyl, CN, OR a11 、S Ra11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR 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 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2Rb11 、S(O)2NR c11 R d11 and oxo;

[0359] Each R Cy1B Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy1B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R Cy1B Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O) Rb11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo; and wherein R Cy1B Each C 6-10 Aryl, 5-10 membered heteroaryl, C3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O) Rb11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo;

[0360] R 16 H, Cy 1C 、C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, which forms R 16 C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of: Cy 1C 、Halogen、CN、OR a11 SR a11 、C(O)R b11 、C(O)NR c11 Rd11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo, provided that R 16 No more than one substituent is Cy 1C ;

[0361] 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; wherein Cy 1C The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

[0362] The formation of Cy 1C Substituted C 6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy1C , halogen, C 1-6 Haloalkyl, CN, OR a11 、S Ra11 、C(O)R b11 、C(O)NR c11 R d11、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O)R b11 NR c11 C(O)NR c11 R d11 NR 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 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo;

[0363] Each R Cy1C Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy1C The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R Cy1C Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a11 SR a11、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O) Rb11 NR c11 C(O)NR c11 R d11 NR c11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo; and wherein R Cy1C Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a11 SR a11 、C(O)R b11 、C(O)NR c11 R d11 、C(O)OR a11 、OC(O)R b11 、OC(O)NR c11 R d11 NR c11 R d11 NR c11 C(O) Rb11 NR c11 C(O)NR c11 R d11 NRc11 C(O)OR a11 、C(=NR e11 )NR c11 R d11 NR c11 C(=NR e11 )NR c11 R d11 、S(O)R b11 、S(O)NR c11 R d11 、S(O)2R b11 NR c11 S(O)2R b11 、S(O)2NR c11 R d11 and oxo;

[0364] R a11 、R b11 、R c11 and R d11 Each independently selected from 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, which forms R a11 、R b11 、R c11 and R d11 The 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, each optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from: C 1-6 Alkyl, halogen, CN, OR a12 SR a12 、C(O)Rb12 、C(O)NR c12 R d12 、C(O)OR a12 、OC(O)R b12 、OC(O)NR c12 R d12 NR c12 R d12 NR c12 C(O)R b12 NR c12 C(O)NR c12 R d12 NR c12 C(O)OR a12 、C(=NR e12 )NR c12 R d12 NR c12 C(=NR e12 )NR c12 R d12 、S(O)R b12 、S(O)NR c12 R d12 、S(O)2R b12 NR c12 S(O)2R b12 、S(O)2NR c12 R d12 and oxo;

[0365] or R attached to the same N atom c11 and R d11 , together with the nitrogen atom to which they are attached, form a 4-, 5-, 6- or 7-membered heterocycloalkyl or a 5-membered heteroaryl, each optionally substituted with 1, 2 or 3 substituents independently selected from: C 1-6 Alkyl, halogen, CN, OR a12 SR a12 、C(O)R b12 、C(O)NR c12 R d12 、C(O)OR a12 、OC(O)R b12 、OC(O)NR c12 R d12 NR c12 R d12 NR c12 C(O)R b12 NR c12 C(O)NR c12 R d12 NR c12 C(O)OR a12 、C(=NRe12 )NR c12 R d12 NR c12 C(=NR e12 )NR c12 R d12 、S(O)R b12 、S(O)NR c12 R d12 、S(O)2R b12 NR c12 S(O)2R b12 、S(O)2NR c12 R d12 and oxo;

[0366] R a12 、R b12 、R c12 and R d12 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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, which forms R a12 、R b12 、R c12 and R d12 The C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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, each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of OH, CN, amino, NH(C 1-6 alkyl), N(C 1-6 Alkyl)2, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 haloalkoxy and oxo;

[0367] or R attached to the same N atom c12 and R d12 , together with the nitrogen atom to which they are attached, form a 4-, 5-, 6- or 7-membered heterocycloalkyl or a 5-membered heteroaryl, each of which is unsubstituted or substituted by 1, 2 or 3 substituents independently selected from the group consisting of OH, CN, amino, NH(C 1-6 alkyl), N(C 1-6 Alkyl)2, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 haloalkoxy and oxo;

[0368] R e11 and Re 12 Each independently is H, CN or NO2;

[0369] Cy 2A is unsubstituted or substituted C 6-10 aryl, or unsubstituted or substituted 5-10 membered heteroaryl; wherein Cy 2A The ring atoms of the 5-10 membered heteroaryl group are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein the ring atoms of the 5-10 membered heteroaryl group are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; 2A Substituted C 6-10 The aryl or substituted 5-10 membered heteroaryl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy2A , halogen, C 1-6 Haloalkyl, CN, OR a21 、S Ra21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)OR a21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O)R b21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21 、C(=NR e21 )NR c21 R d21 、C(=NOR a21 )NR c21 Rd21 、C(=NOC(O)R b21 )NR c21 R d21 、C(=NR e21 )NR c21 C(O)OR a21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21 、S(O)2NR c21 R d21 and oxo;

[0370] Each R Cy2A Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy2A The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, N and S, wherein R Cy2A Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a21 SR a21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)OR a21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O)R b21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21 、C(=NR e21 )NR c21R d21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21 、S(O)2NR c21 R d21 and oxo, which forms R Cy2A Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a21 SR a21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)OR a21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O)R b21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21 、C(=NR e21 )NR c21 R d21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21 、S(O)2NR c21 R d21 and oxo;

[0371] R21 H or C 1-6 Alkyl, C 6-10 Aryl-C 1-6 Alkyl or 5-10 membered heteroaryl-C 1-6 Alkyl, which forms R 21 C 1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, CN, OR a21 SR a21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)OR a21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O)R b21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21 、C(=NR e21 )NR c21 R d21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21 、S(O)2NR c21 R d21 and oxo, and wherein R 21 C 6-10 Aryl-C 1-6 Alkyl or 5-10 membered heteroaryl-C 1-6 The alkyl group is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a21 SR a21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)ORa21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O)R b21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21 、C(=NR e21 )NR c21 R d21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21 、S(O)2NR c21 R d21 and oxo;

[0372] R 22 H or C 1-6 alkyl; or

[0373] R 21 and R 22 Together with the groups to which they are attached, they form a 4-6 membered heterocycloalkyl ring;

[0374] A 23 N or NR 23 ;

[0375] A 24 CR 24 , N and NR 24 ;

[0376] A 26 CR 26 or S;

[0377] The condition is

[0378] A in formula (IIA) 23 、A 24 and A 26 It is chosen so that it contains A 23 、A 24 and A 26 The ring of is a heteroaryl ring, and the symbol represents aromatic ring (normalized) bonds;

[0379] R 23 H or C 1-6 alkyl;

[0380] R 24 H; C 1-6 Alkyl or phenyl;

[0381] 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 Alkynylidene)Cy 2B , where R 25 C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 The alkynylene component is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents each independently selected from halogen, CN, OR a21 SR a21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)OR a21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O)R b21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21 、C(=NR e21 )NR c21 R d21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21、S(O)2NR c21 R d21 and oxo;

[0382] R 26 H or C 1-6 alkyl;

[0383] Each R 25A H or C 1-6 alkyl;

[0384] Each R 25B H or C 1-6 alkyl;

[0385] n25 is 0, 1, or 2;

[0386] 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; wherein Cy 2B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

[0387] The formation of Cy 2B Substituted C 6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy2B , halogen, C 1-6 Haloalkyl, CN, OR a21 、S Ra21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)OR a21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O)R b21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21 、C(=NR e21 )NR c21 R d21 、C(=NORa21 )NR c21 R d21 、C(=NOC(O)R b21 )NR c21 R d21 、C(=NR e21 )NR c21 C(O)OR a21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21 、S(O)2NR c21 R d21 and oxo;

[0388] Each R Cy2B Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy2B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R Cy2B Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a21 SR a21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)OR a21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O) Rb21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21、C(=NR e21 )NR c21 R d21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21 、S(O)2NR c21 R d21 and oxo; and wherein R Cy2B Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a21 SR a21 、C(O)R b21 、C(O)NR c21 R d21 、C(O)OR a21 、OC(O)R b21 、OC(O)NR c21 R d21 NR c21 R d21 NR c21 C(O) Rb21 NR c21 C(O)NR c21 R d21 NR c21 C(O)OR a21 、C(=NR e21 )NR c21 R d21 NR c21 C(=NR e21 )NR c21 R d21 、S(O)R b21 、S(O)NR c21 R d21 、S(O)2R b21 NR c21 S(O)2R b21 、S(O)2NR c21 Rd21 and oxo;

[0389] Each independently selected from 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, which forms R a21 、R b21 、R c21 and R d21 The 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, each optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from: C 1-6 Alkyl, halogen, CN, OR a22 SR a22 、C(O)R b22 、C(O)NR c22 R d22 、C(O)OR a22 、OC(O)R b22 、OC(O)NR c22 R d22 NR c22 R d22 NR c22 C(O)R b22 NR c22 C(O)NR c22 R d22 NR c22 C(O)OR a22 、C(=NR e22 )NR c22 R d22 NR c22 C(=NRe22 )NR c22 R d22 、S(O)R b22 、S(O)NR c22 R d22 、S(O)2R b22 NR c22 S(O)2R b22 、S(O)2NR c22 R d22 and oxo;

[0390] or R attached to the same N atom c21 and R d21 , together with the nitrogen atom to which they are attached, form a 4-, 5-, 6- or 7-membered heterocycloalkyl or a 5-membered heteroaryl, each optionally substituted with 1, 2 or 3 substituents independently selected from: C 1-6 Alkyl, halogen, CN, OR a22 SR a22 、C(O)R b22 、C(O)NR c22 R d22 、C(O)OR a22 、OC(O)R b22 、OC(O)NR c22 R d22 NR c22 R d22 NR c22 C(O)R b22 NR c22 C(O)NR c22 R d22 NR c22 C(O)OR a22 、C(=NR e22 )NR c22 R d22 NR c22 C(=NR e22 )NR c22 R d22 、S(O)R b22 、S(O)NR c22 R d22 、S(O)2R b22 NR c22 S(O)2R b22 、S(O)2NR c22 R d22 and oxo;

[0391] R a22 、R b22 、R c22 and R d22Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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, which forms R a22 、R b22 、R c22 and R d22 The C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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, each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of OH, CN, amino, NH(C 1-6 alkyl), N(C 1-6 Alkyl)2, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 haloalkoxy and oxo;

[0392] or R attached to the same N atom c22 and R d22 , together with the nitrogen atom to which they are attached, form a 4-, 5-, 6- or 7-membered heterocycloalkyl or a 5-membered heteroaryl, each of which is unsubstituted or substituted by 1, 2 or 3 substituents independently selected from the group consisting of OH, CN, amino, NH(C 1-6 alkyl), N(C 1-6 Alkyl)2, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 haloalkoxy and oxo;

[0393] R e21 and Re 22 Each independently is H, CN or NO2;

[0394] Cy 3A is unsubstituted or substituted C 6-10 aryl, or unsubstituted or substituted 5-10 membered heteroaryl; wherein Cy 3A The ring atoms of the 5-10 membered heteroaryl group are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein the ring atoms of the 5-10 membered heteroaryl group are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; 3A Substituted C 6-10 The aryl or substituted 5-10 membered heteroaryl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy3A , halogen, C 1-6 Haloalkyl, CN, OR a31 、S Ra31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR 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 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo;

[0395] Each R Cy3A Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy3A The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, N and S, wherein R Cy3A Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo, and wherein R Cy3A Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo;

[0396] R 31 H or C 1-6 Alkyl, C 6-10 Aryl-C 1-6 Alkyl or 5-10 membered heteroaryl-C 1-6 Alkyl, which forms R 31 C 1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo, and wherein R 31 C 6-10 Aryl-C 1-6 Alkyl or 5-10 membered heteroaryl-C 1-6 The alkyl group is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)Rb31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo;

[0397] R 32 H or C 1-6 alkyl; or

[0398] R 31 and R 32 Together with the groups to which they are attached, they form a 4-6 membered heterocycloalkyl ring;

[0399] 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 Alkynylidene)Cy 3B , where R 35 C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 The alkynylene component is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents each independently selected from halogen, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NRe31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo;

[0400] Each R 33A are independently H or C 1-6 alkyl;

[0401] Each R 33B are independently H or C 1-6 alkyl; or

[0402] or R attached to the same carbon atom 33A and R 33B , does not depend on any other R 33A and R 33B groups, which together can form –(CH2) 2-5 -, thereby forming a 3-6 membered cycloalkyl ring; or

[0403] n33 is 0, 1, 2, or 3;

[0404] 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; wherein Cy 3B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

[0405] The formation of Cy 3B Substituted C 6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from 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 、OC(O)R b31、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR 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 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo;

[0406] Each R Cy3B Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy3B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R Cy3B Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O) Rb31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo; and wherein R Cy3B Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O) Rb31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NRe31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo;

[0407] R 34 Selected from H and C 1-6 alkyl;

[0408] R 35 Selected from H, substituted or substituted C 1-6 Alkyl and Cy 3C , which forms R 35 Substituted C 1-6 The alkyl group is substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of: Cy 3C 、Halogen、CN、OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2Rb31 、S(O)2NR c31 R d31 and oxo; provided that R 35 No more than one substituent is Cy 3C ;

[0409] 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; wherein Cy 3C The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

[0410] The formation of Cy 3C Substituted C 6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy3C , halogen, C 1-6 Haloalkyl, CN, OR a31 、S Ra31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O)R b31 NR c31 C(O)NR c31 R d31 NR 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 NR c31C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo;

[0411] Each R Cy3C Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy3C The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R Cy3C Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O) Rb31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo; and wherein R Cy3C Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a31 SR a31 、C(O)R b31 、C(O)NR c31 R d31 、C(O)OR a31 、OC(O)R b31 、OC(O)NR c31 R d31 NR c31 R d31 NR c31 C(O) Rb31 NR c31 C(O)NR c31 R d31 NR c31 C(O)OR a31 、C(=NR e31 )NR c31 R d31 NR c31 C(=NR e31 )NR c31 R d31 、S(O)R b31 、S(O)NR c31 R d31 、S(O)2R b31 NR c31 S(O)2R b31 、S(O)2NR c31 R d31 and oxo;

[0412] R 36 Selected from H and C 1-6 alkyl;

[0413] R a31 、R b31 、R c31 and R d31 Each independently selected from H, C1-6 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, which forms R a31 、R b31 、R c31 and R d31 The 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, each optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from: C 1-6 Alkyl, halogen, CN, OR a32 SR a32 、C(O)R b32 、C(O)NR c32 R d32 、C(O)OR a32 、OC(O)R b32 、OC(O)NR c32 R d32 NR c32 R d32 NR c32 C(O)R b32 NR c32 C(O)NR c32 R d32 NR c32 C(O)OR a32 、C(=NR e32 )NR c32 R d32 NR c32 C(=NR e32 )NR c32 R d32 、S(O)Rb32 、S(O)NR c32 R d32 、S(O)2R b32 NR c32 S(O)2R b32 、S(O)2NR c32 R d32 and oxo;

[0414] or R attached to the same N atom c31 and R d31 , together with the nitrogen atom to which they are attached, form a 4-, 5-, 6- or 7-membered heterocycloalkyl or a 5-membered heteroaryl, each optionally substituted with 1, 2 or 3 substituents independently selected from: C 1-6 Alkyl, halogen, CN, OR a32 SR a32 、C(O)R b32 、C(O)NR c32 R d32 、C(O)OR a32 、OC(O)R b32 、OC(O)NR c32 R d32 NR c32 R d32 NR c32 C(O)R b32 NR c32 C(O)NR c32 R d32 NR c32 C(O)OR a32 、C(=NR e32 )NR c32 R d32 NR c32 C(=NR e32 )NR c32 R d32 、S(O)R b32 、S(O)NR c32 R d32 、S(O)2R b32 NR c32 S(O)2R b32 、S(O)2NR c32 R d32 and oxo;

[0415] R a32 、R b32 、R c32 and R d32 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-6 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, which forms R a32 、R b32 、R c32 and R d32 The C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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, each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of OH, CN, amino, NH(C 1-6 alkyl), N(C 1-6 Alkyl)2, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 haloalkoxy and oxo;

[0416] or R attached to the same N atom c32 and R d32 , together with the nitrogen atom to which they are attached, form a 4-, 5-, 6- or 7-membered heterocycloalkyl or a 5-membered heteroaryl, each of which is unsubstituted or substituted by 1, 2 or 3 substituents independently selected from the group consisting of OH, CN, amino, NH(C 1-6 alkyl), N(C 1-6 Alkyl)2, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 haloalkoxy and oxo; and

[0417] R e31 and Re 32 Each independently is H, CN or NO2;

[0418] Cy 4Ais unsubstituted or substituted C 6-10 aryl, or unsubstituted or substituted 5-10 membered heteroaryl; wherein Cy 4A The ring atoms of the 5-10 membered heteroaryl group are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein the ring atoms of the 5-10 membered heteroaryl group are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; 4A Substituted C 6-10 The aryl or substituted 5-10 membered heteroaryl is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy4A , halogen, C 1-6 Haloalkyl, CN, OR a41 、S Ra41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR 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 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo;

[0419] Each R Cy4A Independently selected from C1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy4A The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, N and S, wherein R Cy4A Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo, and wherein R Cy4A Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo;

[0420] R 41 H or C 1-6 Alkyl, C 6-10 Aryl-C 1-6 Alkyl or 5-10 membered heteroaryl-C 1-6 Alkyl, which forms R 41 C 1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, CN, OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo, and wherein R 41 C 6-10 Aryl-C 1-6 Alkyl or 5-10 membered heteroaryl-C 1-6 The alkyl group is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo;

[0421] R 42 H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl or Cy 4B ; which forms R 42 Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of: Cy 4B 、Halogen、CN、OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo; provided that no more than one substituent is Cy 4B ;

[0422] Cy 4B is unsubstituted or substituted C 6-10Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C 3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; wherein Cy 4B The ring atoms of the 5-10 membered heteroaryl or the unsubstituted or substituted 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and wherein the Cy 4B Substituted C 6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3-10 The cycloalkyl or 4-10 membered heterocycloalkyl group is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R 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 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR 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 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 Rd41 and oxo;

[0423] Each R Cy4B Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy4B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, and wherein R Cy4B Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo; and forming R Cy4B Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo;

[0424] or R 41 and R 42 Together with the atoms to which they are attached, and the atoms connecting R 41 and R 42 The nitrogen atom of the atom to which it is attached together forms a 4-7 membered heterocycloalkyl ring; which is optionally further substituted with 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R 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 、OC(O)Rb41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR 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 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo;

[0425] R 43 H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl or Cy 4C ; which forms R 43 Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents, each of which is independently selected from: 0, 1, 2, 3, 4 or 5 substituents selected from the group consisting of: Cy 4C 、Halogen、CN、OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo, provided that the formation of R 43 C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 No more than one substituent of an alkynyl group is Cy 4C ;

[0426] 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; wherein Cy 4B The ring atoms of the 5-10 membered heteroaryl or the unsubstituted or substituted 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and wherein the Cy 4C Substituted C 6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C 3-10 The cycloalkyl or 4-10 membered heterocycloalkyl group is substituted by 1, 2, 3, 4 or 5 substituents, each of which is independently selected from R Cy4C , halogen, C 1-6 Haloalkyl, CN, OR a41 、S Ra41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR 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 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo;

[0427] Each R Cy4C Independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R Cy4C The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl are composed of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, wherein R Cy4C Each C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from halogen, CN, OR a41 SR a41 、C(O)R b41、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)OR a41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo; and wherein each R Cy4A Each C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 The cycloalkyl and 4-10 membered heterocycloalkyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, OR a41 SR a41 、C(O)R b41 、C(O)NR c41 R d41 、C(O)OR a41 、OC(O)R b41 、OC(O)NR c41 R d41 NR c41 R d41 NR c41 C(O)R b41 NR c41 C(O)NR c41 R d41 NR c41 C(O)ORa41 、C(=NR e41 )NR c41 R d41 NR c41 C(=NR e41 )NR c41 R d41 、S(O)R b41 、S(O)NR c41 R d41 、S(O)2R b41 NR c41 S(O)2R b41 、S(O)2NR c41 R d41 and oxo;

[0428] R a41 、R b41 、R c41 and R d41 Each independently selected from 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, which forms R a41 、R b41 、R c41 and R d41 The 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, each optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from: C 1-6 Alkyl, halogen, CN, OR a42 SR a42 、C(O)R b42 、C(O)NRc42 R d42 、C(O)OR a42 、OC(O)R b42 、OC(O)NR c42 R d42 NR c42 R d42 NR c42 C(O)R b42 NR c42 C(O)NR c42 R d42 NR c42 C(O)OR a42 、C(=NR e42 )NR c42 R d42 NR c42 C(=NR e42 )NR c42 R d42 、S(O)R b42 、S(O)NR c42 R d42 、S(O)2R b42 NR c42 S(O)2R b42 、S(O)2NR c42 R d42 and oxo;

[0429] or R attached to the same N atom c41 and R d41 , together with the nitrogen atom to which they are attached, form a 4-, 5-, 6- or 7-membered heterocycloalkyl or a 5-membered heteroaryl, each optionally substituted with 1, 2 or 3 substituents independently selected from: C 1-6 Alkyl, halogen, CN, OR a42 SR a42 、C(O)R b42 、C(O)NR c42 R d42 、C(O)OR a42 、OC(O)R b42 、OC(O)NR c42 R d42 NR c42 R d42 NR c42 C(O)R b42 NR c42 C(O)NR c42 R d42 NR c42 C(O)OR a42 、C(=NR e42 )NRc42 R d42 NR c42 C(=NR e42 )NR c42 R d42 、S(O)R b42 、S(O)NR c42 R d42 、S(O)2R b42 NR c42 S(O)2R b42 、S(O)2NR c42 R d42 and oxo;

[0430] R a42 、R b42 、R c42 and R d42 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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, which forms R a42 、R b42 、R c42 and R d42 The C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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, each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of OH, CN, amino, NH(C 1-6 alkyl), N(C 1-6 Alkyl)2, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 haloalkoxy and oxo;

[0431] or R attached to the same N atom c42 and R d42 , together with the nitrogen atom to which they are attached, form a 4-, 5-, 6- or 7-membered heterocycloalkyl or a 5-membered heteroaryl, each of which is unsubstituted or substituted by 1, 2 or 3 substituents independently selected from the group consisting of OH, CN, amino, NH(C 1-6 alkyl), N(C 1-6 Alkyl)2, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 haloalkoxy and oxo; and

[0432] R e41 and Re 42 Each independently is H, CN or NO2.

[0433] In some embodiments, the small molecule is a compound of formula (VA) or (VB):

[0434]

[0435]

[0436] Stereoisomers, tautomers or pharmaceutically acceptable salts thereof;

[0437] in:

[0438] A 1 is selected from -(C=NH)-, -(C=NOR a )–、–[C=NO(C=O)R a ]–、–[C=N[O(C=O)ZR b ]}-, a fused 5- or 6-membered heterocyclyl and a fused 5- or 6-membered heteroaryl;

[0439] When A 1 When it is –(C=NH)–, Y 1 Selected from –NH2, –NH(C=O)R a and –NH(C=O)ZR b ;

[0440] When A 1 =NOR a )–、–[C=NO(C=O)R a ]–or–{C=N[O(C=O)ZR b ]}–, Y 1 is –NH2;

[0441] When A 1When Y is a fused heterocyclic group or heteroaryl group, 1 is –NH2 or a halogen, and A 1 By m other R 1 group substitution;

[0442] Each R a and R b Independently selected from C1-C6 alkyl, C3-C 10 Cycloalkyl, C6-C 10 Aryl and C7-C 12 Arylalkyl; wherein R a having m substituents selected from the group consisting of C1-C6 alkyl, hydroxy, hydroxy(C1-C6 alkyl), C1-C6 alkoxy, C2-C9 alkoxyalkyl, amino, C1-C6 alkylamino, and halogen; or, alternatively, R a and R b connected to form a heterocyclyl ring having m substituents selected from C1-C6 alkyl, hydroxy, C1-C6 alkoxy and halogen;

[0443] Each Z is independently selected from O and S;

[0444] A 2 is a member selected from C3-C6 heteroaryl, C6 aryl and C2-C6 alkyl;

[0445] When A 2 When Y is a C3-C6 heteroaryl group, 2 Selected from -NH2, -CH2NH2, chlorine, -(C=NH)NH2, -(C=NH)NH(C=O)R a 、–(C=NH)NH(C=O)ZR b 、–(C=NOR a )NH2,–[C=NO(C=O)R a ]NH2 and –{C=N[O(C=O)ZR b ]}NH2; and A 2 By m other R 1 group substitution;

[0446] When A 2 When Y is a C6 aryl group, 2 is selected from aminomethyl, hydroxy and halogen, and A 2 By m other R 1 group substitution;

[0447] When A 2 When Y is a C2-C6 alkyl group, 2 Selected from –NH(C=NH)NH2, –NH(C=NH)NH(C=O)R aand –NH(C=NH)NH(C=O)ZR b ;

[0448] Each R 1 is a member independently selected from C1-C6 alkyl, hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino and halogen;

[0449] Each of m and n is an integer independently selected from 0 to 3;

[0450] L is –(O) p –(C(R 2a )(R 2b )) q –,

[0451] Each R 2a or R 2b is a member independently selected from hydrogen and fluorine;

[0452] p is an integer from 0 to 1;

[0453] q is an integer from 1 to 2;

[0454] R 3 is a member selected from hydrogen, C1-C6 alkyl, C1-C6 fluoroalkyl and carboxyl(C1-C6 alkyl); or, alternatively, R 3 and R 4 connected to form an azetidine, pyrrolidine, or piperidine ring; or

[0455] R 4 is a member selected from hydrogen and C1-C6 alkyl; or, alternatively, R 3 and R 4 connected to form an azetidine, pyrrolidine, or piperidine ring;

[0456] R 5 is selected from C3-C7 cycloalkyl, C4-C8 cycloalkylalkyl, heteroaryl and having 0 to 3 R 13 Substituent C7-C 12 a member of an arylalkyl or heteroarylalkyl group; or, alternatively, R 5 and R 6 connected to form a circuit with 0 to 3 R 13 heterocyclic ring of the substituent;

[0457] R 6 is selected from hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, carboxyl (C1-C6 alkyl), having 0 to 3 R 13 Substituent C7-C 12a member of arylalkyl or heteroarylalkyl, amino(C1-C8 alkyl); and amido(C1-C8 alkyl); or, alternatively, R 5 and R 6 connected to form a circuit with 0 to 3 R 13 a substituent heterocyclic ring; and

[0458] Each R 13 are independently selected from C1-C6 alkyl, C6-C 10 Aryl, (C6-C 10 aryl) C1-C6 alkyl, carboxyl (C1-C6 alkoxy), heteroaryl, (C6-C 10 a member of heteroaryl) C1-C6 alkyl, heterocyclyl, hydroxy, hydroxy(C1-C6 alkyl), C1-C6 alkoxy, C2-C9 alkoxyalkyl, amino, C1-C6 amido, C1-C6 alkylamino, and halogen; or, alternatively, two R 13 Groups are connected to form fused C6-C 10 Aryl, C6-C 10 heteroaryl or a C5-C7 cycloalkyl ring.

[0459] In some embodiments, the small molecule is a compound of formula (VIA) or (VIB):

[0460]

[0461] Stereoisomers, tautomers or pharmaceutically acceptable salts thereof;

[0462] in:

[0463] A 1 is selected from -(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;

[0464] When A 1 When it is –(C=NH)–, Y 1 Selected from –NH2, –NH(C=O)R a and –NH(C=O)ZR b ;

[0465] When A 1 =NOR a )–、–[C=NO(C=O)R a ]–or–{C=N[O(C=O)ZR b ]}–, Y1 is –NH2;

[0466] When A 1 When Y is a fused heterocyclic group or heteroaryl group, 1 is –NH2 or a halogen, and A 1 By m other R 1 group substitution;

[0467] Each R a and R b Independently selected from C1-C6 alkyl, C3-C 10 Cycloalkyl, C6-C 10 Aryl and C7-C 12 Arylalkyl; wherein R a having m substituents selected from the group consisting of C1-C6 alkyl, hydroxy, hydroxy(C1-C6 alkyl), C1-C6 alkoxy, C2-C9 alkoxyalkyl, amino, C1-C6 alkylamino, and halogen; or, alternatively, R a and R b connected to form a heterocyclyl ring having m substituents selected from C1-C6 alkyl, hydroxy, C1-C6 alkoxy and halogen;

[0468] Each Z is independently selected from O and S;

[0469] A 2 is a member selected from C3-C6 heteroaryl and C2-C6 alkyl;

[0470] When A 2 When Y is a C3-C6 heteroaryl group, 2 Selected from -NH2, -CH2NH2, chlorine, -(C=NH)NH2, -(C=NH)NH(C=O)R a 、–(C=NH)NH(C=O)ZR b 、–(C=NOR a )NH2,–[C=NO(C=O)R a ]NH2 and –{C=N[O(C=O)ZR b ]}NH2; and A 2 By m other R 1 group substitution;

[0471] When A 2 When Y is a C2-C6 alkyl group, 2 Selected from –NH(C=NH)NH2, –NH(C=NH)NH(C=O)R a and –NH(C=NH)NH(C=O)ZR b ;

[0472] Each R1 is a member independently selected from C1-C6 alkyl, hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino and halogen;

[0473] Each of m and n is an integer independently selected from 0 to 3;

[0474] X and X 2 Each is selected from NR 8 , CH and CR 10 members of

[0475] Each R 8 is a member independently selected from hydrogen and C1-C6 alkyl;

[0476] Each R 10 is independently selected from C1-C6 alkyl, having 0 to 3 R 13 Substituent heteroaryl or C6-C 10 a member of aryl, hydroxy, hydroxy(C1-C6 alkyl), C1-C6 alkoxy, C2-C9 alkoxyalkyl, amino, C1-C6 alkylamino, and halogen; or, alternatively, two R 10 Groups are connected to form a group having 0 to 3 R 13 a fused C6 aryl, heteroaryl, or C5-C7 cycloalkyl ring of a substituent; or

[0477] r is an integer from 0 to 4; and

[0478] Each R 13 are independently selected from C1-C6 alkyl, C6-C 10 a member of aryl, carboxyl (C1-C6 alkoxy), heteroaryl, heterocyclyl, hydroxyl, hydroxyl (C1-C6 alkyl), C1-C6 alkoxy, C2-C9 alkoxyalkyl, amino, C1-C6 amido, C1-C6 alkylamino, and halogen; or, alternatively, two R 13 Groups are connected to form fused C6-C 10 Aryl, C6-C 10 heteroaryl or a C5-C7 cycloalkyl ring.

[0479] In certain embodiments, the small molecule is a compound of formula (VIIA) or (VIIB):

[0480]

[0481] Stereoisomers, tautomers or pharmaceutically acceptable salts thereof;

[0482] in:

[0483] A 1 is selected from -(C=NH)-, -(C=NORa )–、–[C=NO(C=O)R a ]–、–[C=N[O(C=O)ZR b ]}-, a fused 5- or 6-membered heterocyclyl and a fused 5- or 6-membered heteroaryl;

[0484] When A 1 When it is –(C=NH)–, Y 1 Selected from –NH2, –NH(C=O)R a and –NH(C=O)ZR b ;

[0485] When A 1 =NOR a )–、–[C=NO(C=O)R a ]–or–{C=N[O(C=O)ZR b ]}–, Y 1 is –NH2;

[0486] When A 1 When Y is a fused heterocyclic group or heteroaryl group, 1 is –NH2 or a halogen, and A 1 By m other R 1 group substitution;

[0487] Each R a and R b Independently selected from C1-C6 alkyl, C3-C 10 Cycloalkyl, C6-C 10 Aryl and C7-C 12 Arylalkyl; wherein R a having m substituents selected from the group consisting of C1-C6 alkyl, hydroxy, hydroxy(C1-C6 alkyl), C1-C6 alkoxy, C2-C9 alkoxyalkyl, amino, C1-C6 alkylamino, and halogen; or, alternatively, R a and R b connected to form a heterocyclyl ring having m substituents selected from C1-C6 alkyl, hydroxy, C1-C6 alkoxy and halogen;

[0488] Each Z is independently selected from O and S;

[0489] A 2 is a member selected from C3-C6 heteroaryl and C2-C6 alkyl;

[0490] When A 2 When Y is a C3-C6 heteroaryl group, 2 Selected from -NH2, -CH2NH2, chlorine, -(C=NH)NH2, -(C=NH)NH(C=O)Ra 、–(C=NH)NH(C=O)ZR b 、–(C=NOR a )NH2,–[C=NO(C=O)R a ]NH2 and –{C=N[O(C=O)ZR b ]}NH2; and A 2 By m other R 1 group substitution;

[0491] When A 2 When Y is a C2-C6 alkyl group, 2 Selected from –NH(C=NH)NH2, –NH(C=NH)NH(C=O)R a and –NH(C=NH)NH(C=O)ZR b ;

[0492] Each R 1 is a member independently selected from C1-C6 alkyl, hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino and halogen;

[0493] Each of m and n is an integer independently selected from 0 to 3;

[0494] L is –(O) p –(C(R 2a )(R 2b )) q –,

[0495] Each R 2a or R 2b is a member independently selected from hydrogen and fluorine;

[0496] p is an integer from 0 to 1;

[0497] q is an integer from 1 to 2;

[0498] R 3 is a member selected from hydrogen, C1-C6 alkyl and carboxy(C1-C6 alkyl);

[0499] Each R 11 is independently selected from C1-C6 alkyl, hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino, halogen and (R 14 )(R 14 )N(CO)-; or, alternatively, two R 11 Groups are connected to form a group having 0 to 3 R 13 a fused C6 aryl, heteroaryl, or C5-C7 cycloalkyl ring of a substituent; or

[0500] r is an integer from 0 to 4; and

[0501] Each Z is independently selected from O and NR 8 members of

[0502] Each R 8 is a member independently selected from hydrogen and C1-C6 alkyl;

[0503] Each R 12 is independently selected from hydrogen, having 0 to 3 R 13 Substituents of C1-C6 alkyl and C7-C 14 a member of an arylalkyl group;

[0504] Each R 13 is a member independently selected from C1-C6 alkyl, hydroxy, hydroxy(C1-C6 alkyl), C1-C6 alkoxy, C2-C9 alkoxyalkyl, amino, C1-C6 alkylamino and halogen; or, alternatively, two R 13 groups are connected to form a fused C6 aryl, heteroaryl or C5-C7 cycloalkyl ring; and

[0505] Each R 14 are independently selected from hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C4-C8 cycloalkylalkyl, C7-C 14 a member of arylalkyl and heteroaryl(C1-C6 alkyl); or, alternatively, two R 13 The groups are linked to form a fused heterocyclyl ring.

[0506] In some embodiments, the small molecule is a compound having the following structure:

[0507]

[0508] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0509] R 1 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0510] R 2a 、R 2b 、R 2c and R 2d independently selected from hydrogen, halogen, C(=O)OR 5 、OC(=O)R 5 , hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminoalkyl, carboxyalkyl, NR 5 R 6 、C(=O)NR 5 R6 、N(R 5 )C(=O)R 6 NR 5 C(=O)NR 6 、S(O) t SR 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)2R 5 、N(R 5 )S(O)2R 6 、S(O)2NR 5 R 6 , aryl, heteroaryl, heterocyclyl, cycloalkyl and oxo, provided that R 2a 、R 2b 、R 2c or R 2d At least one occurrence of is not hydrogen;

[0511] R 3 NR 3a R 3b ;

[0512] R 3a and R 3b Each is independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH2) n C(=O)OR 6 or (CH2) n P(=O)(OR 6 )2;

[0513] or R 3a and R 3b Together with the nitrogen to which they are attached, they form an optionally substituted 4-7 membered heteroaryl or an optionally substituted 4-7 membered heterocyclyl;

[0514] or R 3a and R 4 Together with the nitrogen and carbon atoms to which they are attached, they each form an optionally substituted 4-7 membered heterocyclyl;

[0515] When n is 2, 3, 4, 5 or 6, R4 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group; or

[0516] When n is 0 or 1, R 4 is a substituted or unsubstituted monocyclic heteroaryl group, or a substituted or unsubstituted heterocyclic group;

[0517] At each occurrence, R 5 、R 6 、R 7 and R 8 are independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl;

[0518] X is a direct key, -CR 2e R 2f -or-CR 2e R 2f -CR 2g R 2h -;

[0519] Y is a direct bond or -CR 2i R 2j -;

[0520] n is an integer from 0 to 6; and

[0521] t is 1-3.

[0522] In some embodiments, the small molecule is a compound having the following structure:

[0523]

[0524] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0525] R 1 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0526] R 2a 、R 2b 、R 2c 、R 2d are independently selected from hydrogen, halogen, OR 5 、C(=O)OR 5 、OC(=O)R 5 , hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminoalkyl, carboxyalkyl, NR 5 R 6 、C(=O)NR 5 R 6、N(R 5 )C(=O)R 6 NR 5 C(=O)NR 6 、S(O) t SR 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)2R 5 、N(R 5 )S(O)2R 6 、S(O)2NR 5 R 6 , aryl, heteroaryl, heterocyclyl, cycloalkyl and oxo, provided that R 2a 、R 2b 、R 2c 、R 2d At least one occurrence of is not hydrogen;

[0527] R 3 NR 3a R 3b ;

[0528] R 3a and R 3b each is independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, -CH2C(C=O)OH, -CH2C(=O)Oalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, or cycloalkyl;

[0529] or R 3a and R 3b Together with the nitrogen to which they are attached, they form an optionally substituted 4-7 membered heteroaryl or an optionally substituted 4-7 membered heterocyclyl;

[0530] When n is 2, 3, 4, 5 or 6, R 4 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group; or

[0531] When n is 0 or 1, R 4 is a substituted or unsubstituted monocyclic heteroaryl group, or a substituted or unsubstituted heterocyclic group;

[0532] At each occurrence, R 5 、R 6 、R 7 and R 8 are independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl;

[0533] X is a direct key, -[C(R 2e )R 2f ]-or-[C(R 2e )R 2f ]-[C(R 2g )R 2h ]-;

[0534] Y is a direct bond or -[C(R 2i )R 2j ]-;

[0535] n is an integer from 0 to 6; and

[0536] t is 1-3,

[0537] The conditions are:

[0538] a) When R 2a 、R 2b 、R 2c 、R 2d When the first occurrence of OH is R 1 Does not have the following structure:

[0539]

[0540] b) When R 2a 、R 2b 、R 2c 、R 2d When one occurrence of is -OH, n is an integer from 2 to 6; and

[0541] c) When R 2a 、R 2b 、R 2c 、R 2d When the first occurrence of R is unsubstituted phenyl, 3a and R 3b Neither has the following structure:

[0542]

[0543] In some embodiments, the small molecule is a compound having the following structure:

[0544]

[0545] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0546] R 17 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0547] R 18a 、R 18b are independently selected from hydrogen, halogen, -OR 21 、C(=O)OR 21 、OC(=O)R 21 , hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminoalkyl, carboxyalkyl, NR 21 R 22 、C(=O)NR 21 R 22 、N(R 21 )C(=O)R 22 NR 21 C(=O)NR 22 、S(O) t SR 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)2R 21 、N(R 21 )S(O)2R 22 、S(O)2NR 21 R 22 , aryl, heteroaryl, heterocyclyl, cycloalkyl and oxo, provided that R 18a 、R 18b At least one occurrence of is not hydrogen;

[0548] R 19 NR 19a R 19b ;

[0549] R 19a and R 19b Each is independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH2) n C(=O)OR 5 or (CH2)n P(=O)(OR 5 )2;

[0550] or R 19a and R 19b Together with the nitrogen to which they are attached, they form an optionally substituted 4-7 membered heteroaryl or an optionally substituted 4-7 membered heterocyclyl;

[0551] R 20 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0552] At each occurrence, R 21 、R 22 、R 23 and R 24 are independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl;

[0553] X is a direct key, -CR 2e R 2f -or-CR 2e R 2f -CR 2g R 2h -;

[0554] Y is a direct bond or -CR 2i R 2j -;

[0555] Z is O or S;

[0556] m is an integer from 0 to 6; and

[0557] t is 1-3.

[0558] In some embodiments, the small molecule is a compound having the following structure:

[0559]

[0560] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0561] R 25 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0562] R 26a 、R 26b are independently selected from hydrogen, halogen, -OR 29 、C(=O)OR 29 、OC(=O)R 29, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminoalkyl, carboxyalkyl, NR 29 R 30 、C(=O)NR 29 R 30 、N(R 29 )C(=O)R 30 NR 29 C(=O)NR 30 、S(O) t SR 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)2R 30 、N(R 29 )S(O)2R 30 、S(O)2NR 29 R 30 , aryl, heteroaryl, heterocyclyl, cycloalkyl and oxo, provided that R 26a 、R 26b At least one occurrence of is not hydrogen;

[0563] R 27 NR 27a R 27b ;

[0564] R 27a and R 27b Each is independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH2) n C(=O)OR 29 or (CH2) n P(=O)(OR 29 )2;

[0565] or R 27a and R 27b Together with the nitrogen to which they are attached, they form an optionally substituted 4-7 membered heteroaryl or an optionally substituted 4-7 membered heterocyclyl;

[0566] R 28is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0567] At each occurrence, R 29 、R 30 、R 31 and R 32 are independently hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl;

[0568] X is a direct key or -CR 26c R 26d -;

[0569] p is an integer from 0 to 6; and

[0570] t is 1-3.

[0571] In some embodiments, the small molecule is a compound having the following structure:

[0572]

[0573] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0574] R 1 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0575] R 2 is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy or cycloalkyl;

[0576] R 3 is hydrogen, alkyl, haloalkyl or cycloalkyl;

[0577] or R 2 and R 3 Together with the carbon and nitrogen to which they are attached, they each form an optionally substituted 4-7 membered heterocyclyl;

[0578] R 4 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0579] R 5a is hydrogen, alkyl, haloalkyl, cycloalkyl, phosphinoalkyl, (CH2) n C(=O)OR 6 、C(=O)R 6 、C(=O)OR 6 or C(=O)NR 6 R7 ;

[0580] R 5b is an electron pair or an alkyl group;

[0581] At each occurrence, R 6 and R 7 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, or arylalkyl;

[0582] R 8 is alkyl, haloalkyl, aminoalkyl, or substituted or unsubstituted arylalkyl; and

[0583] n is 1, 2, 3, 4, 5, 6, 7 or 8,

[0584] The condition is

[0585] A)R 5a Alkyl, haloalkyl, cycloalkyl, phosphinoalkyl, (CH2) n C(=O)OR 6 、C(=O)R 6 、C(=O)OR 6 or C(=O)NR 6 R 7 , or R 1 Selected from substituted heteroaryl, C(=NH)NHC(=O)OR 8 、C(=NOC(=O)R 8 )NH2、C(=NOC(=O)OR 8 )NH2 and C(=NOH)NH2 are substituted with one or more substituents; and

[0586] B) When R 5a is alkyl or (CH2) n C(=O)OR 6 When R 1 Does not have the following structure:

[0587]

[0588] Unless R 2 and R 3 Together with the carbon and nitrogen to which they are attached, they each form an optionally substituted 4-7 membered heterocyclyl.

[0589] In some embodiments, the small molecule is a compound having the following structure:

[0590]

[0591] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0592] R 1 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0593] R 2 is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy or cycloalkyl;

[0594] R 3 is hydrogen, alkyl, haloalkyl or cycloalkyl;

[0595] or R 2 and R 3 Together with the carbon and nitrogen to which they are attached, they each form an optionally substituted 4-7 membered heterocyclyl;

[0596] R 4 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0597] R 5a is hydrogen, alkyl, haloalkyl, cycloalkyl, phosphinoalkyl, (CH2) n C(=O)OR 6 、C(=O)R 6 、C(=O)OR 6 or C(=O)NR 6 R 7 ;

[0598] R 5b is an electron pair or an alkyl group;

[0599] At each occurrence, R 6 and R 7 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, or arylalkyl;

[0600] R 8 is alkyl, haloalkyl, aminoalkyl, or substituted or unsubstituted arylalkyl; and

[0601] n is 1, 2, 3, 4, 5, 6, 7 or 8,

[0602] The condition is

[0603] A)R 5a Alkyl, haloalkyl, cycloalkyl, phosphinoalkyl, (CH2) n C(=O)OR 6 、C(=O)R 6 、C(=O)OR 6 or C(=O)NR 6 R 7 , or R 1Selected from substituted heteroaryl, C(=NH)NHC(=O)OR 8 、C(=NOC(=O)R 8 )NH2、C(=NOC(=O)OR 8 )NH2 and C(=NOH)NH2 are substituted with one or more substituents; and

[0604] B) The compound does not have one of the following structures:

[0605]

[0606]

[0607] In some embodiments, the small molecule is a compound having the following structure:

[0608]

[0609] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0610] R 1 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0611] R 2 is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy or cycloalkyl;

[0612] R 3 is hydrogen, alkyl, haloalkyl or cycloalkyl;

[0613] or R 2 and R 3 Together with the carbon and nitrogen to which they are attached, they each form an optionally substituted 4-7 membered heterocyclyl;

[0614] R 4 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0615] At each occurrence, R 5a and R 5b Independently have one of the following structures:

[0616]

[0617] or R 5a and R 5b Together with the phosphorus atom to which they are attached, they form an optionally substituted 4-7 membered heterocyclyl;

[0618] R6a is alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl;

[0619] At each occurrence, R 6b are independently hydrogen or alkyl;

[0620] At each occurrence, R 7 is independently alkyl, haloalkyl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, or heterocyclylalkyl;

[0621] R 8 is an amino acid side chain; and

[0622] n is 1, 2, 3, 4, 5, 6, 7 or 8.

[0623] In some embodiments, the small molecule is a compound having the following structure:

[0624]

[0625] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0626] represents a double bond or a single bond;

[0627] R 1 is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;

[0628] R 2 is hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, haloalkoxy or cycloalkyl;

[0629] R 3 is hydrogen, alkyl, haloalkyl or cycloalkyl, or R 2 and R 3 Together with the carbon and nitrogen to which they are attached, they each form an optionally substituted 4-7 membered heterocyclyl;

[0630] R 4 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclyl group;

[0631] R 5 is hydrogen, alkyl, haloalkyl, cycloalkyl, phosphinoalkyl, (CH2) m C(=O)OR 6 、C(=O)R 6 、C(=O)OR 6 、(CH2) m NR 6 S(O)2R 7or C(=O)NR 6 R 7 ;

[0632] At each occurrence, R 6 and R 7 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, or arylalkyl;

[0633] L 1 For direct keys, -CR 8a R 8b -、-S(O) t -、NR 8c or -O-;

[0634] R 8a and R 8b are independently hydrogen, alkyl, or R 8a and R 8b Together with the carbon to which they are attached, form an optionally substituted 3-6 membered cycloalkyl;

[0635] R 8c is 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;

[0636] n is 1 or 2;

[0637] m is 1, 2, 3, 4, 5 or 6; and

[0638] t is 0, 1, or 2.

[0639] In some embodiments, the small molecule is a compound having the following structure:

[0640]

[0641] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0642] R 1 is a substituted or unsubstituted heteroaryl group;

[0643] R 2 is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;

[0644] R3 is hydrogen or alkyl;

[0645] R 4 is an alkyl group, a substituted or unsubstituted arylalkyl group, a heterocyclic group substituted by a substituent selected from a substituted or unsubstituted phenyl group or a substituted or unsubstituted pyridyl group, or R 3 and R 4 Together with the nitrogen and carbon atoms to which they are attached, they each form an optionally substituted 4-10 membered heterocyclyl;

[0646] R 5a is hydrogen or halogen;

[0647] R 5b is 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;

[0648] L 1 Direct bond, -CH2-, -S(O) t -、NR 5b , -O-, -C=C- or -C≡C-; and

[0649] t is 0, 1, or 2,

[0650] The conditions are:

[0651] A)R 2 Does not have one of the following structures:

[0652]

[0653] B)R 1 Does not have one of the following structures:

[0654]

[0655] and

[0656] C) When R 2 When it is an unsubstituted phenyl group, R 1 Does not have one of the following structures:

[0657]

[0658] In some embodiments, the small molecule is a compound having the following structure:

[0659]

[0660] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0661] R 6 is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;

[0662] R 7 Alkyl, -SR 10 , substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0663] R 8 is hydrogen, alkyl, haloalkyl or cycloalkyl;

[0664] R 9 is a substituted or unsubstituted arylalkyl group, a substituted or unsubstituted heteroarylalkyl group, or R 8 and R 9 Together with the nitrogen to which they are attached, they form an optionally substituted 4-10 membered heterocyclyl;

[0665] R 10 is hydrogen, alkyl, haloalkyl or cycloalkyl;

[0666] The conditions are:

[0667] A) When R 7 is unsubstituted phenyl, 3-((methylsulfonyl)amino)phenyl, 2-methylphenyl, 3-(dimethylamino)phenyl, 3-(methylamino)phenyl, 3-methylphenyl, 3-aminomethylphenyl, 3-aminophenyl, unsubstituted pyridyl, 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 has one of the following structures:

[0668]

[0669] R 6 Does not have the following structure:

[0670]

[0671] B) When R 7 When it is an unsubstituted phenyl group, R 6 Does not have the following structure:

[0672]

[0673] In some embodiments, the small molecule is a compound having the following structure:

[0674] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein: R 11 Has one of the following structures:

[0675]

[0676] R 12 is methyl or halogen;

[0677] R 13 is a substituted or unsubstituted aryl group; and

[0678] n is 1 or 2

[0679] The conditions are:

[0680] The compound does not have the following structure:

[0681]

[0682] In some embodiments, the small molecule is a compound having the following structure:

[0683]

[0684] or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

[0685] R 14 is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;

[0686] R 15 is a substituted or unsubstituted arylalkyl group, or a substituted or unsubstituted heteroarylalkyl group;

[0687] L 2 is a direct bond, -C(=O) or -S(=O) t -;and

[0688] t is 0, 1, or 2.

[0689] In some embodiments, certain features of compounds and / or formulae are disclosed as groups or ranges. It is specifically contemplated that such disclosures include each and every individual subcombination of the members of such groups and ranges. For example, the term "C 1-6 "C1-C6 alkyl" and "C1-C6 alkyl" are specifically intended to disclose, but are not limited to, methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl, individually.

[0690] At various places in this specification, variables defining a divalent linking group are described. It is specifically contemplated that each linking substituent includes both the forward and reverse forms of the linking substituent. For example, -NR(CR'R") n -Include-NR(CR'R") n -and-(CR'R") n NR - both, and it is intended that each form is disclosed individually. When a structure requires a linking group, the Markush variable listed for that group is understood to be a linking group. For example, if a structure requires a linking group, and the Markush group definition for that variable lists "alkyl" or "aryl," it is understood that "alkyl" or "aryl" represents a linking alkylene or arylene group, respectively.

[0691] The term "substituted" means that an atom or a group of atoms formally replaces hydrogen as a "substituent" attached to another group. Unless otherwise indicated, the term "substituted" refers to any substitution level, such as mono-, di-, tri-, tetra- or penta-substitution, when such substitutions are allowed. Substituents are independently selected, and substitutions can be at any chemically accessible position. It should be understood that substitutions at a given atom are limited by valence. The phrase "optionally substituted" means substituted or unsubstituted. The term "substituted" means that a hydrogen atom is formally removed and replaced by a substituent. A single divalent substituent, such as oxo, can replace two hydrogen atoms.

[0692] The term "C" where n and m are integers n-m " and "C n -C m " indicates a group containing n to m carbon atoms. Examples include C 1-4 、C 1-6 Etc. This term is intended to explicitly disclose every member within the scope, i.e., C n 、C n+1 、C n+2 …C m-2 、C m-1 、C m For example, C 1-6 It is expected that C1, C2, C3, C4, C5 and C6 are disclosed. n-m "Will and" C n -C m "same.

[0693] The term "alkyl" used alone or in combination with other terms refers to a saturated hydrocarbon group which may be straight or branched. n-m Alkyl" and "C n -C m "Alkyl" refers to an alkyl group having n to m carbon atoms. For example, C1-C 12Indicates that the group may have 1 to 12 (inclusive) carbon atoms therein. If not otherwise specified, it is an alkyl group of about 1 to about 20 carbon atoms. Alkyl formally corresponds to an alkane with one C-H bond replaced as the point of attachment of the alkyl group to the remainder of the compound. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Exemplary alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologues such as 2-methyl-1-butyl, 1,1-dimethylpropyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, and the like. The term "lower alkyl" refers to an alkyl group having 1-6 carbon atoms in the chain. "Substituted alkyl" is an alkyl group substituted by one or more substituents.

[0694] The term "alkenyl", taken alone or in combination with other terms, refers to a straight or branched hydrocarbon radical corresponding to an alkyl radical having one or more double carbon-carbon bonds. Alkenyl formally corresponds to an alkene with one C—H bond replaced as the point of attachment of the alkenyl radical to the remainder of the compound. n-m Alkenyl" and "C n -C m "Alkenyl" refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Exemplary alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

[0695] The term "alkynyl", taken alone or in combination with other terms, refers to a straight or branched hydrocarbon radical corresponding to an alkyl radical having one or more triple carbon-carbon bonds. Alkynyl formally corresponds to an alkyne with one C—H bond replaced as the point of attachment of the alkyl radical to the remainder of the compound. n-m Alkynyl" and "C n -C m "Alkynyl" refers to an alkynyl group having n to m carbon atoms. Exemplary alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0696] The term "alkylene" taken alone or in combination with other terms refers to a divalent alkyl linking group. Alkylene formally corresponds to an alkane with two C—H bonds replaced as points of attachment of the alkylene to the remainder of the compound. n-m"Cn-m alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, methylene, ethane-1,2-diyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, and the like. In some embodiments, "Cn-m alkylene" may refer to a chain of n to m methylene (CH2) groups, -(CH2) n-m -, for example, -CH2-, -CH2CH2-, -CH2CH2CH2-, etc.

[0697] The term "alkoxy" employed alone or in combination with other terms refers to a radical of the formula -O-alkyl, wherein alkyl is as defined above. n-m "Alkoxy" refers to an alkoxy group whose alkyl group has n to m carbons. Exemplary alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0698] The term "alkoxyalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced by an alkoxy group. n-m Alkoxy-C p-q "Alkyl" refers to a C n-m Alkoxy-substituted C p-q Alkyl. In some embodiments, the hydroxyalkyl group has one alkoxy group. In some embodiments, the alkoxyalkyl group has one or two alkoxy groups, each on a different carbon atom. Examples may include, but are not limited to, methoxymethyl, ethoxymethyl, 3-ethoxyethyl, and 1-methoxyethyl.

[0699] The term "amino" refers to a group of formula -NH2.

[0700] The term "carbamoyl" refers to a group of formula -C(O)NH2.

[0701] The term "carbonyl," employed alone or in combination with other terms, refers to a -C(=O)- group, which may also be written as C(O).

[0702] The term "cyano" or "nitrile" refers to a group of formula -C≡N, which may also be written as -CN.

[0703] The term "halo" or "halogen," used alone or in combination with other terms, refers to fluorine, chlorine, bromine, and iodine. In some embodiments, "halogen" refers to a halogen atom selected from F, Cl, or Br. In some embodiments, halogen is F.

[0704] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced by a halogen atom. n-m"Haloalkyl" refers to a C group having n to m carbon atoms and at least one to at most {2(n to m)+1} halogen atoms. n-m In some embodiments, the alkyl radicals are halogenated alkyl radicals, which may be identical or different. In some embodiments, the halogen atom is a fluorine atom. In some embodiments, the haloalkyl radical has 1 to 6 or 1 to 4 carbon atoms. Exemplary haloalkyl radicals include CF 3 , C 2 F 5 , CHF 2 , CCl 3 , CHCl 2 , C 2 Cl 5 or the like. In some embodiments, the haloalkyl radical is a fluoroalkyl radical.

[0705] The term "haloalkoxy" employed alone or in combination with other terms refers to a radical of the formula -O-haloalkyl, wherein haloalkyl is as defined above. n-m "Haloalkoxy" refers to a haloalkoxy group whose haloalkyl group has n to m carbon atoms. Exemplary haloalkoxy groups include trifluoromethoxy and the like. In some embodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0706] The term "hydroxyalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced by a hydroxy group. n-m "Hydroxyalkyl" refers to a C group having n to m carbon atoms and at least one hydroxyl group. n-m Alkyl. In some embodiments, the hydroxyalkyl group has one alcohol group. In certain aspects, the hydroxyalkyl group has one or two alcohol groups, each on a different carbon atom. In certain aspects, the hydroxyalkyl group has 1, 2, 3, 4, 5, or 6 alcohol groups. Examples may include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, and 1-hydroxyethyl.

[0707] The term "oxo" refers to an oxygen atom as a divalent substituent, forming a carbonyl when attached to carbon, or forming a sulfoxide or sulfone group or an N-oxide group when attached to a heteroatom.

[0708] The term "sulfido" refers to a sulfur atom as a divalent substituent which when attached to carbon forms a thiocarbonyl (C=S).

[0709] The term "n-membered," where n is an integer, generally describes a moiety in which the number of ring atoms is n. The term "nm-membered," where n and m are integers, describes a range in which the number of ring atoms is n to m. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydronaphthalene is an example of a 10-membered cycloalkyl.

[0710] The term "aromatic" refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings having aromatic character (ie, having (4n+2) delocalized π (pi) electrons, where n is an integer).

[0711] The term "aryl", taken alone or in combination with other terms, refers to an aromatic hydrocarbon group which may be monocyclic or polycyclic (eg, having 2, 3, or 4 fused rings). n-m "Aryl" refers to an aromatic radical having n to m ring carbon atoms. Aryl groups include, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, tetracenyl, and the like. In some embodiments, an aryl group has 6 to about 20 carbon atoms, 6 to about 18 carbon atoms, 6 to about 15 carbon atoms, or 6 to about 10 carbon atoms. In some embodiments, an aryl group is phenyl.

[0712] The term "arylalkyl" or "aralkyl" or "alkylaryl" taken alone or in combination with other terms refers to a radical of the formula -alkylene-aryl and refers to an alkyl group as defined herein in which at least one hydrogen has been replaced by an aryl group as defined herein. In some embodiments, the arylalkyl group is C 6-10 Aryl-C 1-3 In some embodiments, arylalkyl is C 6-10 Aryl-C 1-4 In some embodiments, arylalkyl is C 6-10 Aryl-C 1-3 In some embodiments, arylalkyl is phenyl-C 1-3 Examples include, but are not limited to, benzyl, 1-phenylethyl, 4-methylbenzyl, and 1,1,-dimethyl-1-phenylmethyl. In some embodiments, arylalkyl is benzyl.

[0713] The term "heteroaryl" or "heteroaromatic," alone or in combination with other terms, refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. An "n-membered heteroaryl" or "n-membered heteroaromatic," where n is an integer, refers to a heteroaryl having n ring atoms. An "nm-membered heteroaryl" or "nm-membered heteroaromatic," where n and m are integers, refers to a heteroaryl having n to m ring atoms. The number of carbon atoms in the ring is less than the number of heteroatoms. Thus, in some embodiments, an n-membered heteroaryl may have n-1, n-2, n-3, or n-4 ring carbon atoms, while an nm-membered heteroaryl may have n-1, n-2, n-3, or n-4 ring carbon atoms to m-1, m-2, m-3, or m-4 ring carbon atoms. In some embodiments, an nm-membered heteroaryl may have 1 to m-1 ring carbon atoms. In some embodiments, the heteroaryl ring has 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-10 ring atoms, including carbon atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring. In other embodiments, the heteroaryl is an eight-membered, nine-membered or ten-membered fused bicyclic heteroaryl ring. Exemplary heteroaryl groups include, but are not limited to, pyridine, pyrimidine, pyrazine, pyridazine, pyrrole, pyrazole, oxazolyl, oxazole, isoxazole, thiazole, isothiazole, imidazole, furan, thiophene, quinoline, isoquinoline, naphthyridine (including 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, and 2,6-naphthyridine), indole, azaindole, benzothiophene, benzofuran, benzisoxazole, benzimidazole, imidazo[1,2-b]thiazole, purine, furazan, triazole, tetrazole, 1,2,4-thiadiazole, quinazoline, phthalazine, imidazo[1,2-a]pyridine, imidazo[2,1]-b]thiazolyl, and the like.

[0714] A five-membered heteroaryl ring is a heteroaryl group having five ring atoms, wherein one or more (e.g., 1, 2, or 3) of the ring atoms are independently selected from N, O, and S. Exemplary five-membered heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

[0715] A six-membered heteroaryl ring is a heteroaryl group having six ring atoms, wherein one or more (e.g., 1, 2, or 3) of the ring atoms are independently selected from N, O, and S. Exemplary six-membered ring heteroaryl groups are pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

[0716] The term "heteroarylalkyl" taken alone or in combination with other terms refers to a radical of the formula -alkylene-heteroaryl. The term "n-membered heteroarylalkyl" where n is an integer refers to a heteroarylalkyl radical where the heteroaryl radical is n-membered. The term "nm-membered C p-q "-alkyl" refers to heteroarylalkyl groups where the heteroaryl group is n to m membered and the alkyl group has p to q carbon atoms. In some embodiments, the heteroarylalkyl group is a 5-10 membered heteroaryl-C 1-3 Alkyl or C 1-9 Heteroaryl-C 1-3 wherein the heteroaryl moiety is monocyclic or bicyclic and has 1, 2, 3, 4, or 5 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, heteroarylalkyl is C 1-9 Heteroaryl-C 1-4 Alkyl, wherein the heteroaryl portion is monocyclic or bicyclic and has 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. Examples include pyridylmethyl, such as 2-pyridylmethyl, 3-pyridylmethyl or 4-pyridylmethyl.

[0717] The term "cycloalkyl" taken alone or in combination with other terms refers to a non-aromatic, saturated, monocyclic, bicyclic or polycyclic hydrocarbon ring system. The term includes cyclized alkyl and alkenyl groups. The term "C n-m "Cycloalkyl" refers to a cycloalkyl group having n to m ring member carbon atoms. Cycloalkyl groups may include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocyclic groups. Cycloalkyl groups may have 3, 4, 5, 6, or 7 ring carbon atoms (C 3-7 In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to 5 ring members, or 3 to 4 ring members. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. In some embodiments, the cycloalkyl group is C 3-6Monocyclic cycloalkyl. The ring-forming carbon atoms of the cycloalkyl group may optionally be oxidized to form an oxo or thio group. Cycloalkyl also includes cycloalkylene. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Also included in the definition of cycloalkyl are moieties having one or more aromatic rings fused to the cycloalkyl ring (i.e., having a common bond therewith), such as benzo or thienyl derivatives of cyclopentane, cyclohexane, etc. Cycloalkyl groups containing fused aromatic rings may be attached by any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4,4-dimethylcyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, etc. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0718] The term "cycloalkylalkyl" employed alone or in combination with other terms refers to a radical of the formula -alkylene-cycloalkyl. The term C wherein n, m, p and q are integers n-m Cycloalkyl-C p-q Alkyl refers to a cycloalkyl group having n to m carbon atoms attached to an alkyl group having p to q carbon atoms. In some embodiments, the cycloalkylalkyl group is C 3-7 Cycloalkyl-C 1-3 Alkyl, wherein the cycloalkyl portion is monocyclic or bicyclic. Examples include cyclopropylmethyl, cyclobutylmethyl, cyclopentanemethyl, and cyclohexylmethyl.

[0719] The term "heterocycloalkyl," taken alone or in combination with other terms, refers to a non-aromatic ring or ring system that may optionally contain one or more alkenylene groups as part of a ring structure having at least one heteroatom ring member independently selected from nitrogen, sulfur, and oxygen. An "n-membered heterocycloalkyl," wherein n is an integer, refers to a heteroaryl group having n ring atoms. An "nm-membered heterocycloalkyl," wherein n and m are integers, refers to a heterocycloalkyl group having n to m ring atoms. The number of carbon atoms in the ring is less than the number of heteroatoms. Thus, in some embodiments, an n-membered heterocycloalkyl group may have n-1, n-2, n-3, or n-4 ring carbon atoms, while an nm-membered heterocycloalkyl group may have n-1, n-2, n-3, or n-4 ring carbon atoms to m-1, m-2, m-3, or m-4 ring carbon atoms. In some embodiments, an nm-membered heterocycloalkyl group may have 1 to m-1 ring carbon atoms. In some embodiments, heterocycloalkyl has 4-12 ring members, 4-10 ring members, 4-7 ring members or 4-6 ring members. Included in heterocycloalkyl are monocyclic 4-, 5-, 6- and 7-membered heterocycloalkyls. Heterocycloalkyl can include monocyclic or bicyclic (e.g., having two fused rings or bridged rings) ring systems. In some embodiments, heterocycloalkyl is a monocyclic group having 1, 2 or 3 heteroatoms independently selected from nitrogen, sulfur and oxygen. The ring-forming carbon atoms and heteroatoms of heterocycloalkyl can be optionally oxidized to form oxo or thio groups or other oxidative bonds (e.g., C(O), S(O), C(S) or S(O) 2, N-oxides, etc.), or the nitrogen atom can be quaternized. Heterocycloalkyl can be attached through ring-forming carbon atoms or ring-forming heteroatoms. In some embodiments, heterocycloalkyl contains 0 to 3 double bonds. In some embodiments, heterocycloalkyl contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties having one or more aromatic rings fused to (i.e., having a common bond with) the heterocycloalkyl ring, such as benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. Heterocycloalkyl groups containing fused aromatic rings may be attached via any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Examples of heterocycloalkyl groups include azetidine, azepane, dihydrobenzofuran, dihydrofuran, dihydropyran, morpholine, 3-oxa-9-azaspiro[5.5]undecane, 1-oxa-8-azaspiro[4.5]decane, piperidine, piperazine, pyran, pyrrolidine, quinuclidine, tetrahydrofuran, tetrahydropyran, 1,2,3,4-tetrahydroquinoline, tropane, and thiomorpholine.

[0720] As used herein, the term "heterocycloalkylalkyl" employed alone or in combination with other terms refers to a radical of the formula -alkylene-heterocycloalkyl. The term "n-membered heterocycloalkylalkyl" wherein n is an integer refers to a heteroarylalkylalkyl wherein the heterocycloalkyl radical is n-membered. The term "n-membered C p-q-alkyl" refers to heterocycloalkylalkyl, wherein the heterocycloalkyl is n to m membered and the alkyl has p to q carbon atoms. In some embodiments, the heterocycloalkylalkyl is a 4-10 membered heterocycloalkyl-C 1-3 Alkyl or C 1-9 Heterocycloalkyl-C 1-3 wherein the heterocycloalkyl moiety is monocyclic or bicyclic and has 1, 2, 3, 4, or 5 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, heterocycloalkylalkyl is C 2-9 Heterocycloalkyl-C 1-4 Alkyl or C 2-9 Heterocycloalkyl-C 1-3 Alkyl, wherein the heterocycloalkyl portion is monocyclic or bicyclic and has 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen.

[0721] In some places, definition or embodiment can refer to specific ring (such as azetidine ring, pyridine ring etc.).Unless otherwise indicated, these rings can be connected to any ring member, provided that the valence of the atom is not exceeded.For example, azetidine ring can be connected at any position of the ring, and azetidine-3-yl ring is connected at 3 position.

[0722] When any two groups or two instances of the same substituent are "independently selected" from a list of alternatives, those groups may be the same or different. For example, if R a and R b are independently selected from alkyl, fluoro, amino and hydroxyalkyl, then there are two R a group and two R b The molecule of the group may have all groups that are alkyl groups (e.g., four different alkyl groups). Alternatively, the first R a Can be an alkyl group, the second R a It can be fluorine, the first R b It can be a hydroxyalkyl group, and the second R b It may be amino (or any other substituent selected from this group). Alternatively, R a and the first R b Both can be fluorine, and the second R b can be alkyl (i.e., some pairs of substituents can be identical, while other pairs can be different). Unless otherwise stated, if two or more groups having the same definition are present, but that definition provides alternatives, it is understood that each occurrence of the same group is independently selected from the possible alternatives. For example, if two or more R a group, and R a The definition of R a It can be A, B or C, then it should be understood that each R present in the compounda The groups are independently selected from A, B and C such that R a The groups may be the same or different.

[0723] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are contemplated. Compounds described herein containing asymmetrically substituted carbon atoms can be separated in optically active or racemic forms. Methods for preparing optically active forms from optically inert raw materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of alkenes, C=N double bonds, and the like may also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be separated as a mixture of isomers or as separated isomeric forms.

[0724] The resolution of the racemic mixture of compound can be carried out by any of numerous methods known in the art.A kind of method comprises the fractional recrystallization using chiral resolution acid, and described chiral resolution acid is optically active, salified organic acid.Suitable resolving agent for fractional recrystallization method is such as optically active acid, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or various optically active camphorsulfonic acid such as β-camphorsulfonic acid of D and L form.Other resolving agents that are suitable for fractional crystallization method comprise the stereoisomerically pure form (such as S and R form, or diastereoisomerically pure form) of α-methylbenzylamine, 2-phenyl glycol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane etc.

[0725] The resolution of the racemic mixture can also be carried out by elution on a column equipped with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by those skilled in the art.

[0726] In some embodiments, the compounds of the present invention have the (R)-configuration. In other embodiments, the compounds have the (S)-configuration. In compounds with more than one chiral center, unless otherwise indicated, each chiral center in the compound can independently be (R) or (S).

[0727] Compounds described herein may also include tautomeric forms. Tautomeric forms arise from the exchange of a single bond with an adjacent double bond along with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Exemplary prototropic tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactam pairs, enamine-imine pairs, and cyclic forms in which a proton can occupy two or more positions of a heterocyclic ring system, such as 1H- and 3H-imidazoles, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles, and 1H- and 2H-pyrazoles. Tautomeric forms may be in equilibrium or spatially locked into one form by appropriate substitution. This disclosure contemplates all such tautomers of the compound.

[0728] The compounds described herein may also include all isotopes of atoms present in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0729] As used herein, the term "compound" is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure.

[0730] The compounds described herein may include acidic and / or basic groups and may be capable of forming salts. It should be understood that the present disclosure contemplates all salts of compounds capable of forming salts, regardless of whether the possible presence of a salt is explicitly described, including both acidic and basic salts of the compound. Furthermore, when a compound is described as a salt, it should be understood that the disclosure of that compound contemplates all forms of that compound, including the free base or free acid, as well as alternative salt forms thereof. The term "salt" refers to a derivative of a disclosed compound in which the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The terms "a salt thereof," "salts thereof," or "salts thereof" may apply to any preceding member of the relevant Markush group. For example, a group consisting of A, B, C, and their salts includes within its scope embodiments in which the compound is a salt of A, embodiments in which the compound is a salt of B, and embodiments in which the compound is a salt of C.

[0731] The salts of the compounds disclosed herein include pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a non-toxic salt of a parent compound formed, for example, from a non-toxic inorganic or organic acid. The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19, and Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). In some embodiments, the compounds described herein include N-oxide forms. Additional information on suitable pharmaceutically acceptable salts can be found in Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, PA, which references are incorporated herein by reference.

[0732] Compounds and salts thereof, including pharmaceutically acceptable salts, may be found together with other substances such as water and solvents (e.g., hydrates and solvates), or may be isolated. When in the solid state, the compounds described herein and their salts may exist in various forms and may, for example, take the form of solvates, including hydrates. The compounds may be in any solid state form, such as a polymorph or solvate, and therefore, unless expressly stated otherwise, references to compounds and their salts should be understood to encompass any solid state form of the compound.

[0733] In some embodiments, the compounds described herein, or their salts, are substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it is formed or detected. Partial separation can include, for example, a composition enriched in the compounds of the invention. Substantially separated can include a composition containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the invention, or its salt.

[0734] The compounds described herein, including salts, hydrates and / or solvates thereof, can be prepared by any suitable known organic synthesis technique and can be synthesized according to any of a number of possible synthetic routes, such as those described 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 herein by reference in its entirety.

[0735] The reactions for preparing the compounds described herein can be carried out in suitable solvents that can be easily selected by those skilled in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), intermediates, or products at temperatures at which the reactions are carried out, for example, at temperatures that can range from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in one solvent or in a mixture of more than one solvent. Depending on the specific reaction step, a suitable solvent for a particular reaction step can be selected by those skilled in the art.

[0736] The preparation of the compounds of the present disclosure may involve the protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be readily determined by those skilled in the art. The chemistry of protecting groups is described, for example, in Kocienski, Protecting Groups, (Thieme, 2007); Robertson, Protecting Group Chemistry, (Oxford University Press, 2000); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6th edition (Wiley, 2007); Peturssion et al., "Protecting Groups in Carbohydrate Chemistry," J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groups in Organic Synthesis, 4th edition (Wiley, 2006).

[0737] The reaction can be monitored by any suitable method known in the art. For example, product formation can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g. 1 H or 13C), infrared spectroscopy, spectrophotometry (eg, UV-visible), mass spectrometry, or chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0738] Those skilled in the art will appreciate that formulations can be modified or optimized using general knowledge of organic chemistry to prepare various compounds within the scope of the present disclosure.

[0739] Starting materials, reagents, and intermediates whose synthesis is not described herein are either commercially available, known in the literature, or can be prepared by methods known to those skilled in the art.

[0740] Those skilled in the art will appreciate that the methods are not the only means by which the compounds of the present disclosure can be synthesized, and that a wide library of synthetic organic reactions is available to potentially be used in synthesizing the compounds of the present disclosure. Those skilled in the art will know how to select and implement appropriate synthetic routes. Suitable synthetic methods for starting materials, intermediates, and products can be identified by reference to the literature, including reference sources such as: Advances in Heterocyclic Chemistry, Vols. 1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry, Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira et al. (eds.) Science of Synthesis, Vols. 1-48 (2001-2010) and Knowledge Updates KU 2010 / 1-4; 2011 / 1-4; 2012 / 1-2 (Thieme, 2001-2012); Katritzky et al. (eds.) Comprehensive Organic Functional Group Transformations, (Pergamon Press, 1996); Katritzky et al. (eds.); Comprehensive Organic Functional Group Transformations II (Elsevier, 2nd ed., 2004); Katritzky et al. (eds.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984); Katritzky et al., Comprehensive Heterocyclic Chemistry II (Pergamon Press, 1996); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6th ed. (Wiley, 2007); Trost et al. (eds.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

[0741] In some embodiments, the compounds of the present disclosure selectively inhibit MASP-2 over thrombin. In some embodiments, the selectivity ratio for MASP-2:thrombin inhibition is at least 1.1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, or 30:1. In some embodiments, in addition to inhibiting MASP-2, the compounds of the present disclosure may also inhibit one or more coagulation proteases, such as thrombin.

[0742] The MASP-2 inhibitory activity of the small molecule compounds of the present disclosure can be determined by methods known in the art, for example, an enzymatic MASP-2 assay utilizing a fluorogenic substrate as disclosed in PCT Application No. PCT / US19 / 34220, which is incorporated herein by reference in its entirety. Similarly, the thrombin inhibitory activity of the compounds of the present disclosure can be determined using methods known in the art. Exemplary assay methods and MASP-2 inhibitory activity of representative small molecule compounds are described in Examples 23 to 25 herein.

[0743] In some embodiments, the compounds disclosed herein inhibit activation of the lectin pathway. The lectin pathway inhibitory activity of the compounds of the present invention can be determined by methods known in the art, for example, using the lectin pathway activation (LPA) assay in human serum as described in PCT Application No. PCT / US19 / 34220, which is incorporated herein by reference in its entirety.

[0744] In some embodiments, the compounds have a Ki value for MASP-2 inhibition of less than about 25 μM, less than about 10 μM, less than about 2.5 μM, less than about 1 μM, less than about 0.5 μM, or less than about 0.1 μM, as determined, for example, by the enzymatic MASP-2 assay described above. In some embodiments, the compounds have a lectin pathway inhibition IC of less than about 50 μM, less than about 5 μM, less than about 0.5 μM, or less than about 0.05 μM, as determined, for example, by the lectin pathway activation (LPA) assay described above. 50 In some embodiments, the compound has greater than about 5.0-fold, greater than about 25-fold, greater than about 50-fold, or greater than about 100-fold selectivity for MASP-2 inhibition relative to thrombin (as determined, for example, by the ratio of the respective Ki).

[0745] In some embodiments, the compound is a compound of Tables 4A-4E below.

[0746] Table 4A. Exemplary compounds

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795]

[0796]

[0797]

[0798]

[0799]

[0800]

[0801]

[0802]

[0803]

[0804]

[0805]

[0806]

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833] TABLE 4B Exemplary MASP-2 Inhibitory Compounds

[0834]

[0835]

[0836]

[0837]

[0838]

[0839] TABLE 4C Exemplary MASP-2 Inhibitory Compounds

[0840]

[0841]

[0842]

[0843]

[0844]

[0845]

[0846]

[0847]

[0848]

[0849] TABLE 4D Exemplary MASP-2 Inhibitory Compounds

[0850]

[0851]

[0852]

[0853]

[0854]

[0855]

[0856]

[0857]

[0858]

[0859]

[0860]

[0861]

[0862]

[0863]

[0864]

[0865]

[0866]

[0867]

[0868]

[0869]

[0870]

[0871] TABLE 4E Exemplary MASP-2 Inhibitory Compounds

[0872]

[0873]

[0874]

[0875]

[0876]

[0877]

[0878]

[0879]

[0880]

[0881]

[0882]

[0883]

[0884]

[0885]

[0886]

[0887]

[0888]

[0889]

[0890]

[0891]

[0892]

[0893]

[0894]

[0895]

[0896]

[0897]

[0898]

[0899]

[0900]

[0901]

[0902]

[0903]

[0904]

[0905]

[0906]

[0907]

[0908] In some embodiments, the MASP-2 inhibitory agent is a compound of any one of Tables 4A, 4B, 4C, 4D, and 4E. In some embodiments, the MASP-2 inhibitory agent is compound 1230, 1231, III-91, or I-89. In some embodiments, the MASP-2 inhibitory agent is a small molecule with a molecular weight of 200 Da to 2,000 Da. In some embodiments, the MASP-2 inhibitory agent is a small molecule with a molecular weight of 250 Da to 2,000 Da. In some embodiments, the MASP-2 inhibitory agent is a small molecule with a molecular weight of 350 Da to 2,000 Da. In some embodiments, the MASP-2 inhibitory agent is a small molecule with a molecular weight of 350 Da to 1,500 Da. In some embodiments, the MASP-2 inhibitory agent is a small molecule with a molecular weight of 350 Da to 1,200 Da.

[0909] In some embodiments, the MASP-2 inhibitory agent is a small molecule having only one basic group selected from guanidino and benzamidinyl. In some embodiments, the MASP-2 inhibitory agent is a small molecule that does not include a basic group selected from guanidino and benzamidinyl.

[0910] Inhibitors of MASP-2 expression

[0911] In another embodiment of this aspect of the invention, the MASP-2 inhibitory agent is a MASP-2 expression inhibitor that is capable of inhibiting MASP-2-dependent complement activation. In the practice of this aspect of the 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.

[0912] Antisense RNA and DNA molecules act to block the translation of MASP-2 mRNA by hybridizing to it and preventing the translation of the MASP-2 protein. Antisense nucleic acid molecules can be constructed in a variety of different ways, provided that they are capable of interfering with the expression of MASP-2. For example, an antisense nucleic acid molecule can be constructed by reversing the coding region (or a portion thereof) of the MASP-2 cDNA (SEQ ID NO:4) relative to its normal transcriptional direction to allow transcription of its complement.

[0913] Antisense nucleic acid molecules are generally substantially identical to at least a portion of one or more target genes. However, nucleic acids do not need to be identical to inhibit expression. Generally, higher homology can be used to compensate for the use of shorter antisense nucleic acid molecules. The minimum percent identity is generally greater than about 65%, but higher percent identity can exert more effective suppression on the expression of endogenous sequences. Generally, substantially higher percent identities greater than about 80% are preferred, although generally about 95% to absolute identity is most preferred.

[0914] Antisense nucleic acid molecules do not need to have the same intron or exon pattern as the target gene, and noncoding segments of the target gene can be just as effective as coding segments in achieving antisense suppression of target gene expression. DNA sequences of at least about 8 or so nucleotides can be used as antisense nucleic acid molecules, although longer sequences are preferred. A representative example of a useful MASP-2 inhibitory agent in the present invention is an antisense MASP-2 nucleic acid molecule that is at least 90 percent identical to the complement of the MASP-2 cDNA consisting of the nucleic acid sequence set forth in SEQ ID NO:4. The nucleic acid sequence set forth in SEQ ID NO:4 encodes the MASP-2 protein consisting of the amino acid sequence set forth in SEQ ID NO:5.

[0915] Targeting MASP-2 mRNA with antisense oligonucleotides is another mechanism that can be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptor has been inhibited by antisense oligonucleotides directed against their respective mRNA sequences (U.S. Patent No. 5,739,119 to Cheng and U.S. Patent No. 5,759,829 to Shewmaker). In addition, examples of antisense inhibition have been demonstrated for the following: nuclear cell cycle proteins, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA AReceptor and human EGF (see, e.g., U.S. Patent No. 5,801,154 to Baracchini; U.S. Patent No. 5,789,573 to Baker; U.S. Patent No. 5,718,709 to Considine; and U.S. Patent No. 5,610,288 to Reubenstein).

[0916] A system has been described that allows one of ordinary skill to determine which oligonucleotides can be used in the present invention and involves probing for suitable sites in a target mRNA using RNase H cleavage as an indicator of sequence accessibility within the transcript. Scherr, M. et al., Nucleic Acids Res. 26:5079-5085, 1998; Lloyd et al., Nucleic Acids Res. 29:3665-3673, 2001. A mixture of antisense oligonucleotides complementary to certain regions of the MASP-2 transcript is added to an extract of cells expressing MASP-2, such as hepatocytes, and hybridized to generate sites susceptible to RNase H. This approach can be combined with computer-assisted sequence selection, which can predict the optimal sequence selection for an antisense composition based on their relative ability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to the target mRNA in the host cell. Can use 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), carry out these secondary structure analysis and target site selection consideration.Preferably comprise the length of about 8 to about 50 nucleotides for the antisense compounds of target sequence.It is particularly preferred that the antisense oligonucleotide comprising about 9 to about 35 nucleotides.The inventor has considered all oligonucleotide compositions (that is, the composition of length 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) within the scope of 9 to 35 nucleotides, is highly preferred for the practice of the method based on antisense oligonucleotide of the present invention. Highly preferred target regions of the MASP-2 mRNA are those at or near the AUG translation start codon, and 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 below:

[0917] SEQ ID NO:30 (nucleotides 22-680 of SEQ ID NO:4): Nucleic acid sequence of the MASP-2 cDNA (SEQ ID NO:4) encoding CUBIEGF.

[0918] SEQ ID NO:31 (5'CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC3): nucleotides 12-45 of SEQ ID NO:4 including the MASP-2 translation start site (sense).

[0919] SEQ ID NO:32 (5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3'): nucleotides 361-396 of SEQ ID NO:4 encoding the region encompassing the MASP-2 MBL binding site (sense).

[0920] SEQ ID NO:33 (5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3'): nucleotides 610-642 of SEQ ID NO:4 encoding a region comprising the CUBII domain

[0921] As noted above, as used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or its analogs. The term also encompasses the oligonucleotide bases composed of naturally occurring nucleotides, sugars, and covalent nucleoside (main chain) bonds, as well as oligonucleotides with non-natural modifications. These modifications allow for the introduction of certain desired properties not provided by naturally occurring oligonucleotides, such as reduced toxicity, increased stability for nuclease degradation, and enhanced cellular uptake. In illustrative embodiments, the antisense compounds of the present invention are different from natural DNA in the modification of the phosphodiester backbone, to extend the lifespan of antisense oligonucleotides, in which the phosphate substituent is replaced by thiophosphate. Similarly, one or both ends of an oligonucleotide can be substituted by one or more acridine derivatives, which are embedded between the adjacent base pairs in the nucleic acid chain.

[0922] Another alternative to antisense is to use " RNA interference " (RNAi). Double-stranded RNA (dsRNA) can induce gene silencing in mammals. The natural function of RNAi and co-suppression seems to be to protect the genome from invasion by mobile genetic elements, such as retrotransposons and viruses, which, when they become active, produce abnormal RNA or dsRNA in host cells (see, for example, Jensen, J. et al., Nat. Genet. 21: 209-12, 1999). Double-stranded RNA molecules can be prepared by synthesizing two RNA chains that can form double-stranded RNA molecules, each RNA chain having a length of about 19 to 25 (e.g., 19 23 nucleotides). For example, the dsRNA molecules that can be used in the method of the present invention can include RNA corresponding to the sequences listed herein and their complements (e.g., SEQ ID NO: 30 to SEQ ID NO: 33). Preferably, at least one chain of RNA has a 3' overhang of 1-5 nucleotides. The synthetic RNA chains are combined under conditions that form double-stranded molecules. The RNA sequence may comprise at least an 8-nucleotide portion of SEQ ID NO: 4 with a total length of 25 nucleotides or less. Design of siRNA sequences for a given target is within the ordinary skill of those skilled in the art. Commercial services for designing siRNA sequences and ensuring at least 70% expression knockdown are available (Qiagen, Valencia, Calf).

[0923] dsRNA can be administered as a pharmaceutical composition and by known methods, wherein the nucleic acid is introduced into the desired target cell. Common gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection, and viral methods. Such methods are taught in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

[0924] Ribozymes can also be used to reduce the amount and / or biological activity of MASP-2, for example, ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molecules that can cleave nucleic acid molecules having sequences that are completely or partially homologous to the ribozyme's sequence. Ribozyme transgenes can be designed that encode RNA ribozymes that specifically pair with target RNA and cleave the phosphodiester backbone at specific locations, thereby functionally inactivating the target RNA. While performing this cleavage, the ribozyme itself remains unchanged and is therefore able to recycle and cleave other molecules. Including a ribozyme sequence within an antisense RNA confers RNA-cleaving activity, thereby increasing the activity of the antisense construct.

[0925] Ribozymes useful in the practice of the present invention generally comprise a hybridizing region of at least about nine nucleotides that is complementary in nucleotide sequence to at least a portion of a target MASP-2 mRNA and a catalytic region that is suitable for cleaving the target MASP-2 mRNA (see generally EPA No. 0321201; WO 88 / 04300; Haseloff, J. et al., Nature 334:585-591, 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).

[0926] Ribozymes can be directly targeted to cells in the form of RNA oligonucleotides incorporating ribozyme sequences, or 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.

[0927] Antisense RNA and DNA, ribozymes and RNAi molecules that can be used in the methods of the present invention can be prepared by any method known in the art for synthesizing DNA and RNA molecules. These include techniques well known in the art for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of a DNA sequence encoding the antisense RNA molecule. Such DNA sequence can be incorporated into a wide variety of vectors that incorporate a suitable RNA polymerase promoter, such as a T7 or SP6 polymerase promoter. Alternatively, depending on the promoter used, an antisense cDNA construct that synthesizes antisense RNA constitutively or inducibly can be stably introduced into a cell line.

[0928] Various well-known modifications of DNA molecules can be introduced as a means of increasing stability and half-life. Useful modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides at the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

[0929] V. Pharmaceutical Compositions and Delivery Methods

[0930] Drug administration

[0931] In another aspect, the present invention provides a composition for inhibiting the adverse effects of MASP-2-dependent complement activation in a subject suffering from a disease or condition as disclosed herein, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. The MASP-2 inhibitory agent can be administered to a subject in need thereof at a therapeutically effective dose to treat or ameliorate a condition associated with MASP-2-dependent complement activation. A therapeutically effective dose refers to an amount of the MASP-2 inhibitory agent sufficient to result in an amelioration of symptoms associated with the disease or condition.

[0932] Toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical procedures using experimental animal models, such as the murine MASP-2- / - mouse model expressing a human MASP-2 transgene, as described in Experimental Example 1. Using such animal models, the NOAEL (no observed adverse effect level) and MED (minimum effective dose) can be determined using standard methods. The dose ratio between the NOAEL and the MED effect is the therapeutic ratio, expressed as the ratio NOAEL / MED. MASP-2 inhibitory agents that exhibit large therapeutic ratios or indices are most preferred. Data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for use in humans. The dosage of the MASP-2 inhibitory agent preferably lies within a range of circulating concentrations that includes the MED with little or no toxicity. The dosage can vary within this range, depending on the dosage form employed and the route of administration utilized.

[0933] In some embodiments, the therapeutic efficacy of a MASP-2 inhibitory agent for treating, inhibiting, reducing or preventing fibrosis in a mammalian subject having or at risk of developing a disease or condition caused by 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 soluble markers of inflammation and fibrotic renal disease released into the urine and plasma (e.g., by measurement of renal excretory function).

[0934] For any compound formulation, animal models can be used to estimate the therapeutically effective dose. For example, a dose can be formulated in an animal model to achieve a circulating plasma concentration range that includes the MED. Quantitative levels of the MASP-2 inhibitory agent in plasma can also be measured, for example, by high performance liquid chromatography.

[0935] In addition to toxicity studies, effective doses can also be estimated based on the amount of MASP-2 protein present in living subjects and the binding affinity of the MASP-2 inhibitory agent. MASP-2 levels in normal human subjects have been shown to be present in serum at low levels in the range of 500 ng / ml, and the MASP-2 level in a particular subject can be determined using a quantitative assay for MASP-2, which is described in Moller-Kristensen M. et al., J. Immunol. Methods 282:159-167, 2003.

[0936] Generally, the dosage of the administered composition comprising a MASP-2 inhibitory agent will vary depending on such factors as the subject's age, weight, height, sex, general medical condition, and prior medical history. Illustratively, a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, can be administered at a dose ranging from about 0.010 to 10.0 mg / kg, preferably 0.010 to 1.0 mg / kg, and more preferably 0.010 to 0.1 mg / kg of the subject's body weight. In some embodiments, the composition comprises a combination of an anti-MASP-2 antibody and a MASP-2 inhibitory peptide.

[0937] The therapeutic efficacy and appropriate dosage of the MASP-2 inhibitory compositions and methods of the present invention in a given subject can be determined based on complement assays well known to those skilled in the art. Complement produces numerous specific products. Over the past decade, 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 new antigens (neoantigens) exposed on the fragments, but not with antigens on the native proteins from which they are formed, making these assays very simple and specific. Although radioimmunoassays are sometimes still used for C3a and C5a, most rely on ELISA technology. These latter assays measure both the unprocessed fragments and their 'desArg' fragments, which are the predominant forms found in circulation. Unprocessed fragments and C5a desArg It is rapidly cleared by binding to cell surface receptors and is therefore present in very low concentrations, whereas C3a desArgIt does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complement activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Detection of sC5b-9, a liquid-phase product of membrane attack pathway activation, provides evidence that complement has been activated to completion. Since both the lectin pathway and the classical pathway generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of the two pathways has generated the activation products.

[0938] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in a component of the complement system that occurs as a result of administration of a MASP-2 inhibitory agent according to the methods of the invention: inhibition of the generation or production of the MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (e.g., as measured as described in Example 2), reduction in C4 cleavage and C4b deposition (e.g., as measured as described in Example 10), or reduction in C3 cleavage and C3b deposition (e.g., as measured as described in Example 10).

[0939] Additional reagents

[0940] In certain embodiments, methods for preventing, treating, reversing, and / or inhibiting fibrosis and / or inflammation comprise administering a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) as part of a treatment regimen in conjunction with one or more other drugs, biologics, or therapeutic interventions suitable for inhibiting fibrosis and / or inflammation. In certain embodiments, the additional drug, biologic, or therapeutic intervention is suitable for a specific symptom associated with a disease or condition caused by or exacerbated by fibrosis and / or inflammation. For example, a MASP-2 inhibitory antibody can be administered as part of a treatment regimen in conjunction with one or more immunosuppressants (e.g., methotrexate, cyclophosphamide, azathioprine, and mycophenolate mofetil). As a further example, a MASP-2 inhibitory antibody can be administered as part of a treatment regimen in conjunction with one or more agents designed to increase blood flow (e.g., nifedipine, amlodipine, diltiazem, felodipine, or nicardipine). As a further example, a MASP-2 inhibitory antibody can be administered as part of a treatment regimen in conjunction with one or more agents intended to reduce fibrosis (e.g., d-penicillamine, colchicine, PUVA, relaxin, cyclosporin, a TGFβ blocker, or a p38 MAPK blocker). As a further example, a MASP-2 inhibitory antibody can be administered as part of a treatment regimen in conjunction with a steroid or bronchodilator.

[0941] Compositions and methods comprising a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) may optionally comprise one or more additional therapeutic agents that can potentiate the activity of the MASP-2 inhibitory agent or provide related therapeutic functions in an additive or synergistic manner. For example, in the context of treating a subject suffering from a disease or condition caused by or exacerbated by fibrosis and / or inflammation, one or more MASP-2 inhibitory agents may be administered in combination (including co-administration) with one or more additional anti-fibrotic agents, and / or one or more antiviral agents and / or anti-inflammatory agents and / or immunosuppressive agents.

[0942] MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) can be used in combination with other therapeutic agents, such as general antiviral drugs, or immunosuppressive drugs such as corticosteroids, immunosuppressive or cytotoxic agents, and / or anti-fibrotic agents.

[0943] In some embodiments of the methods described herein, a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody or a small molecule inhibitor of MASP-2) is used as a monotherapy for treating a subject with a coronavirus, such as COVID-19 or influenza. In some embodiments of the methods described herein, a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody or a small molecule inhibitor of MASP-2) is used in combination with other therapeutic agents, such as antiviral agents, therapeutic antibodies, corticosteroids, and / or other agents shown to be effective for treating a subject with a coronavirus or influenza. In some embodiments, a pharmaceutical composition comprises a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody or a small molecule inhibitor of MASP-2) and at least one additional therapeutic agent, such as an antiviral agent (e.g., remdesivir), a therapeutic antibody directed against a target other than MASP-2, a corticosteroid, an anticoagulant such as a low molecular weight heparin (e.g., enoxaparin), and an antibiotic (e.g., azithromycin).

[0944] In such combination therapies, the MASP-2 inhibitory agent can...

Claims

1. Use of an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation in the preparation of a medicament for treating a human subject suffering from COVID-19-induced acute respiratory distress syndrome (ARDS) or pneumonia, comprising administering to the subject an amount of the MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation.

2. The method of claim 1, wherein: (i) the subject was on a mechanical ventilator prior to treatment, and the MASP-2 inhibitory agent is administered at a dose and for a period of time sufficient to discontinue the need for mechanical ventilation; and / or (ii) the subject required supplemental oxygen prior to treatment, and the MASP-2 inhibitory agent is administered at a dose and for a period of time sufficient to discontinue the use of supplemental oxygen.

3. The use of claim 1, wherein the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:

6.

4. The use of claim 3, wherein the antibody or 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.

5. The use of claim 3, wherein the MASP-2 inhibitory antibody selectively inhibits lectin pathway complement activation while not substantially inhibiting C1q-dependent complement activation.

6. The use of claim 3, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 10% human serum, wherein IC 50 30nM or less.

7. The use of claim 1, wherein the MASP-2 inhibitory agent is a small molecule MASP-2 inhibitory compound.

8. The use of claim 1, wherein the MASP-2 inhibitory agent is an inhibitor of the expression of MASP-2.

9. Use of an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation in the preparation of a medicament for treating, preventing, or reducing the severity of coagulation or thrombosis in a human subject infected by SARS-CoV-2, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to treat, prevent, or reduce the severity of coagulation or thrombosis in the subject.

10. The use of claim 9, wherein the subject has a D-dimer level above the normal range prior to treatment and the MASP-2 inhibitory agent is administered in an amount and for a time sufficient to reduce the subject's D-dimer level to within the normal range for healthy subjects.

11. The use according to claim 9, wherein the MASP-2 inhibitory agent provides an anticoagulant and / or antithrombotic effect without affecting hemostasis.

12. The use according to claim 9, wherein the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:

6.

13. The use according to claim 9, wherein the MASP-2 inhibitory agent is a small molecule MASP-2 inhibitory compound.

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