Composition for treating lung inflammation

Abstract

This article provides a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the Isthmin 1 (ISM1) protein or its GRP78-activating fragment, and an expressible nucleic acid encoding said polypeptide. The use of such agents, and methods for inducing alveolar macrophage apoptosis and / or for treating, improving, or preventing inflammation or lung diseases such as chronic obstructive pulmonary disease (COPD), emphysema, asthma, acute lung injury (ALI), pulmonary fibrosis, and / or acute respiratory distress syndrome.

Description

Technical Field

[0001] The present invention generally relates to the treatment of inflammation. More specifically, the present invention relates to the treatment of lung inflammation based on Isthmin 1 (ISM1). Background Art

[0002] Lung inflammation, whether acute or chronic, can have serious health consequences. According to the World Health Organization (WHO), chronic obstructive pulmonary disease (COPD) was the third most common cause of death in 2016. It is considered a major socioeconomic and health burden, with direct healthcare costs reaching US$20 billion in the United States alone in 2004. In Singapore, in 2010, COPD was estimated to cost patients and healthcare providers S$165 million annually, with over 10,000 hospitalizations, placing a significant burden on the public health system. COPD can be characterized by progressive emphysema (irreversible dilation of the alveoli or destruction of their walls) and chronic airway inflammation, leading to a severe decline in lung function. Currently, there is no effective treatment that can slow or reverse emphysema. Smoking may be a major risk factor for COPD and is closely associated with its progression and exacerbation. Other risk factors may include air pollution.

[0003] In fact, COPD is currently the third leading cause of death worldwide, with an estimated cumulative lifetime risk of 25% and a high socioeconomic burden (Gershon, Warner et al., 2011, Mortality & Causes of Death, 2016). The pathogenesis of COPD is believed to involve perturbations in lung homeostasis and a dysregulated immune response to exogenous factors from the environment, with cigarette smoke (CS), biomass fuel exposure, and air pollution as major risk factors (Singh, Agusti et al., 2019). Hallmark features of COPD include emphysema and chronic obstructive bronchitis (inflammation of the airways). COPD patients present with persistent respiratory symptoms accompanied by progressive, long-term lung function impairment. However, traditional pharmacological interventions only provide symptom relief to patients and do not target the underlying tissue damage and inflammation, so they are not effective in preventing COPD progression or reducing mortality. Therefore, there is an urgent unmet need for new COPD therapies.

[0004] The respiratory tract is constantly exposed to an external environment containing dust and microorganisms. To avoid inflammatory responses to environmental stimuli, healthy airways and lungs have mechanisms that suppress immune responses and inflammation. In addition, in response to injury or pathogens, acute lung inflammatory responses protect the host from systemic infection and restore tissue homeostasis. However, when acute inflammation is not limited in amplitude or duration, it can lead to lung diseases characterized by excessive or chronic inflammation, including asthma and / or COPD. Asthma mainly affects the large airways, while COPD affects the small airways and lung parenchyma. The molecular mechanisms of COPD are still poorly understood.

[0005] Meanwhile, severe acute lung inflammation due to infection or injury, such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), is a serious clinical syndrome with a mortality rate as high as 50% without effective drug treatment. There is an unmet need to develop therapies for acute and chronic lung inflammation.

[0006] There is a need for alternative, additional and / or improved treatments and / or therapeutic methods for inflammation-related diseases or disorders, and particularly those affecting the lung. Summary of the Invention

[0007] Healthy adults have the ability to regulate and maintain lung homeostasis under ambient conditions to prevent aseptic inflammation. The immune response may be triggered by injury and / or infection, leading to acute inflammation that eventually subsides to allow wound healing and recovery. Failure to subside acute inflammation may lead to chronic inflammation and / or inflammation-related diseases such as chronic obstructive pulmonary disease (COPD), emphysema, chronic obstructive bronchitis and / or pulmonary fibrosis. Acute lung diseases such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are also associated with lung inflammation.

[0008] As described in detail herein, the inventors have now developed polypeptides, nucleic acids, compositions and methods for treating inflammation, such as lung inflammation, which are derived from Isthmin 1, a secreted protein that studies described herein have shown to play a role in inhibiting, preventing and / or resolving inflammation, particularly lung inflammation. In the studies described herein, supplementation of the lungs with exogenous recombinant ISM1 protein (rISM1) suppressed the lung inflammation phenotype in ISM1-deficient lungs, and the results suggest that ISM1 may help resolve inflammation by inducing apoptosis in alveolar macrophages. The results suggest that ISM1 may play an important role in inhibiting and / or resolving sterile lung inflammation and / or inflammation triggered by infection and / or injury.

[0009] In one embodiment, the present invention provides a composition comprising:

[0010] A polypeptide or peptide, or an expressible nucleic acid encoding said polypeptide or peptide, comprising a polypeptide or peptide having at least 70% affinity to Isthmin 1 (ISM1) protein or its GRP78-activating fragment

[0011] amino acid sequences with sequence identity; and

[0012] a pharmaceutically acceptable carrier, diluent or excipient;

[0013] The composition is formulated for administration to the lungs of a subject.

[0014] In another embodiment of the above composition, the composition may be formulated for intratracheal administration, intranasal administration or inhalation administration.

[0015] In yet another embodiment of any one or more of the above compositions, the composition may be formulated for administration as an aerosol, inhaler, or nebulizer.

[0016] In yet another embodiment of any one or more of the above compositions, the composition can be formulated as a dry powder for administration to the lungs by aerosolization, or as a liquid for administration to the lungs by nebulization.

[0017] In another embodiment of any one or more of the above compositions, the composition can be used to target cell surface GRP78 (csGRP78) in a subject in need thereof.

[0018] In yet another embodiment of any one or more of the above compositions, the composition can be used to induce apoptosis in proinflammatory cells in a subject in need thereof.

[0019] In yet another embodiment of any one or more of the above compositions, the composition can be used to induce apoptosis of alveolar macrophages (AMs), or to reduce AM levels, in a subject in need thereof.

[0020] In another embodiment of any one or more of the above compositions, the composition can be used to treat, ameliorate or prevent lung inflammation in a subject in need thereof.

[0021] In yet another embodiment of any one or more of the above compositions, the composition can be used to treat, ameliorate or prevent a lung disease or condition associated with lung inflammation in a subject in need thereof.

[0022] In yet another embodiment of any one or more of the above compositions, the composition can be used to treat, improve or prevent chronic obstructive pulmonary disease (COPD), chronic obstructive bronchitis, asthma or emphysema in a subject in need thereof.

[0023] In another embodiment of any one or more of the above compositions, the composition can be used to treat, improve or prevent acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof.

[0024] In yet another embodiment of any one or more of the above compositions, the composition can be used to prevent or reduce excessive proliferation of type II (AE2) cells on the surface of alveolar walls in a subject in need thereof.

[0025] In yet another embodiment of any one or more of the above compositions, the composition can be used to treat, improve, or prevent pulmonary fibrosis in a subject in need thereof.

[0026] In yet another embodiment, provided herein is a method for regulating GRP78 activity in a subject in need thereof using a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide.

[0027] In yet another embodiment, provided herein is a use of a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide for inducing proinflammatory cell apoptosis in a subject in need thereof.

[0028] In yet another embodiment, provided herein is a use of a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, for inducing apoptosis of alveolar macrophages (AMs) or for reducing AM levels in a subject in need thereof.

[0029] In yet another embodiment, provided herein is a method for treating, ameliorating, or preventing lung inflammation in a subject in need thereof using a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide.

[0030] In yet another embodiment, provided herein is a method for treating, ameliorating, or preventing a lung disease or condition associated with lung inflammation in a subject in need thereof, comprising a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide.

[0031] In yet another embodiment, provided herein is a method for treating, ameliorating, or preventing chronic obstructive pulmonary disease (COPD), chronic obstructive bronchitis, asthma, or emphysema in a subject in need thereof, comprising a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide.

[0032] In yet another embodiment, provided herein is a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide for treating, ameliorating or preventing acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof.

[0033] In yet another embodiment, provided herein is a method for preventing or reducing the hyperproliferation of type II (AE2) cells on the alveolar wall surface in a subject in need thereof, comprising a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide.

[0034] In yet another embodiment, provided herein is a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with the Isthmin 1 (ISM1) protein or its GRP78-activating fragment, or an expressible nucleic acid encoding the polypeptide or peptide for treating, ameliorating or preventing pulmonary fibrosis in a subject in need thereof.

[0035] In yet another embodiment of any one or more of the above uses, the polypeptide or peptide or nucleic acid may be administered to the lungs of a subject.

[0036] In another embodiment of any one or more of the above uses, the polypeptide or peptide or nucleic acid may be administered intratracheally, intranasally or by inhalation to a subject.

[0037] In yet another embodiment of any one or more of the above uses, the polypeptide or peptide or nucleic acid may be administered as an aerosol, inhaler or nebulizer.

[0038] In yet another embodiment of any one or more of the above uses, the polypeptide or peptide or nucleic acid may be formulated as a dry powder for administration to the lungs by aerosolization, or as a liquid for administration to the lungs by nebulization.

[0039] In another embodiment, provided herein is a method for modulating GRP78 activity, targeting and binding GRP78, or both, in a subject in need thereof, the method comprising:

[0040] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, is administered to a subject in need thereof.

[0041] In another embodiment, provided herein is a method for inducing apoptosis of proinflammatory cells in a subject in need thereof, the method comprising:

[0042] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, is administered to a subject in need thereof.

[0043] In another embodiment, provided herein is a method for inducing apoptosis of alveolar macrophages (AMs) or reducing AM levels in a subject in need thereof, the method comprising:

[0044] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, is administered to a subject in need thereof.

[0045] In another embodiment, provided herein is a method for treating, ameliorating or preventing lung inflammation in a subject in need thereof, the method comprising:

[0046] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, is administered to a subject in need thereof.

[0047] In another embodiment, provided herein is a method for treating, ameliorating or preventing a pulmonary disease or condition associated with pulmonary inflammation in a subject in need thereof, the method comprising:

[0048] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, is administered to a subject in need thereof.

[0049] In another embodiment, provided herein is a method for treating, ameliorating or preventing chronic obstructive pulmonary disease (COPD), chronic obstructive bronchitis, asthma or emphysema in a subject in need thereof, the method comprising:

[0050] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide is administered to the subject in need thereof.

[0051] In another embodiment, provided herein is a method for treating, ameliorating or preventing acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof, the method comprising:

[0052] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, is administered to a subject in need thereof.

[0053] In another embodiment, provided herein is a method for preventing or reducing excessive proliferation of type II (AE2) cells on the surface of alveolar walls in a subject in need thereof, the method comprising:

[0054] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide is administered to the subject in need thereof.

[0055] In another embodiment, provided herein is a method for treating, ameliorating or preventing pulmonary fibrosis in a subject in need thereof, the method comprising:

[0056] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide is administered to the subject in need thereof.

[0057] In yet another embodiment of any one or more of the above methods, the polypeptide or peptide or nucleic acid may be administered to the lungs of the subject in need thereof.

[0058] In yet another embodiment of any one or more of the above methods, the polypeptide or peptide or nucleic acid may be administered intratracheally, intranasally, or by inhalation to the subject.

[0059] In another embodiment of any one or more of the above methods, the polypeptide or peptide or nucleic acid may be administered as an aerosol, inhaler or nebulizer.

[0060] In yet another embodiment of any one or more of the above methods, the polypeptide or peptide or nucleic acid may be formulated as a dry powder for administration to the lungs by aerosolization, or as a liquid for administration to the lungs by nebulization.

[0061] In yet another embodiment of any one or more of the above methods, the method may further comprise the following steps:

[0062] determining the level of ISM1 in the subject, determining the level of GRP78 protein in the subject, or both; and

[0063] The administering step is performed or repeated if a decreased level of ISM1 is determined in the subject relative to healthy control levels or relative to low severity disease control levels; if an increased level of GRP78 protein is determined in the subject relative to healthy control levels or relative to low severity disease control levels; or both.

[0064] In yet another embodiment, provided herein is a pulmonary drug delivery device comprising a polypeptide or peptide, or an expressible nucleic acid encoding the polypeptide or peptide, comprising an amino acid sequence having at least 70% sequence identity to the Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0065] In yet another embodiment of the above-mentioned pulmonary drug delivery device, the pulmonary drug delivery device may comprise a composition as defined herein.

[0066] In yet another embodiment of any one or more of the foregoing pulmonary drug delivery devices, the pulmonary drug delivery device may be an intratracheal drug delivery device, an intranasal drug delivery device, or an inhaled drug delivery device.

[0067] In yet another embodiment of any one or more of the above pulmonary drug delivery devices, the pulmonary drug delivery device may be an aerosol, an inhaler, or a nebulizer.

[0068] In another embodiment of any one or more of the above pulmonary drug delivery devices, the pulmonary drug delivery device can be an aerosol and the polypeptide or peptide or nucleic acid can be formulated as a dry powder, or wherein the pulmonary drug delivery device can be a nebulizer and the polypeptide or peptide or nucleic acid can be formulated as a liquid.

[0069] In yet another embodiment of any one or more of the foregoing pulmonary drug delivery devices, the pulmonary drug delivery device can be used to modulate GRP78 activity in a subject in need thereof.

[0070] In yet another embodiment of any one or more of the foregoing pulmonary drug delivery devices, the pulmonary drug delivery device may be used to induce apoptosis in proinflammatory cells in a subject in need thereof.

[0071] In another embodiment of any one or more of the above-described pulmonary drug delivery devices, the pulmonary drug delivery device can be used to induce apoptosis of alveolar macrophages (AMs) or reduce AM levels in a subject in need thereof.

[0072] In yet another embodiment of any one or more of the foregoing pulmonary drug delivery devices, the pulmonary drug device may be used to treat, ameliorate, or prevent pulmonary inflammation in a subject in need thereof.

[0073] In yet another embodiment of any one or more of the foregoing pulmonary drug delivery devices, the pulmonary drug device may be used to treat, ameliorate, or prevent a pulmonary disease or condition associated with pulmonary inflammation in a subject in need thereof.

[0074] In another embodiment of any one or more of the above pulmonary drug delivery devices, the pulmonary drug device may be used to treat, ameliorate, or prevent chronic obstructive pulmonary disease (COPD), chronic obstructive bronchitis, asthma, or emphysema in a subject in need thereof.

[0075] In yet another embodiment of any one or more of the aforementioned pulmonary drug delivery devices, the pulmonary drug device may be used to treat, ameliorate, or prevent acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof.

[0076] In yet another embodiment of any one or more of the aforementioned pulmonary drug delivery devices, the pulmonary drug device can be used to prevent or reduce hyperproliferation of type II (AE2) cells on the surface of alveolar walls in a subject in need thereof.

[0077] In another embodiment of any one or more of the above-described pulmonary drug delivery devices, the pulmonary drug device may be used to treat, ameliorate, or prevent pulmonary fibrosis in a subject in need thereof.

[0078] In yet another embodiment of any one or more of the foregoing pulmonary drug delivery devices, the pulmonary drug delivery device may be a nebulizer, a metered dose inhaler (MDI), or a dry powder inhaler (DPI).

[0079] In yet another embodiment of any one or more of the above compositions, the composition may further comprise an agent for preventing or reducing lung inflammation.

[0080] In yet another embodiment of any one or more of the above uses, the polypeptide or peptide or nucleic acid may be used in combination with an agent for preventing or reducing lung inflammation.

[0081] In another embodiment of any one or more of the above methods, the method may further comprise the step of administering to the subject an agent for preventing or reducing lung inflammation in combination with the polypeptide or peptide or nucleic acid, simultaneously or sequentially.

[0082] In yet another embodiment of any one or more of the foregoing pulmonary drug delivery devices, the pulmonary drug delivery device may further comprise an agent for preventing or reducing pulmonary inflammation.

[0083] In another embodiment, provided herein is a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, for use in treating, ameliorating or preventing a disease or condition associated with macrophage-mediated inflammation in a subject in need thereof.

[0084] In another embodiment, provided herein is a method for treating, ameliorating, or preventing a disease or condition associated with macrophage-mediated inflammation in a subject in need thereof, comprising a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide.

[0085] In another embodiment, provided herein is a method for treating, ameliorating or preventing a disease or disorder associated with macrophage-mediated inflammation in a subject in need thereof, the method comprising:

[0086] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, is administered to a subject.

[0087] In another embodiment, provided herein is a use of a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity with an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide in the manufacture of a medicament.

[0088] In another embodiment, provided herein is a method for treating, ameliorating, or preventing a pulmonary disease or disorder associated with pulmonary inflammation in a subject in need thereof, the method comprising:

[0089] A GRP78-activator is administered to the lungs of a subject.

[0090] In another embodiment, provided herein is a method for identifying a subject having or at risk of developing a pulmonary disease or condition associated with pulmonary inflammation, the method comprising:

[0091] determining the level of ISM1 in the subject, determining the level of GRP78 protein in the subject, or both; and

[0092] If a decreased level of ISM1 is determined in the subject relative to healthy control levels or relative to low severity disease control levels; if an increased level of GRP78 protein is determined in the subject relative to healthy control levels or relative to low severity disease control levels; or both, the subject is identified as having, or at risk for developing, a lung disease or condition associated with lung inflammation.

[0093] In another embodiment, provided herein is a method for identifying a candidate subject for treatment with a method as defined herein, the method comprising:

[0094] determining the level of ISM1 in the subject, determining the level of GRP78 protein in the subject, or both; and

[0095] The subject is identified as a candidate for treatment if it is determined that the ISM1 level in the subject is decreased relative to the healthy control level or relative to the low severity disease control level; if it is determined that the GRP78 protein level is increased relative to the healthy control level or relative to the low severity disease control level; or both.

[0096] In another embodiment, provided herein is a method for maintaining lung homeostasis and / or reducing lung inflammation and / or promoting lung repair while reducing remodeling in a subject in need thereof, comprising a polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide.

[0097] In another embodiment, provided herein is a method for maintaining lung homeostasis and / or resolving lung inflammation and / or promoting lung repair while reducing remodeling in a subject in need thereof, the method comprising:

[0098] A polypeptide or peptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide or peptide, is administered to the lungs of a subject.

[0099] In another embodiment of any of the above compositions, uses, methods, pulmonary drug delivery devices or polypeptides, the polypeptide may be or may comprise an ISM1 protein (precursor or mature). In another embodiment, the polypeptide may be or comprise a human ISM1 protein or a mouse ISM1 protein (precursor or mature).

[0100] In another embodiment of any of the above compositions, uses, methods, pulmonary drug delivery devices, or polypeptides or peptides, the polypeptide or peptide may comprise or may consist of the following amino acid sequence:

[0101] FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE(SEQ ID NO:26); or

[0102] FEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE(SEQ ID NO:27)

[0103] or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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%, or 100% sequence identity thereto.

[0104] In another embodiment of any of the above compositions, uses, methods, pulmonary drug delivery devices, or polypeptides or peptides, the polypeptide or peptide may comprise or may consist of the following amino acid sequence:

[0105] FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY (SEQ ID NO: 24 – Mouse ISM1 287-461); or

[0106] FEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY (SEQ ID NO: 25–Human ISM1 290-464);

[0107] or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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%, or 100% sequence identity thereto.

[0108] In another embodiment of any of the above compositions, uses, methods, pulmonary drug delivery devices, or polypeptides or peptides, the polypeptide or peptide may comprise or may consist of the sequence of endogenous mature ISM1.

[0109] In another embodiment of any of the foregoing compositions, uses, methods, pulmonary drug delivery devices, or polypeptides or peptides, the polypeptide or peptide may not be an endogenous precursor or mature ISM1. In certain embodiments, the polypeptide or peptide may be longer or shorter than an endogenous precursor or mature ISM1. In certain embodiments, the polypeptide or peptide may comprise at least one substitution or mutation not found in an endogenous precursor or mature ISM1. In certain embodiments, the polypeptide or peptide may comprise an RKD to RAA mutation, or an RKD to AAA mutation, in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] These and other features will become more apparent from the following description with reference to the accompanying drawings, in which:

[0111] Figure 1 AN shows that ISM1 deficiency leads to pulmonary emphysema. Figure 1 Figures A and B show 1-month, 2-month, 6-month, and 9-month-old FVB / NTac WT and Ism1 Δ / Δ Representative micrographs of the peripheral left lung lobe stained with hematoxylin and eosin of mice ( Figure 1 A) and mean linear intercept (MLI) ( Figure 1 B). n = 3-4 mice / group. Scale bar 200 μm. Figure 1 C and D show 6-month-old FVB / NTac WT and Ism1 Δ / Δ The left lung lobe of mice ( Figure 1 C) and elastin / collagen labeled left lung lobe ( Figure 1 D) Representative whole-mount stereoscopic images. n = 3 mice / group. Scale bar 500 μm ( Figure 1 C) and 200μm( Figure 1 D). Figure 1 E shows a 9-month-old FVB / NTac Ism1 + / Δ Representative micrographs of the peripheral left lung lobe of mice stained with hematoxylin and eosin. n = 4 mice / group. Scale bar 200 μm. Figure 1 F shows 9-month-old FVB / NTac WT, Ism1 + / Δ and Ism1 Δ / Δ MLI of mice. n = 4 mice / group. Figure 1 G–N show 2-month-old FVB / NTac WT and Ism1 Δ / Δ Spirometry of mice. n = 4 mice / group. Total lung capacity (TLC) ( Figure 1 G), functional residual capacity (FRC) ( Figure 1H), residual volume (RV) ( Figure 1 I), static compliance (Cchord) ( Figure 1 J), dynamic compliance (Cdyn) ( Figure 1 K), forced expiratory volume at 100ms (FEV 100 )( Figure 1 L), Tiffeneau–Pinelli index (FEV 100 / FVC)( Figure 1 M) and airway resistance (RI) ( Figure 1 Data are mean ± sem and were compared by unpaired two-tailed Student's t test ( Figure 1 B, G–N) and one-way ANOVA with Tukey post hoc test ( Figure 1 F) was analyzed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0112] Figure 2 Show Ism1 Δ / Δ Mouse lungs exhibit upregulated COPD mediators. (A) Shows representative micrographs of hematoxylin and eosin-stained lungs showing alveolar macrophages (AMs) in 2-month-old FVB / NTac Ism1 Δ / Δ Accumulation in 2-month-old FVB / NTac WT and Ism1 mice. n = 4 mice / group. Scale bar 20 μm. (B) and (C) show the accumulation of WT and Ism1 cells from 2-month-old FVB / NTac WT and Ism1 cells. Δ / Δ Liu-stained cytospin preparations (B) and quantification (C) of bronchoalveolar lavage fluid (BALF) cells from mouse lungs. n = 4 mice / group. (B)-(D) show cytospin preparations and flow cytometric analysis of bronchoalveolar lavage fluid (BALF) cells to determine Ism1 expression in BALF cells compared to WT mice. Δ / Δ AMs increased in the lungs of mice. (E) Shows the increase in AMs in 2-month-old FVB / NTac WT and Ism1 Δ / Δ Western blot (top) and fold change (bottom; AU, arbitrary units) of MMP-12, MMP-9, and NF-κB p65 in mouse lungs, with β-actin as a loading control. n = 4 mice / group. (F) shows 2-month-old FVB / NTac WT and Ism1 Δ / Δ Representative immunohistochemical staining of MMP-12 and MMP-9 in AM of mouse lungs. n = 4 mice / group. Scale bar 20 μm. (G) Shows the expression of MMP-12 and MMP-9 in AM of 2-month-old FVB / NTac WT and Ism1 mice. Δ / ΔRepresentative immunofluorescence staining of NF-κB p65 (green) and nuclei (DAPI; blue) isolated from mice (left) and quantification of primary AMs (right). n = 3 average measurements from 3 mice / group. Scale bar 20 μm. 250-350 alveolar macrophages were quantified per mouse. (H) Shows the quantification of primary AMs in 2-month-old FVB / NTac WT and Ism1 Δ / Δ Western blot (top) and fold change (bottom; AU, arbitrary units) of GM-CSF in mouse lungs, with β-actin as a loading control. n = 4 mice / group. (I) and (J) show 2-month-old FVB / NTac WT and Ism1 Δ / Δ Representative immunohistochemical staining of ISM1 (I) and GRP78 (J) in mouse AM. n = 4 mice / group. Scale bar 20 μm. (K) and (L) show WT primary AM treated with 1 μM recombinant ISM1 (rISM1) (K) and untreated WT and Ism1 AM. Δ / Δ IncuCyte quantification of apoptosis in primary AM (L). The analysis was performed in triplicate or quadruplicate wells, and 4 images were taken per well for quantification. (M) shows the apoptosis of WT and Ism1 Δ / Δ Proliferation assay of primary AM. Analyses were performed in triplicate. n = 4 mice / group. Data are mean ± sem and analyzed by unpaired two-tailed Student's t-test (C, E, G, H, K, L, and M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0113] Figure 3 showed that exogenous rISM1 alleviated Ism1 Δ / Δ (A) shows emphysema in 2-month-old FVB / NTacIsm1 mice after treatment with vehicle (PBS), 1 μg rISM1, 5 μg rISM1, and liposome-clodronate (CLO). Δ / Δ AM counts in mice. n = 4 mice / group. (B) shows AM counts in 2-month-old FVB / NTac Ism1 mice after treatment with vehicle (PBS), 5 μg rISM1, or liposome-clodronate (CLO). Δ / Δ Representative micrographs of hematoxylin and eosin-stained lungs of mice. n = 4 mice / group. Scale bar 50 μm. (C) and (D) show the difference between untreated 2-month-old FVB / NTac WT and Ism1 Δ / Δ MLI (C) and FEV in the treated mice group (B) were compared. 100Quantification of FVC (D). n = 3–4 mice / group. (E) and (F) Show the experimental design of the 2-week (E) and 8-week (F) cigarette smoke-induced COPD model in WT BALB / c AnNTac (WT BALB / c) mice. Room air-exposed WT BALB / c mice (Sham) and cigarette smoke-exposed WT BALB / c mice (CS) were treated with vehicle (CS + PBS) or rISM1 (CS + 10 μg rISM1) at the indicated frequencies and intervals. n = 5 mice / group. (G) Shows quantification of bronchoalveolar lavage fluid (BALF) cells from the experimental groups in (E). n = 4–5 mice / group. (H) Shows representative hematoxylin and eosin-stained lung micrographs from the experimental groups in (F), showing immune cell infiltration. n = 5 mice / group. Scale bar 50 μm. (I)–(L) show AM (I), MMP-12 expression (J), MLI (K), and FEV of the experimental group in (F). 100 Quantification of FVC (L). n = 5 mice / group. Data are mean ± sem and were compared by unpaired two-tailed Student's t-test (C, D, untreated 2-month-old FVB / NTac WT and Ism1 Δ / Δ Mice) and one-way ANOVA with Tukey's post hoc test (C, D, G, I to L) were performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #: no significant difference compared with the Sham group;

[0114] Figure 4Human ISM1 expression is associated with AM apoptosis. (A) and (B) Show representative immunohistochemical staining of human ISM1 (hISM1) (A) and immunofluorescent staining of CD68 (red), hISM1 (green), and nuclei (DAPI, blue) in AM from excised human lung tissue sections. Scale bar 20 μm. (C) Shows a matrix table of hISM1 expression derived from IHC staining intensity (A) and expression frequency in human AM (B). hISM1 expression is annotated by matrix scores 1–3: +, 4–6: ++, 9: +++. (D) Shows the percentage distribution of hISM1 expression in patients stratified by smoking and COPD status. (E) and (F) Show the correlation between hISM1 expression and smoking (E) and AM apoptosis (F). (G) Shows representative immunohistochemical staining of GRP78 in non-COPD and COPD patients. Scale bar 20 μm. (H) Shows the percentage of apoptotic AM in COPD patients stratified by COPD status and hISM1 expression. Data are mean ± sem and analyzed by point biserial correlation (E), Pearson correlation (F), and one-way ANOVA with Tukey's post hoc test (H). *P < 0.05. Patient sample size is depicted on the graph;

[0115] Figure 5 Ism1 is shown Δ / Δ Characterization of mice. (A) and (B) show 2-month-old FVB / NTac (A) and C57BL / 6J (B) WT and Ism1 Δ / Δ Pathological grading of emphysema in mice. n = 4 mice / group. (C) and (D) show 2-month-old, 6-month-old, and 9-month-old C57BL / 6JWT and Ism1 Δ / Δ Representative micrographs (C) and mean linear intercepts (MLI) (D) of the peripheral left lung lobes of mice stained with hematoxylin and eosin. n = 4 mice / group. Scale bar 200 μm. (E) and (F) show 2-month-old FVB / NTac Ism1 Δ / Δ Representative micrographs of lungs stained with Van Hoeff-Van Gieson (VVG) staining, showing loss of elastin (black) and collagen (red) (E), and representative micrographs of airways stained with Periodic Acid-Schiff (PAS) staining, showing mucus hypersecretion (red) (F) in mice. n = 4 mice / group. Scale bar 20 μm. (G) Shows 2-month-old FVB / NTac WT and Ism1 Δ / Δ Measurement of bronchial epithelial cell height (left) and cell number (right) in mice. n = 34–38 average measurements collected from 8–10 small airways / mouse, 4 mice / group. (H) Shown are the mean values ​​of bronchial epithelial cell height (left) and cell number (right) in 2-month-old FVB / NTac WT and Ism1Δ / Δ Western blot (top) and fold change (bottom; AU, arbitrary units) of TGF-β1, VEGF-A, neutrophil elastase (NE), and α-1-antitrypsin (A1AT) in mouse lungs, with β-actin as a loading control. n = 4 mice / group. (I) Shown are 2-month-old FVB / NTac WT and Ism1 Δ / Δ Representative immunohistochemical staining of TGF-β1 and VEGF-A in AM of mouse lung. n = 4 mice / group. Scale bar 20 μm. (J) Shows 2-month-old FVB / NTac WT and Ism1 Δ / Δ Quantification of relative reactive oxygen species (ROS) in mouse lungs. Brain tissue (positive for ISM1 expression) was used as a control to demonstrate the specificity of ISM1 deficiency in the lungs. n = 3 mice / group. (K) shows 2-month-old FVB / NTac WT and Ism1 Δ / Δ Heatmap of relative cytokine expression between mouse lungs. n = 3 WT mice and 6 Ism1 Δ / Δ (L) shows P1FVB / NTac WT and Ism1 Δ / Δ Representative immunofluorescence staining of GM-CSF (red) and nuclei (DAPI; blue) in mouse lungs (left). n = 4 mice / group. (M) Shows the expression of GM-CSF in P1 FVB / NTac WT and Ism1 Δ / Δ Western blot (top) and fold change (bottom; AU, arbitrary units) of GM-CSF in mouse lungs, with β-actin as a loading control. n = 8-9 mice / group. Data are mean ± sem and analyzed by unpaired two-tailed Student's t-test (D, G, H, J, and M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (N) shows 1-month-old Ism1 Δ / Δ In mouse lungs, MMP-12 increases before GM-CSF upregulation;

[0116] Figure 6ISM1 is shown to induce apoptosis of alveolar macrophages through cell surface GRP78. (A) Shows representative immunofluorescence staining of AM (CD68, red) and nuclei (DAPI, blue) expressing ISM1 (green) in the lungs of 2-month-old FVB / NTac WT mice. Scale bar 10 μm. (B) Shows representative in situ hybridization using antisense ISM1 in the lungs of 2-month-old WT BALB / cAnNTac mice, with ISM1 expression in bronchial epithelium and alveolar macrophages depicted in the inset. (C)-(E) Show representative confocal images of primary AMs showing colocalization of rISM1 (red) and GRP78 (green) (C and D) and cleaved caspase-3 (CCP3) (E) after treatment with 1 μM rISM1. Nuclei are stained with DAPI (blue). Scale bar 5 μm. (F) and (G) show IncuCyte quantification of apoptosis in MH-S cells after thapsigargin (TG) treatment and 1 μM rISM1 treatment (F) and GRP78 antibody neutralization (G). Treatment conditions are as shown. Analyses were performed in triplicate wells, and four images were taken per well for quantification. Data are mean ± sem and analyzed by one-way ANOVA with Tukey's post hoc test (F and G). *P < 0.05, **P < 0.01, ***P < 0.001;

[0117] Figure 7 showed that exogenous rISM1 alleviates Ism1 Δ / Δ (A) and (B) show PBS and rISM1-treated FVB / NTac Ism1 Δ / Δ Representative immunostaining of rISM1 (A) and cleaved caspase-3 (CCP3, green) (B) in mouse AM. n = 4 mice / group. Scale bars 10 μm (A) and 20 μm (B). (C) and (D) show the expression of rISM1 in FVB / NTac AM after treatment with PBS, 5 μg rISM1, or liposome-clodronate (CLO). Δ / ΔRepresentative immunofluorescence staining of mouse lungs for surfactant protein C (SP-C, red), PCNA (green), and nuclei (DAPI, blue) (C) and quantification (D). n = 34–40 fields / group, 9–10 random images per mouse lung. Scale bar 20 μm. (E) Representative hematoxylin and eosin-stained lung micrographs of room air-exposed (Sham) and cigarette smoke-exposed WT BALB / cAnNTac (WT BALB / c) mice (CS) treated with vehicle (CS + PBS) or rISM1 (CS + 10 μg rISM1). n = 5 mice / group. Scale bar 100 μm. (F) Representative immunofluorescence staining of CCP3 (green) in the lungs of PBS- and rISM1-treated CS mice shown in (E). n = 5 mice / group. Scale bar 20 μm. (G)–(J) show quantification of lung neutrophil counts (G) and spirometry (H to J) of COPD mice in (E). Total lung capacity (TLC) (H), static compliance (Cchord) (I), and work of breathing (WOB) (J). Data are mean ± sem and analyzed by one-way ANOVA with Tukey's post hoc test (D, G to J). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. #: No significant difference compared with the Sham group;

[0118] Figure 8 ISM1 expression in COPD patients and cigarette smoke-exposed mice is shown. (A) Shows representative immunohistochemistry (top panel) and immunofluorescence (bottom panel) staining using anti-hISM1 or mouse IgG isotype control in empty vector (EV) or hISM1-overexpressing (hISM1-OE) HEK293FT cells. Scale bar 20 μm. (B) and (C) Show representative immunohistochemistry staining of ISM1 in the bronchial epithelium of room air-exposed (Sham) and cigarette smoke-exposed WT BALB / cAnNTac (WT BALB / c) mice (CS). n = 5 mice / group (B); and non-COPD and COPD patients (C). Scale bar 20 μm. (D) Shows the percentage distribution of hISM1 expression in patients stratified by current smoking status. Patient sample size is depicted on the graph. (E) and (F) Show representative immunohistochemical staining of ISM1 in AMs (E) and non-polymorphonuclear leukocytes or lymphocytes (F) of room air-exposed (Sham) and cigarette smoke-exposed WT BALB / c mice (CS). n = 5 mice / group. Scale bar 20 μm.

[0119] Figure 9 AF showed that ISM1 deficiency leads to increased leukocyte infiltration in the lung under non-pathological conditions. Figure 9 A shows Ism1 at 2 months old Δ / Δ Increased immune cell infiltration in the mouse lungs was observed, as shown by H&E staining. Figure 9 B shows Ism1 Δ / Δ and quantification of total leukocytes in wild-type lungs. Figure 9 C shows Ism1 at 2 months old Δ / Δ The mice had increased macrophages and neutrophils in their lungs, as detected by IHC staining for CD68 and NIMP-R14, respectively. Figure 9 D and E show differential immune cell counts, showing Ism1 Δ / Δ There is an increase in macrophages and neutrophils in the lungs. Figure 9 F shows Ism1 Δ / Δ Peripheral blood analysis of mice showed an increase in total white blood cells (WBC), neutrophils (NE), and lymphocytes (LY) at 2 months. * represents p < 0.05; n = 5 animals / group in (9 AE). Figure 9 F) n = 7 animals / group;

[0120] Figure 10 AG showed that ISM1 deficiency leads to an enhanced immune response to intratracheal LPS in the lung. Figure 10 AE shows the time course of immune response to intratracheal LPS challenge by quantifying total leukocytes, neutrophils, macrophages, T cells and B cells, Ism1 Δ / Δ Lungs show an enhanced immune response to LPS. LPS (2 mg / kg) was administered once intratracheally, and single cell suspensions were isolated from the lungs on days 1, 3, 5, and 7 after LPS administration. Figure 10 F shows that 1 day after LPS challenge, LPS expression in Ism1 was significantly increased compared with that in wild-type mice. Δ / Δ This triggered an enhanced increase in lung permeability (higher BAL protein) in mice. Figure 10 G shows representative H&E-stained lung sections showing the extent of immune cell infiltration into lung airspaces (acute inflammation) 1 day after LPS challenge (left) and immunofluorescent staining of neutrophils for NIMP-R14 (a neutrophil marker) (right). * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; n = 3 animals / group.

[0121] Figure 11 AG showed that intratracheally delivered rISM1 inhibited LPS-induced lung inflammation. Figure 11Figure A is a schematic diagram showing the treatment of LPS-induced acute lung inflammation in mice with rISM1. Mice were pretreated with 50 μg rISM1 via intratracheal delivery one day before receiving a single dose of LPS (2 mg / kg). Mice were treated continuously with 50 μg rISM1 once daily until day 3 after LPS. BAL fluid was then isolated and separated into BAL fluid protein and cellular components. Figure 11 B shows that total BAL fluid protein was reduced in rISM1-treated mice 1 day after LPS challenge. Figure 11 Figure CG shows total and differential immune cell counts performed using cells from BAL fluid of mice treated with PBS or rISM1 one day after LPS challenge. rISM1 significantly reduced the number of total leukocytes, neutrophils, and macrophages. T and B cell counts were also reduced in response to rISM1, although this was not statistically significant, as these two cell types were significantly altered in PBS-treated mice. * indicates p < 0.05; n = 5 animals / group.

[0122] Figure 12 AE showed that ISM1 deficiency led to lung tissue remodeling and fibrosis after recovery from LPS-induced acute lung injury. Figure 12 A shows representative images of H&E-stained lung sections showing the expression of wild-type and Ism1 cells 9 days after LPS challenge. Δ / Δ The extent of tissue fibrosis in mice. Figure 12 BC showed that collagen deposition in Ism1 was detected by Picro-Sirius staining. Δ / Δ Higher in mouse lungs. Figure 12 DE showed that α-smooth muscle actin (α-SMA) immunofluorescence staining indicated that Ism1 Δ / Δ Increased myofibroblasts within fibrotic lesions in mouse lungs. ** indicates p < 0.01. Quantification was performed using n = 3 lungs / group, 2 sections / lung, and 5 microscopic fields / section.

[0123] Figure 13 AB showed that ISM1 deficiency increased the proliferation of alveolar epithelial type 2 cells (AEC2) in the lung. Figure 13 A shows that Ism1 Δ / Δ Representative images of SP-C and PCNA double immunofluorescence stained lung sections from wild-type and wild-type mice. Figure 13 B shows the quantification of SP-C and PCNA double-positive cells in lung tissue sections on day 9 after PLS challenge. Δ / ΔThe number of proliferating AEC2 cells was significantly increased in mice. *** indicates p < 0.001. Quantification was performed using n = 3 lungs / group, 2 sections / group, and 5 microscopic fields / section.

[0124] Figure 14 AC revealed that ISM1 deficiency resulted in higher levels of the pro-fibrotic cytokine TGF-β in the lung. Figure 14 A is the Ism1 9 days after LPS challenge Δ / Δ Representative images of TGF-β immunofluorescence-stained lung sections from wild-type and WT mice. Figure 14 B shows the expression of Ism1 using Western blotting and whole lung lysates 9 days after LPS challenge. Δ / Δ The expression levels of TGF-β in the lungs of wild-type and human mice. Figure 14 C showed that after 9 days of LPS challenge, Ism1 Δ / Δ Quantification of lung TGF-β protein levels in mice. ** indicates p < 0.01. n = 3 lungs / group;

[0125] Figure 15 Figures A to C show that ISM1 deficiency alters the inflammatory cytokine / chemokine profile of the lung 1 day after LPS challenge. Δ / Δ Lung homogenates from wild-type and wild-type mice were analyzed for cytokines and chemokines using an inflammatory cytokine antibody array. Figure 15 Panel A shows relative changes in cytokines / chemokines analyzed by antibody array. * indicates p < 0.05. n = 4 lungs / group. Figure 15 BC showed that Ism1 was expressed in the 1-day LPS challenged cells by Western blot analysis. Δ / Δ TNF-α expression in the lungs was increased. ** indicates p < 0.01. n = 3 lungs / group;

[0126] Figure 16 AC showed that ISM1 deficiency enhanced the expression level and nuclear translocation of p65 NF-κB. Figure 16 A is the Ism1 after 1 day of LPS challenge Δ / Δ Representative images of immunofluorescence staining of NF-κB p65 subunit in lung sections from wild-type and WT mice. Δ / Δ Increased p65 NF-κB (red) signal colocalized with DAPI (nuclei, blue) was present in lung sections from mice. Figure 16 B shows that Ism1 Δ / Δ p65 NF-κB levels were increased in lung homogenates of mice, as shown by Western blotting. Figure 16 C shows the quantitative amount of p65 NF-κB relative to β-actin. n = 3 lungs / group;

[0127] Figure 17 AD shows the generation of Ism1 Δ / Δ C57BL / 6J mice. Figure 17 Figure A is a schematic diagram of CRISPR / Cas9 targeting Ism1 via guide RNA pairs, gRNA1 and gRNA3. P1 and P2 represent primers used for T7E1 assay and genotyping. Figure 17 B shows Ism1 Δ / Δ The DNA sequence of the knockout line shows the 23-bp deletion that results in a premature stop codon and no ISM1 protein production. The red arrow indicates the Cas9 cleavage site, and the yellow region refers to the two overlapping gRNA target regions. Figure 17 C shows the expression of C56BL / 6J WT, Ism1 + / Δ 、Ism1 Δ / Δ RT-PCR of Ism1 mRNA. Figure 17 D shows C57BL / 6JWT and Ism1 Δ / Δ Representative immunohistochemical staining of ISM1 (brown) and nuclei (hematoxylin, blue) in mouse lung sections. Br, bronchus; Al, alveoli. Scale bar 20 μm;

[0128] Figure 18 A model comparing normal cells (right) and cells under stress (left) is shown, wherein stressed cells (e.g., tumor cells, ECs, activated inflammatory cells) have increased cell surface GRP78 (csGRP78), which can interact with ISM1 and / or its fragments, leading to cell death (providing a therapeutic effect, for example). Similarly, changes in csGRP78 levels and / or ISM1 interaction therewith can provide diagnostic and / or prognostic information. As shown in the figure, it is expected that csGRP78 may be a therapeutic target and / or diagnostic biomarker in diseases such as inflammatory diseases;

[0129] Figure 19 The AB display is ISM1 C (287-461) instead of ISM1 N Supports EC adhesion. Figure 19 A shows that the AMOP domain in ISM1 is important in mediating EC adhesion. C Can support cell adhesion as well as ISM1, as there is no significant difference in confluence between the two proteins over time. N Has a reduced ability to support cell adhesion as the chance of confluence becomes slower over time. **p<0.01, n=3. Figure 19 B shows mutant ISM1 RKD341RAA(C) and ISM1RKD340AAA(C) It has a reduced ability to support cell adhesion, as the cell adhesion rate is significantly lower than that of ISM1C. **p<0.01, n=3;

[0130] Figure 20 AB showing internalized ISM1 C (287-461) triggers EC apoptosis. Figure 20 AB depicts ISM1 C Instead of ISM1 N Significantly triggers EC apoptosis. Error bars represent SD. *P<0.05, **P<0.01, N=3;

[0131] Figure 21 This study demonstrates that gelatin, but not BSA, supports EC adhesion. Dynamic changes in cell confluence were monitored. Gelatin supports EC adhesion, with confluence increasing over time. BSA does not support cell adhesion, as confluence remains low or even decreases over time. **P < 0.01, N = 3;

[0132] Figure 22 CRISPR / Cas9 targeting and generation of FVB / N Ism1 is shown Δ / Δ Mice. (A) Schematic diagram of CRISPR / Cas9 targeting Ism1 via guide RNA pairs gRNA1 and gRNA2. (B) and (C) show FVB / NTac (B) and C57BL6 / J (C) WT, Ism1 + / Δ and Ism1 Δ / Δ Genotyping PCR of mice. (D) shows FVB / NTac WT and Ism1 Δ / Δ Representative immunofluorescence staining of ISM1 (red) and nuclei (DAPI, blue) in mouse airway epithelium. n = 4 mice / group. Scale bar 20 μm. (E) shows C57BL / 6J WT and Ism1 Δ / Δ Representative immunohistochemical staining of ISM1 in mouse lung. n = 4 mice / group. Scale bar 20 μm.

[0133] Figure 23Figure 3 shows alveolar macrophage apoptosis in COPD associated with cell surface GRP78 expression. (A) Shows representative immunohistochemical staining of cleaved caspase-3 (CCP3) in the patient population. Scale bar 50 μm. (B) Shows the correlation between hISM1 expression and AM apoptosis stratified by smoking status. Patient sample size depicted on the graph. Data were analyzed by Pearson correlation. (C) and (D) show representative immunofluorescence staining of GRP78 (red), cleaved caspase-3 (CCP3, green) and nuclei (DAPI, blue) in the lungs of indoor air-exposed (Sham) and cigarette smoke-exposed (CS) WT BALB / cAnNTac mice. n=5 mice / group. Scale bar 20 μm (C); and non-COPD and COPD patients. Scale bar 10 μm (D);

[0134] Figure 24 Without wishing to be bound by theory, the proposed mechanism of ISM1 in regulating AM apoptosis and lung homeostasis and inflammation is shown. (A) Left, autocrine / paracrine ISM1 specifically targets AMs with high csGRP78 and induces apoptosis. AM numbers remain controlled, inflammation is regulated, and lung homeostasis is maintained. Right, no / low ISM1 leads to AM accumulation in the alveolar space and the onset of emphysema, with a gradual decline in lung function. (B) Schematic diagram of the proposed mechanism of ISM1 in regulating AM apoptosis and lung homeostasis is shown, without wishing to be bound by theory. Left, autocrine / paracrine ISM1 specifically targets AMs with high csGRP78 and induces apoptosis. AM numbers remain controlled, inflammation is regulated, and lung homeostasis is maintained. Right, loss of ISM1 leads to AM accumulation in the alveolar space and the onset of emphysema, with a gradual decline in lung function. Emphysema mediators including ROS, NF-κB signaling, MMP-12, and MMP-9 are upregulated;

[0135] Figure 25 Gating strategy for flow cytometric analysis of cells from bronchoalveolar lavage fluid (BALF) is shown. Representative gating strategy for flow cytometric analysis and quantification of alveolar macrophages is shown;

[0136] Figure 26The timeline of allergen exposure in the HDM-induced allergic asthma mouse model is shown. The disease model was generated according to the protocols described in Hammad et al. (2009) and Peh et al. (2015). Briefly, female C57BL / 6J mice (6-8 weeks) were anesthetized using isoflurane and sensitized using 40 μL of 100 μg HDM extract (house dust mite) via the intratracheal route on days 0, 7, and 14. 2 hours after the challenge, on days 15 and 16 (red arrows), a single daily dose of bacterially produced recombinant ISM1 (2 mg / kg, 40 μg / mouse) or an equal volume of vehicle (normal saline) was continuously administered. All animals were euthanized on day 17, and bronchoalveolar lavage fluid (BALF) was collected for immune cell infiltration analysis. A naive group consisting of five healthy mice was used as a control;

[0137] Figure 27 Figures show inflammatory cell counts in bronchoalveolar lavage fluid (BALF) 24 hours after the last treatment [naive, n=5; HDM, n=5; saline, n=7; ISM1, n=7]. (A) Differential cell counts were performed on ten different observation fields to identify eosinophils (Eos), alveolar macrophages (AM), neutrophils (Neu), and lymphocytes (Lym). Compared with the naive group, challenge with house dust mite (HDM) extract significantly increased total cell counts, eosinophils, alveolar macrophages, and slightly increased neutrophil and lymphocyte counts. On the other hand, the Isthmin 1 (ISM1) treatment group showed a 70% decrease in total cell counts (P=0.0053), with significant decreases in eosinophil (P=0.0062) and lymphocyte (P=0.0381) counts. No changes in the total counts of alveolar macrophages and neutrophils were observed. Values ​​are shown as mean ± SEM. Significantly different from HDM. (B) Representative photomicrographs of Liu staining showing immune cell infiltration. Scale bar 40 μm;

[0138] Figure 28 The results showed that rISM1 treatment significantly reduced total IgE levels compared with the HDM treatment group. TM Total IgE levels were analyzed using a mouse IgE ELISA kit (N = 5). Values ​​are shown as mean ± SEM. Mean differences between groups were compared using one-way ANOVA, *P < 0.05; **P < 0.005;

[0139] Figure 29The C-terminal AMOP domain alone is shown to retain the full pro-apoptotic activity of ISM1. Recombinant protein constructs are shown on the left, and their pro-apoptotic activities are shown on the right. Mam: mammalian-produced; bac: E. coli-produced. Mouse ISM1 protein was used. Both mammalian- and E. coli-produced rISM1 287-461 fragments retained the full pro-apoptotic activity of full-length ISM1.

[0140] Figure 30 Shown is a co-IP assay using purified GRP78 and ISM1 proteins, demonstrating their direct binding through the AMOP domain 287-461 region;

[0141] Figure 31 The expression and purification of recombinant mouse ISM1 protein are shown. (A) Schematic diagram of the mammalian expression construct of ISM1. The mouse Igκ1 leader sequence is included at the N-terminus to enhance secretion efficiency. The c-Myc tag and the hexa-histidine tag are included at the C-terminus for protein detection and purification. (B) Schematic diagram of the workflow for expressing and purifying mammalian rISM1. (C) The purified mammalian recombinant ISM1 protein migrates at approximately 70 kDa on denaturing SDS-PAGE. (D) Schematic diagram of the bacterial expression construct of ISM1. The hexa-histidine tag is included at the N-terminus for protein detection and purification. The SUMO-tag at the N-terminus facilitates protein solubility. (E) Schematic diagram of the workflow for expressing and purifying bacterial rISM1. (F) The final purified bacterial recombinant ISM1 protein, with the SUMO-tag removed, migrates at approximately 55 kDa on denaturing SDS-PAGE;

[0142] Figure 32 Mammalian recombinant ISM1 is shown to be densely deposited with heterogeneous glycans. (A) Schematic diagram of the two N-glycosylation sites present in ISM1. (B) Mammalian recombinant ISM1 protein was incubated with PNGaseF to remove N-linked glycans. (C) Wild-type and N-glycan mutants of ISM1 were expressed and analyzed in HEK293T, HEK293FT, and HeLa cells. Protein expression and secretion were analyzed using Western blotting. (D) Summary of O- and C-linked glycan deposition on ISM1. Red residues indicate glycan deposition sites. The first underlined sequence refers to the TSR domain, and the second underlined sequence corresponds to the AMOP domain. (E) O- and C-glycan mutants of ISM1 were expressed and analyzed in HEK293FT cells;

[0143] Figure 33The AMOP domain of ISM1 that mediates its receptor interaction is shown. (A) Schematic diagram of the recombinant protein constructs of ISM1 and GRP78 used for Co-IP experiments. mamISM1 represents mammalian recombinant ISM1 protein; bacGRP78 represents bacterial recombinant GRP78 protein. (B) Coomassie blue-stained SDS-PAGE shows the quality of the purified recombinant proteins. (C) Co-IP analysis confirmed that the AMOP domain mediated the ISM1-GRP78 interaction. (D) Co-IP analysis confirmed that the AMOP domain mediated the ISM1-integrin αvβ5 interaction. (E) Co-IP analysis confirmed that ISM1 without the AMOP domain abolished the interaction with integrin αvβ5; and

[0144] Figure 34 Demonstration that AMOP domain boundaries influence their pro-apoptotic activity. (A) Schematic diagram of the ISM1 AMOP construct used for mammalian recombinant protein production and purification. (B) Coomassie blue-stained SDS-PAGE demonstrating the quality of the purified recombinant protein. (C) Apoptosis assay structure for two mammalian AMOP truncations. (D) Schematic diagram of the ISM1 AMOP construct used for bacterial recombinant protein production and purification. (E) Coomassie blue-stained SDS-PAGE demonstrating the quality of the purified recombinant protein. (F) Comparison of the pro-apoptotic activity of different bacterial recombinant AMOP truncations. (G) Comparison of the pro-apoptotic activity of mammalian and bacterial recombinant AMOP truncations. DETAILED DESCRIPTION

[0145] Described herein are peptides, polypeptides, nucleic acids, compositions and methods for treating diseases or conditions associated with inflammation, particularly lung inflammation. It should be understood that the embodiments and examples are provided for illustrative purposes for those skilled in the art and are not intended to be limiting in any way.

[0146] Healthy adults regulate and maintain lung homeostasis under ambient conditions to prevent aseptic inflammation. Immune response may be triggered by injury, environmental stressors and / or infection, resulting in acute inflammation. Ideally, this inflammation is ultimately resolved to allow wound healing and recovery, and to prevent inflammation-induced illness and / or injury. Failure to fully resolve inflammation may result in chronic inflammation and / or inflammation-related diseases, such as chronic obstructive pulmonary disease (COPD), emphysema, chronic obstructive bronchitis and / or pulmonary fibrosis. Acute lung diseases such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are also relevant to lung inflammation.

[0147] As described in detail herein, the inventors have now developed polypeptides, nucleic acids, compositions, pulmonary drug delivery devices and methods for treating inflammation, such as lung inflammation, that are derived from and / or based on Isthmin 1 (ISM1), a secreted protein that studies described herein show plays a role in inhibiting, preventing and / or resolving inflammation, particularly lung inflammation. In studies described in detail below, supplementation of the lungs with exogenous recombinant ISM1 protein (rISM1) suppressed the lung inflammation phenotype in ISM1-deficient lungs, and the results showed that ISM1 can help resolve inflammation by inducing apoptosis of alveolar macrophages. As described herein, for example, recombinant ISM1 (rISM1) can block cigarette smoke-induced COPD and can inhibit LPS-induced acute lung inflammation and injury. The results show that ISM1 plays an important role in inhibiting and / or resolving sterile lung inflammation and / or inflammation triggered by infection and / or injury.

[0148] Therapeutic compositions, formulations and devices for treating diseases or conditions associated with inflammation

[0149] For example, provided herein are therapeutic compositions, peptides, polypeptides, nucleic acids, formulations, and devices for treating, ameliorating, or preventing diseases or conditions associated with inflammation, particularly pulmonary inflammation.

[0150] Isthmin 1 (ISM1, sometimes referred to as ISM) is a secreted protein found in several different vertebrate species. The ISM1 protein includes a thrombospondin type 1 repeat (TSR) domain and an adhesion-associated domain in MUC4 and other proteins (AMOP) domains. Glucose-regulated protein 78 kDa (GRP78) and αvβ5 integrin are known receptors for ISM1. Isthmin 1 (ISM1) has previously been associated with angiogenesis inhibition in mice (see Xiang, W. et al., 2011, Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumor growth in mice, Journal of Cellular and Molecular Medicine, 15(2): 359-374, the entire contents of which are incorporated herein by reference).

[0151] The sequence of the full-length Isthmin 1 expressed in humans (ie, including the N-terminal signal peptide) is as follows:

[0152] Isthmin-1 precursor [human]

[0153] NCBI reference sequence: NP_543016.1

[0154]

[0155] (SEQ ID NO: 1)

[0156] The full-length sequence of Isthmin 1 expressed in mice (i.e., including the N-terminal signal peptide) is as follows:

[0157] Isthmin-1 precursor [Mus musculus]

[0158] NCBI reference sequence: NP_001263418.1

[0159]

[0160] (SEQ ID NO: 2)

[0161] Because ISM1 is a secreted protein, these human (SEQ ID NO: 1) and mouse (SEQ ID NO: 2) ISM1 sequences include an N-terminal signal peptide sequence. This signal peptide sequence is underlined above and is typically removed in the mature secreted form of ISM1. The TSR domain is shown in bold, and the AMOP domain is shown in italics in the above sequences.

[0162] Mature ISM1 in humans and mice with the signal peptide sequence removed is shown below:

[0163] Isthmin-1 (mature) [human]

[0164] GSGAADGPDAAAGNASQAQLQNNLNVGSDTTSETSFSLSKEAPREHLDHQAAHQPFPRPRFRQETGHPSLQRDFPRSFLLDLPNFPDLSKADINGQNPNIQVTIEVVDGPDSEADKDQHPENKPSWSVPSPDWRAWWQRSLSLARANSGDQDYKYDSTSDDSNFLNPPRGWDHTAPGHRTFETKDQPEYDSTDGEGDWSLWSVCSVTCGNGNQKRTRSCGYACTATESRTCDRPNCPGIEDTFRTAATEVSLLAGSEEFNATKLFEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY

[0165] (SEQ ID NO:3)

[0166] Isthmin-1 (mature) [Mus musculus]

[0167] GSGASDRQDAAAGNVSGSQLQNNLNLESDSTSETSFPLSKEAPEEHQVVHQPFPRQRFPPETGHPSLQRDGPRSFLLDLPNFPDLSKADINGQNPNIQVTIEVVDGPDSEAEKDQHPENKPSWSLPAPDWRAWWQRSLSLARTNSGDQDDKYDSTSDDSNFLSVPRGWDRPAPGHRTFETKEQPEYDSTDGEGDWSLWSVCSVTCGNGNQKRTRSCGYACIATESRTCDRPNCPGIEDTFRTAATEVSLLAGSEEFNATKLFEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY

[0168] (SEQ ID NO:4)

[0169] As will be appreciated, in certain embodiments, references herein to the Isthmin 1 (ISM1) protein may be understood as references to full-length ISM1 (i.e., with the signal peptide present) or mature ISM1 (i.e., with the signal peptide removed). Because secreted ISM1 is typically present in a mature form (i.e., lacking the signal peptide), the signal peptide sequence may be omitted in certain embodiments. Generally, in certain embodiments, mature ISM1 may be preferred.

[0170] As will also be understood, in certain embodiments, references herein to the Isthmin 1 (ISM1) protein may include ISM1 found in any suitable species that expresses ISM1 or a homolog, ortholog, paralog, or functional equivalent thereof. ISM1 is expressed by many different species. The ISM1 sequence is further described in WO2009 / 113965, the entire contents of which are incorporated herein by reference. In Xiang, W. et al., 2011, Journal of Cellular and Molecular Medicine, 15(2):359-374 (the entire contents of which are incorporated herein by reference), a sequence comparison is provided showing the amino acid alignment of the ISM1 found in humans, mice, Xenopus laevis, and zebrafish. The pairwise alignment scores calculated by NCBI using HomoloGene for human ISM1 (precursor) and the corresponding ISM1 sequences of several different species are shown below:

[0171] Pairwise comparison scores

[0172]

[0173] As can be seen, while ISM1 is somewhat similar between species, sequence variation is observed. In terms of protein sequence, the above scores indicate sequence identities up to 30% divergence from the human sequence (i.e., the zebrafish sequence has 70% sequence identity with human ISM1).

[0174] In certain embodiments, provided herein is a peptide or polypeptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof. In certain embodiments, the polypeptide can comprise an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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%, or 100% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, which can be any suitable ISM1 protein from any particular species, with or without an N-terminal signal peptide. In certain embodiments, the polypeptide may comprise any suitable polypeptide, peptide, or peptide-based or polypeptide-based molecule or group of molecules, which may or may not be further modified to include one or more additional protein or non-protein moieties, such as purification tags, linkers, fluorophores, signal peptides, targeting or delivery sequences, cell penetrating peptides, additional active agents (such as another drug for targeting lung inflammation), or other moieties suitable for a particular application. In certain embodiments, the polypeptide may be modified at one or more amino acid side chains, the N-terminus, the C-terminus, or any combination thereof. In certain embodiments, in addition to the amino acid sequence having at least 70% sequence identity to the Isthmin 1 (ISM1) protein or its GRP78-activating fragment, the polypeptide may include one or more additional amino acid sequences, which may be located at the N-terminus or C-terminus of the sequence having identity to ISM1.

[0175] In certain embodiments, Isthmin The GRP78-activating fragment of the 1 (ISM1) protein can include at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least

[0015] The present invention also provides any suitable peptide or polypeptide having at least 7%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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%, or 100% sequence identity to ISM1, wherein the GRP78-activating fragment is capable of binding to and activating GRP78 with substantially the same efficacy as ISM1, or binding to and activating GRP78 with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% as compared to ISM1.

[0176] In the studies described herein, ISM1 has been shown to be a pro-apoptotic ligand of GRP78, binding to GRP78 to trigger apoptosis. Therefore, ISM1 can be considered an agonist of GRP78. As a signaling receptor, GRP78 can bind to many different ligands, which may occur through different regions of the protein (a review of GRP78 can be found in Ni, et al., Biochem J., 2011, 434(2): 181-188, the entire contents of which are incorporated herein by reference). Some ligands may be pro-proliferative, others may be pro-apoptotic, etc., and each may have different intracellular signaling pathways. In certain embodiments, the interaction between ISM1 and GRP78 may involve intracellular signaling pathways, which may involve ISM1 internalization and / or caspase activation. Therefore, in certain embodiments, a GRP78-activating fragment may include any suitable peptide or polypeptide that can act as a pro-apoptotic ligand for GRP78 and / or can bind to GRP78 and trigger apoptosis. In certain embodiments, a GRP78-activating fragment of an ISM1 protein can include any suitable peptide or polypeptide capable of triggering apoptosis via GRP78, which may or may not involve internalization of the fragment and / or caspase activation.

[0177] In certain embodiments, references herein to GRP78 or a GRP78 receptor may be understood to include cell surface GRP78 (csGRP78). Extracellular ISM1 interacts with cell surface GRP78 (csGRP78), and thus, those skilled in the art, having considered the teachings herein, will understand that, where appropriate, references herein to GRP78 may be understood to refer to csGRP78 (i.e., in certain embodiments, references herein to GRP78 may be understood to refer to csGRP78).

[0178] It is anticipated that, in certain embodiments, the AMOP domain may mediate the binding of ISM1 to GRP78 and / or may mediate the pro-apoptotic activity of ISM1. It is further anticipated that, in certain embodiments, the TSR domain of ISM1 may have little or no involvement in the binding of ISM1 to GRP78 and / or the pro-apoptotic effects of ISM1. Indeed, results indicate that the AMOP domain may be responsible for GRP78 binding and triggering apoptosis (see Example 3 below for further details). Thus, in certain embodiments, the peptide or polypeptide may generally comprise any suitable peptide or polypeptide or peptidyl or polypeptide-based molecule having at least the AMOP domain of ISM1, or a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto. In embodiments where the polypeptide comprises more than one domain or region from ISM1, the domains or regions may be organized generally in the same manner as the NC-terminal sequence in ISM1, or may be rearranged relative to ISM1. ISM1 domains and fragments are described in more detail in WO 2009 / 113965, entitled "Isthmin Derivatives for use in Treating Angiogenesis," the entire contents of which are incorporated herein by reference.

[0179] In certain embodiments, provided herein is a peptide or polypeptide comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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%, or 100% sequence identity to any one of SEQ ID NO: 10 or 11:

[0180] SEQ ID NO:10—Human ISM1 AMOP domain

[0181] FEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDED

[0182] SEQ ID NO:11—Mouse ISM1 AMOP domain

[0183] FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDED

[0184] In certain embodiments, provided herein is a peptide or polypeptide comprising at least 70 amino acids identical to any one of SEQ ID NOs: 1-4, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, or 440 amino acids thereof. %, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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%, or 100% sequence identity.

[0185] In certain embodiments, provided herein is a polypeptide comprising or consisting of any one of SEQ ID NOs: 1-4. In certain embodiments, provided herein is a polypeptide that is an Isthmin 1 protein, or comprises an Isthmin 1 protein. In certain embodiments, provided herein is a polypeptide that is a human ISM1 protein, or comprises a human ISM1 protein.

[0186] In certain embodiments, the present invention provides a polypeptide or peptide comprising or consisting of the following amino acid sequence:

[0187] FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE(SEQ ID NO:26);

[0188] or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0189] The structure described in Example 3 indicates that the polypeptide sequence SEQ ID NO: 26 is sufficient to bind to GRP78. Example 3 shows effective results in the absence of the EVSLLAGSEEFNATKL sequence preceding SEQ ID NO: 26 (i.e., positions 271-286) in ISM1. Therefore, in certain embodiments, the polypeptide or peptide or GRP78-activating fragment thereof may include a polypeptide or peptide or GRP78-activating fragment thereof comprising or consisting of the following amino acid sequence: FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE (SEQ ID NO: 26). NO:26); or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, or a GRP78-activating fragment thereof.

[0190] In certain embodiments, the present invention provides a polypeptide or peptide comprising or consisting of the following amino acid sequence:

[0191] FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY(SEQ ID NO:24);

[0192] or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0193] The results described in Example 3 indicate that the polypeptide sequence SEQ ID NO: 24 may be sufficient to bind GRP78, or to support EC adhesion, to be internalized, to be localized in mitochondria, and to induce apoptosis. Example 3 shows effective results in the absence of the EVSLLAGSEEFNATKL sequence preceding SEQ ID NO: 26 (i.e., positions 271-286) in ISM1. Thus, in certain embodiments, the polypeptide or peptide or its GRP78-activating fragment may include a polypeptide or peptide or its GRP78-activating fragment comprising or consisting of the following amino acid sequence: FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY (SEQ ID NO: NO:24); or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, or a GRP78-activating fragment thereof.

[0194] The amino acid sequence of SEQ ID NO: 26 is the N-terminal region of the C-terminal region of mouse ISM1, in which the AMOP domain is located, and contains sequences from the N-terminal portion of the AMOP domain (ISM1 C-N, residues 2287-373). The amino acid sequence of SEQ ID NO: 24 is the C-terminal region of mouse ISM1, containing the AMOP domain but not the TSR domain (ISM1 C , residues 287-461). Each of SEQ ID NOs: 26 and 24 is derived from a mouse ISM1 sequence, but it will be appreciated that in certain embodiments, a corresponding sequence / region of ISM1 from another species may be used. For example, in certain embodiments, provided herein is a peptide or polypeptide or GRP78-activating fragment thereof comprising or consisting of the following amino acid sequence: a region 287-373 (SEQ ID NO: 26) or a region 287-461 (SEQ ID NO: 26) found in human ISM1 that corresponds to a region 287-373 (SEQ ID NO: 46) or a region 287-461 (SEQ ID NO: 46) of mouse ISM1. NO:25), or an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:25 (see the corresponding human sequence, e.g., region 290-464 in human), or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, or a GRP78-activating fragment thereof.

[0195] FEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE (SEQ ID NO: 27; human ISM1 C-N )

[0196] FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY (SEQ ID NO: 24 – Mouse ISM1 287-461); or

[0197] FEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY (SEQ ID NO: 25–Human ISM1 290-464)

[0198] In certain embodiments, the aforementioned peptide or polypeptide or GRP78-activating fragment thereof may or may not further comprise an N-terminal signal peptide sequence of ISM1.

[0199] In certain embodiments, the peptide or polypeptide or GRP78-activating fragment thereof is not full-length or mature ISM1 (i.e., in certain embodiments, the peptide or polypeptide or GRP78-activating fragment thereof may be an exogenous peptide or polypeptide that is not naturally expressed in a cell or subject). In certain embodiments, the peptide or polypeptide or GRP78-activating fragment thereof may be longer or shorter than full-length or mature ISM1. In certain embodiments, the peptide or polypeptide or GRP78-activating fragment thereof may have a primary amino acid sequence that differs from naturally expressed ISM1 in at least one residue (i.e., for example, the peptide or polypeptide or GRP78-activating fragment thereof may contain one or more amino acid additions, deletions, or substitutions compared to naturally expressed ISM1 from humans or other species). In certain embodiments, for example, the peptide or polypeptide or GRP78-activating fragment thereof may contain one or more conservative amino acid substitutions compared to naturally expressed ISM1.

[0200] In certain embodiments, the peptide or polypeptide or its GRP78-activating fragment may comprise or consist of the following amino acid sequence:

[0201] FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE(SEQ ID NO:26);

[0202] FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY(SEQ ID NO:24);

[0203] FEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE(SEQ ID NO:27); or

[0204] FEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY (SEQ ID NO: 25–Human ISM1 290-464);

[0205] or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, or a GRP78-activating fragment thereof.

[0206] In another embodiment of the above peptide, polypeptide, or GRP78-activating fragment, the peptide, polypeptide, or GRP78-activating fragment does not contain the amino acid sequence EVSLLAGSEEFNATKL at the N-terminus of any of SEQ ID Nos: 24-27, or does not contain the sequence EVSLLAGSEEFNATKL at all. In another embodiment, the peptide, polypeptide, or GRP78-activating fragment does not contain or is not the following sequence:

[0207] EVSLLAGSEEFNATKLFEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE(SEQ ID NO:12);

[0208] EVSLLAGSEEFNATKLFEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLE(SEQ ID NO:20);

[0209] EVSLLAGSEEFNATKLFEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY(SEQ ID NO:13); or

[0210] EVSLLAGSEEFNATKLFEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY(SEQ ID NO:21).

[0211] In certain embodiments, the aforementioned peptide or polypeptide or GRP78-activating fragment thereof may comprise an isolated peptide or polypeptide or GRP78-activating fragment thereof.

[0212] In certain embodiments, the polypeptides or peptides, or GRP78-activating fragments thereof, as described herein (including those having at least 70% sequence identity with an ISM1 protein or GRP78-activating fragment thereof) may include polypeptides or peptides, or GRP78-activating fragments thereof, having at least one mutation (i.e., an amino acid addition, deletion, or substitution) compared to full-length or mature ISM1 naturally expressed in a particular species. In certain embodiments, the at least one mutation may or may not comprise an amino acid substitution. In certain embodiments, the at least one mutation may or may not comprise a conservative amino acid substitution, wherein an amino acid is replaced with another amino acid residue that is at least somewhat similar to the original residue in at least one of size, charge, hydrophilicity / hydrophobicity, hydrogen bonding, or any combination thereof. In certain embodiments, the at least one mutation may or may not comprise a conservative amino acid substitution, wherein an amino acid residue is replaced with another amino acid residue that is generally considered inconspicuous at the replacement position or does not interfere with the desired function, such as, for example, glycine or alanine.

[0213] The studies in Example 3 include the discussion of polypeptides with KD341AA and RKD340AAA mutations (ISM1 C-N Mutants). These mutations can cause ISM1 to lose its binding affinity to the αvβ5 integrin receptor, but as discussed in Example 3, these mutations do not disrupt binding to GRP78, suggesting that such mutant sequences can provide more specific targeting of the target GRP78 receptor. Therefore, in certain embodiments, the polypeptide or peptide or GRP78-activating fragment thereof may include a KD341AA mutation or a RKD340AAA mutation. It will be understood that the "341" and "340" numbering is provided relative to the mouse sequence and may vary between species. For example, when referring to the numbering in the human ISM1 sequence, the numbering of KD-AA and RKD-AAA may vary (e.g., the corresponding human mutations may be KD343AA and RKD342AAA).

[0214] Thus, in certain embodiments, the polypeptide or peptide or GRP78-activating fragment thereof may include a polypeptide or peptide or GRP78-activating fragment thereof comprising or consisting of the following amino acid sequence: FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKRAAFRWKDASGPKEKLEIYKPTARYCIRSMLSLE (SEQ ID NO:14, the N-terminal portion of the C-terminal region contains the mouse AMOP domain, residues 287-373, with the KD341AA mutation), or the corresponding mutant region from another species, or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, or a GRP78-activating fragment thereof.

[0215] In certain embodiments, the polypeptide or peptide or GRP78-activating fragment thereof may include a polypeptide or peptide or GRP78-activating fragment thereof comprising or consisting of the following amino acid sequence: FEVDMDSCERWMSCKSEFLKKYMHKVINDLPSCPCSYPTEVAYSTADIFDRIKAAAFRWKDASGPKEKLEIYKPTARYCIRSMLSLE (SEQ ID NO:15, the N-terminal portion of the C-terminal region contains the mouse AMOP domain, residues 287-373, with the RKD340AAA mutation), or the corresponding mutant region from another species, or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, or a GRP78-activating fragment thereof.

[0216] As discussed in Example 3, the results indicate that the C-terminal AMOP domain alone is sufficient to mediate the pro-apoptotic activity of ISM1. Indeed, as discussed in Example 3 below, structure-function relationship studies using truncations of the ISM1 protein indicate that the C-terminal AMOP domain from 287-461 retains the full pro-apoptotic activity of the full-length ISM1 protein and that the AMOP domain does not require post-translational modification to mediate the pro-apoptotic activity of ISM1. Thus, in certain embodiments, the polypeptide or peptide, or GRP78-activating fragment thereof, may include a polypeptide or peptide, or GRP78-activating fragment thereof, comprising or consisting of the following amino acid sequence:

[0217] OR

[0218] FEVDTDSCERWMSCKSEFLKKYMHKVMNDLPSCPCSYPTEVAYSTADIFDRIKRKDFRWKDASGPKEKLEIYKPTARYCIRSMLSLESTTLAAQHCCYGDNMQLITRGKGAGTPNLISTEFSAELHYKVDVLPWIICKGDWSRYNEARPPNNGQKCTESPSDEDYIKQFQEAREY (SEQ ID NO: 25; human);

[0219] or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0220] SEQ ID NOs: 24 and 25 are derived from mouse and human ISM1 sequences, respectively; however, it will be appreciated that in certain embodiments, the corresponding sequence / region of ISM1 from another species may be used.

[0221] In certain embodiments, the polypeptide or peptide or its GRP78-activating fragment may include a chemically modified polypeptide or peptide, or a peptide mimetic derived from or based on the polypeptide or peptide. For example, in certain embodiments, the polypeptide or peptide or its GRP78-activating fragment may include a chemically modified polypeptide or peptide, or a peptide mimetic derived from or based on the polypeptide or peptide, which may include a cyclized derivative, a derivative comprising one or more non-natural amino acid residues (such as a D-amino acid), and / or a peptide mimetic or one or more other constructs comprising one or more different chemical bonds or connections different from a peptide bond, such as peptoids and β-peptides. In certain embodiments, examples of peptide-derived agents from ISM1 may include those described in Kao et al., EBioMedicine 33 (2018) 22-32 (the entire contents of which are incorporated herein by reference), such as BC71.

[0222] In certain embodiments, the peptides or polypeptides as described herein can be formulated for administration to the lungs of subjects in need. In view of the teachings herein, those skilled in the art will recognize that a variety of suitable methods and techniques are used to formulate medicaments for administration to the lungs (i.e., for pulmonary delivery). Delivery of drugs (especially proteins) to the lungs has always been the subject of important research, such as, for example, Bodier-Montagutelli, E., et al., 2018, Designing inhaled protein therapeutics for topical lung delivery: what are the next steps? , Expert Opinion on Drug Delivery, 15 (8): 729-736; and Labiris, NR, et al., 2003, Pulmonary Drug Delivery. Part II: The role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications, Br J Clin Pharmacol, 56: 600-612, each of which is incorporated herein by reference in its entirety.

[0223] In certain embodiments, the polypeptides, peptides, or nucleic acids described herein can be used for local administration to the lungs. For example, in certain embodiments, the polypeptides, peptides, or nucleic acids described herein can be administered directly or locally to the lungs via intratracheal, intranasal, or inhalation administration.

[0224] In certain embodiments, the peptides or polypeptides described herein can be formulated for intratracheal administration, intranasal administration, or inhalation administration to a subject in need thereof. In certain embodiments, the polypeptides can be formulated for administration as an aerosol, inhaler, or nebulizer. In certain embodiments, the polypeptides can be formulated as a dry powder for administration to the lungs by aerosolization, or as a liquid for administration to the lungs by nebulization.

[0225] In certain embodiments, the polypeptides described herein can be used to modulate GRP78 activity in a subject in need thereof; including inducing apoptosis of alveolar macrophages (AMs) or other immune cells; treating, improving or preventing lung inflammation in a subject in need thereof; treating, improving or preventing lung diseases or conditions associated with lung inflammation in a subject in need thereof; treating, improving or preventing chronic obstructive pulmonary disease (COPD) or emphysema in a subject in need thereof; treating, improving or preventing asthma in a subject in need thereof; treating, improving or preventing acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof; treating, improving or preventing pulmonary fibrosis in a subject in need thereof; or any combination thereof.

[0226] In certain embodiments, it is contemplated that peptides or polypeptides as described herein may be optionally covalently or non-covalently conjugated or compounded (e.g., optionally via a biodegradable linker) with one or more additional agents for preventing or reducing lung inflammation in a subject in need thereof, or may be optionally used in combination, simultaneously or sequentially with the one or more additional agents. In view of the teachings herein, conventional agents for preventing or reducing lung inflammation will be known to those skilled in the art, and may include, for example, steroids.

[0227] For example, conventional medicaments for treating or controlling COPD can include the LAMA of LABA+ inhalation of combination inhalation, which can improve the lung function of COPD patients and reduce exacerbations. LABA (long-acting β2-agonist) and LAMA (long-acting muscarinic antagonist) are bronchodilators that provide symptom control. The example of LABA is salmeterol and formoterol. The example of LAMA is tiotropium bromide and glycopyrrolate. In most COPD, patients show a poor response to corticosteroids, and due to their side effect spectrum, anti-inflammatory inhaled corticosteroids (ICS) are not usually given alone. It is usually combined with LABA, such as formoterol+beclomethasone or salmeterol+fluticasone. Triple inhalation therapy can also be used, combining LABA+LAMA+ICS for very severe COPD and reducing COPD exacerbations. A newer anti-inflammatory agent, roflumilast, a phosphodiesterase 4 (PDE4) inhibitor, can improve lung function and reduce moderate and severe exacerbations. However, this drug is associated with major dose-limiting side effects (see the GOLD guidelines or NICE guidelines available online).

[0228] In certain embodiments, provided herein is a peptide or polypeptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide, for example, for treating, ameliorating or preventing a disease or condition associated with macrophage-mediated inflammation in a subject in need thereof.

[0229] In another embodiment, the present invention provides a nucleic acid encoding a peptide or polypeptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or its GRP78-activating fragment. The nucleotide codons corresponding to a given amino acid are well known, and a skilled person, having considered the teachings herein, will be able to easily select an appropriate nucleic acid to encode a given polypeptide. In certain embodiments, the nucleic acid sequence can be selected so that the coding region uses codons optimized for expression in a specific target organism (e.g., when a nucleic acid is used to produce a polypeptide in Escherichia coli, codons can be optimized for expression in Escherichia coli, or when a nucleic acid is to be administered to humans to promote in vivo expression of the polypeptide, it can be optimized for expression in humans). In certain embodiments, the nucleic acid can be an expressible nucleic acid (i.e., when introduced into or present in a given cell, the nucleic acid can be designed to result in expression of the polypeptide). In certain embodiments, the nucleic acid can be DNA or RNA. In certain embodiments, the nucleic acid can be a plasmid, an expression vector, or mRNA (which, in certain embodiments, can include sequences suitable for translation in the target cell, such as a start codon, a poly-A tail, an RBS sequence, etc.), with appropriate upstream and / or downstream sequences such that translation, or transcription and translation, of the nucleic acid can occur once the nucleic acid is introduced into the cell, thereby providing the polypeptide. In embodiments where the nucleic acid is to be introduced into lung cells of a subject for expression of the polypeptide therein, it is contemplated that, in certain embodiments, the polypeptide encoded by the nucleic acid can include a signal peptide sequence such that the polypeptide is secreted.

[0230] Suitable expression vector technologies for overexpressing a specific polypeptide or introducing a specific polypeptide into a cell are known in the art (see, for example, Molecular Cloning: A Laboratory Manual (4th Ed.), 2012, Cold Spring Harbor Laboratory Press). As known to those skilled in the art, the nucleotide sequence for expressing a specific polypeptide can encode or include features as described in "Genes VII", Lewin, B. Oxford University Press (2000) or "Molecular Cloning: A Laboratory Manual", Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition (2001). The nucleotide sequence encoding the specific polypeptide can be incorporated into a suitable vector, such as a commercially available vector. Standard molecular biology techniques such as those described in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)) can also be used to construct or modify the vector separately. Those skilled in the art will recognize that the vector can include a nucleotide sequence encoding the desired elements, which are operably linked to the nucleotide sequence encoding the polypeptide. Such nucleotide sequences encoding the desired elements may include transcriptional promoters (e.g., constitutive or inducible promoters), transcriptional enhancers, transcriptional terminators, and / or origins of replication. The selection of an appropriate vector may depend on several factors, including but not limited to the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational control elements desired, the desired expression level, the desired copy number, whether chromosomal integration is desired, the type of selection process desired, or the host cell or host range to be transformed.

[0231] In the embodiment that described nucleic acid will be introduced into experimenter or cell to produce described polypeptide therein (for example, in the manufacture of polypeptide, or for external or in vivo therapeutic application), it is contemplated that described nucleic acid can be compound with suitable nucleic acid delivery vector or transfection reagent that is suitable for introducing nucleic acid into cell.In some embodiments, described nucleic acid can be incorporated in virus to be delivered to cell, and described nucleic acid may or may not be integrated into the genome of cell.Considering that those skilled in the art of this paper instruction will know various delivery vectors, transfection reagent and / or virus delivery construct, they can be selected to deliver nucleic acid as herein described to given cell or to the experimenter in need thereof.

[0232] In another embodiment, provided herein is a nucleic acid sequence that is fully or partially complementary to any nucleic acid sequence described herein.

[0233] As used herein, percent identity (%) or % sequence identity relative to a particular sequence or a particular portion thereof can be defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a target sequence (or a specified portion thereof), after the sequences are aligned and gaps are introduced (if necessary) to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0 with search parameters set to default values ​​(Altschul et al., J. Mol. Biol. (1990) 215: 403-410; website: blast.wustl.edu / blast / README.html). For example, the % identity value can be determined by dividing the number of matching identical nucleotides or amino acids by the length of the sequence for which the percent identity is reported. The percent amino acid sequence similarity (%) can be determined by the same calculation used to determine % amino acid sequence identity, but in addition to identical amino acids in the calculation, conservative amino acid substitutions can also be included, for example. Sequence identity % can be calculated using an oligonucleotide or amino acid alignment algorithm such as, for example, BLAST (GenBank; using default parameters).

[0234] In another embodiment, provided herein is a composition comprising:

[0235] A polypeptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide; and

[0236] a pharmaceutically acceptable carrier, diluent or excipient;

[0237] The composition is formulated for administration to the lungs of a subject.

[0238] In certain embodiments, the polypeptide or expressible nucleic acid may comprise a polypeptide or expressible nucleic acid as described above or elsewhere herein.

[0239] In certain embodiments, pharmaceutically acceptable carrier, diluent or excipient may include any suitable carrier, diluent or excipient known to those skilled in the art after considering the teachings herein. The example of a pharmaceutically acceptable excipient may include, but is not limited to, cellulose derivatives, sucrose and starch. Those skilled in the art will recognize that a pharmaceutically acceptable excipient may include suitable fillers, adhesives, lubricants, buffers, glidants, dispersants and / or dispersants well known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2006)). Examples of pharmaceutically acceptable carriers, diluents and excipients can be found in, for example, Remington's Pharmaceutical Sciences (2000- 20th edition) and the U.S. Pharmacopeia published in 1999: National Formulary (USP 24NF19). Those skilled in the art will understand that suitable pharmaceutically acceptable carriers, diluents and excipients for use in the lungs of subjects in need thereof will be formulated into polypeptides and / or nucleic acids as described herein after considering the teachings herein. In certain embodiments, the polypeptides and / or nucleic acids and / or compositions described herein can be formulated with a propellant or carrier gas, which may or may not be pressurized, as a pharmaceutically acceptable carrier.

[0240] In certain embodiments, aerosol delivery of macromolecules such as proteins or peptides can be given by devices such as dry powder inhalers (DPI) or nebulizers. Excipients commonly used for such macromolecules may include any one or more of surfactants, saccharides (e.g., sucrose, trehalose) and / or polyols (e.g., PEG). Amino acids (e.g., glycine, lysine) are typically used as stabilizers. See, for example, Expert Opin Drug Deliv (2018) 15: 729-736 and Adv Drug Deliv Rev (2015), 93: 79-94, the entire contents of which are incorporated herein by reference.

[0241] In certain embodiments, the polypeptides, nucleic acids, and / or compositions as described herein can be formulated for pulmonary administration to a subject in need thereof. Those skilled in the art considering the teachings herein will recognize a variety of suitable methods and techniques for formulating agents for pulmonary administration (i.e., for pulmonary delivery). The delivery of agents to the lungs and particularly proteins has been the subject of significant research, as described, for example, in Bodier-Montagutelli, E., et al., 2018, Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?, Expert Opinion on Drug Delivery, 15(8):729-736; Labiris, N.R., et al., 2003, Pulmonary Drug Delivery. Part II: The role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications, Br J Clin Pharmacol, 56:600-612; and Ibrahim, M., et al., 2015, Inhalaton drug delivery devices: technology update, Med Devices (Auckl), 8:131-9, each of which is incorporated herein by reference in its entirety. In certain embodiments, the polypeptides, nucleic acids, and / or compositions as described herein can be delivered to the lungs using a suitable drug delivery device. In certain embodiments, the drug delivery device can be in the form of a pulmonary device, such as an inhaler, nebulizer, aerosolizer, puffer, nasal spray, or other suitable delivery device for pulmonary administration. Examples of pulmonary delivery devices are described, for example, in US5983893, US6732732, US20070295332, US5007419, US4832015, US20040244794, US20100065048, US20030235555, US20050201951, and US20090000615, each of which is incorporated herein by reference in its entirety.

[0242] In certain embodiments, the compositions described herein can be formulated for intratracheal administration, intranasal administration, or inhalation administration to a subject in need thereof. In certain embodiments, the compositions can be formulated for administration as an aerosol, inhaler, or nebulizer. In certain embodiments, the compositions can be formulated as a dry powder for administration to the lungs by aerosolization, or as a liquid for administration to the lungs by atomization.

[0243] In certain embodiments, the compositions described herein can be used to modulate GRP78 activity in a subject in need thereof; to induce apoptosis of proinflammatory cells in a subject in need thereof; to induce apoptosis of alveolar macrophages (AMs); to reduce AM levels in a subject in need thereof; to treat, improve or prevent lung inflammation in a subject in need thereof; to treat, improve or prevent lung diseases or conditions associated with lung inflammation in a subject in need thereof; to treat, improve or prevent chronic obstructive pulmonary disease (COPD), chronic obstructive bronchitis or emphysema in a subject in need thereof; to treat, improve or prevent asthma in a subject in need thereof; to treat, improve or prevent acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof; to prevent or reduce excessive proliferation of type II (AE2) cells on the surface of the alveolar wall in a subject in need thereof; to treat, improve or prevent pulmonary fibrosis in a subject in need thereof; or any combination thereof.

[0244] In certain embodiments, proinflammatory cells may include any one or more of innate and / or adaptive immune cells, such as macrophages, neutrophils, T and B lymphocytes, NK cells, etc. In the lungs, macrophages may include alveolar macrophages, interstitial macrophages, or both.

[0245] In certain embodiments, the lung disease or condition associated with lung inflammation may include any one or more of COPD, idiopathic pulmonary fibrosis (IPF), ALI, ARDS, asthma, chronic bronchitis, emphysema, pneumonia, and the like.

[0246] In certain embodiments, it is contemplated that the compositions as described herein may optionally further comprise one or more additional agents, or may optionally be used in combination, simultaneously or sequentially with one or more additional agents for preventing or reducing lung inflammation in a subject in need thereof. Conventional agents for preventing or reducing lung inflammation will be known to those skilled in the art having considered the teachings herein, and may include, for example, steroids. Examples of conventional agents have been described above.

[0247] In yet another embodiment, provided herein is a pulmonary drug delivery device comprising a polypeptide or an expressible nucleic acid encoding the polypeptide, the polypeptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof. In certain embodiments, the pulmonary drug delivery device may contain or otherwise be loaded with a polypeptide, nucleic acid, or composition as described herein. In certain embodiments, the pulmonary drug delivery device may be configured to have a replaceable or non-replaceable cartridge loaded with a polypeptide, nucleic acid, or composition as described herein. As will be appreciated, the pulmonary drug delivery device may generally comprise any medical device suitable for administering a polypeptide, nucleic acid, or composition as described herein to the lungs of a subject in need thereof. Those skilled in the art, having considered the teachings herein, will appreciate various devices that can be configured to deliver the polypeptides, nucleic acids, and / or compositions described herein. In certain embodiments, the pulmonary drug delivery device may comprise, for example, an intratracheal drug delivery device, an intranasal drug delivery device, or an inhaled drug delivery device. Those skilled in the art, having considered the teachings herein, will appreciate various medical device designs suitable for administering an agent to the lungs (i.e., for pulmonary delivery of an active agent). Delivery of pharmaceutical agents, and in particular proteins, to the lungs has been the subject of significant research, as described, for example, in Bodier-Montagutelli, E., et al., 2018, Designing inhaled protein therapeutics for topical lung delivery: what are the next steps? , Expert Opinion on Drug Delivery, 15(8): 729-736; Labiris, NR, et al., 2003, Pulmonary Drug Delivery. Part II: The role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications, Br J Clin Pharmacol, 56: 600-612; and Ibrahim, M., et al., 2015, Inhalation drug delivery devices: technology update, Med Devices (Aukl), 8: 131-9, each of which is incorporated herein by reference in its entirety. In certain embodiments, the pulmonary drug delivery device may comprise any suitable drug delivery device for delivery to the lungs.As non-limiting example, in some embodiments, the pulmonary drug delivery device can include aerosols, nasal sprays, inhalers, puffs, nebulizers or another delivery device suitable for each pulmonary administration. For example, the example of pulmonary delivery device is described in US5983893, US6732732, US20070295332, US5007419, US4832015, US20040244794, US20100065048, US20030235555, US20050201951 and US20090000615, which are each incorporated herein by reference in their entirety. In some embodiments, it is contemplated that, for example, the pulmonary drug delivery device can include aerosols and the polypeptide or nucleic acid can be formulated as dry powder, or the pulmonary drug delivery device can include nebulizers and the polypeptide or nucleic acid can be formulated as liquid. In certain embodiments, the pulmonary drug delivery device can comprise a nebulizer, a metered dose inhaler (MDI), or a dry powder inhaler (DPI) loaded with a correspondingly formulated polypeptide, nucleic acid, or composition described herein.

[0248] In certain embodiments, it is contemplated that a pulmonary drug delivery device as described herein may optionally further comprise one or more additional agents, or may optionally be used in combination with one or more additional agents, simultaneously or sequentially, for preventing or reducing lung inflammation in a subject in need thereof. Conventional agents for preventing or reducing lung inflammation will be known to those skilled in the art having considered the teachings herein, and may include, for example, steroids. Examples of conventional agents have been described above.

[0249] In certain embodiments, for example, the pulmonary drug delivery devices described herein can be used to modulate GRP78 activity in a subject in need thereof; induce apoptosis of proinflammatory cells in a subject in need thereof; induce apoptosis of alveolar macrophages (AMs); reduce AM levels in a subject in need thereof; treat, improve or prevent lung inflammation in a subject in need thereof; treat, improve or prevent a lung disease or condition associated with lung inflammation in a subject in need thereof; treat, improve or prevent chronic obstructive pulmonary disease (COPD), chronic obstructive bronchitis or emphysema in a subject in need thereof; treat, improve or prevent asthma in a subject in need thereof; treat, improve or prevent acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof; prevent or reduce excessive proliferation of type II (AE2) cells on the surface of the alveolar wall in a subject in need thereof; or treat, improve or prevent pulmonary fibrosis in a subject in need thereof, or a combination thereof.

[0250] Methods and uses for treating diseases or conditions associated with inflammation

[0251] For example, the present invention also provides uses and methods for treating, improving or preventing diseases or conditions associated with inflammation and particularly pulmonary inflammation. In certain embodiments, such uses and methods can utilize one or more of the polypeptides, nucleic acids, compositions and / or pulmonary drug delivery devices as described herein, such as those described above.

[0252] In some embodiments of the pulmonary administration or delivery of a peptide, polypeptide, nucleic acid and / or composition as described herein, the peptide or polypeptide or nucleic acid or composition can be delivered by, for example, intratracheal administration, intranasal administration or inhalation (e.g., oral inhalation) administration or delivery. In some embodiments, for example, the peptide, polypeptide, nucleic acid or composition can be administered as an aerosol, inhaler or nebulizer. In some embodiments, the peptide or polypeptide can be formulated as a dry powder and administered to the lungs by aerosolization, or can be formulated as a liquid and administered to the lungs by atomization. In some embodiments, a pulmonary drug delivery device as described herein can be used for administration. It has been considered that those skilled in the art will understand that various methods and techniques suitable for pulmonary administration (i.e., for pulmonary delivery) will be understood. Delivery of medicaments and particularly proteins to the lungs has always been the subject of important research, as for example Bodier-Montagutelli, E., et al., 2018, Designing inhaled protein therapeutics for topical lung delivery: what are thenext steps? , Expert Opinion on Drug Delivery, 15(8):729-736;Labiris, NR, et al., 2003, Pulmonary Drug Delivery. Part II: The role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications, Br J Clin Pharmacol, 56:600-612; and Ibrahim M., et al., 2015, Inhalation drug delivery devices: technology update, Med Devices (Auckl), 8:131-9, each of which is incorporated herein by reference in its entirety. In certain embodiments, any suitable pulmonary drug delivery device for administration to the lungs can be used to perform administration of the polypeptides, nucleic acids and / or compositions described herein as part of the methods described herein. In certain embodiments, the drug delivery device can take the form of a pulmonary device, such as an inhaler, nebulizer, aerosol, puff, nasal spray, or other suitable delivery device for administration to the lungs.Examples of pulmonary delivery devices are described, for example, in US5983893, US6732732, US20070295332, US5007419, US4832015, US20040244794, US20100065048, US20030235555, US20050201951, and US20090000615, each of which is herein incorporated by reference in its entirety.

[0253] For simplicity, the term polypeptide is generally used herein to describe any protein, polypeptide, peptide or other amino acid sequence of generally any length. The term polypeptide can be understood to refer to a protein, polypeptide or peptide, depending on the length of the amino acid sequence used in a particular embodiment and / or application. For example, in embodiments where the polypeptide is less than about 30 amino acids in length, the polypeptide can be considered a peptide. Unless otherwise indicated, the term polypeptide as used herein is intended to include peptides, polypeptides and proteins.

[0254] In certain embodiments where a nucleic acid encoding a polypeptide is to be administered to the lungs of a subject, the nucleic acid can be delivered by, for example, intratracheal administration, intranasal administration, or oral inhalation. In certain embodiments, the nucleic acid can be administered as an aerosol, inhaler, or nebulizer. In certain embodiments, the nucleic acid can be formulated as a dry powder and administered to the lungs by aerosolization, or can be formulated as a liquid and administered to the lungs by atomization. In certain embodiments, a pulmonary drug delivery device as described herein can be used for the administration. A person skilled in the art, having considered the teachings herein, will understand various suitable methods and techniques for administering to the lungs (i.e., for pulmonary delivery). In certain embodiments where a nucleic acid is to be administered to a subject, the nucleic acid can be introduced into a subject or cell to produce the polypeptide encoded therein. In certain embodiments, the nucleic acid can comprise an expression vector or expression cassette. In certain embodiments, the nucleic acid can comprise a DNA vector. In certain embodiments, it is contemplated that the nucleic acid can be compounded with a suitable nucleic acid delivery vector or transfection reagent suitable for introducing the nucleic acid into a cell. In certain embodiments, the nucleic acid can be incorporated into a virus for delivery to a cell, and the nucleic acid may or may not be integrated into the genome of the cell. Those skilled in the art who have considered the teachings herein will be aware of various delivery vectors, transfection reagents and / or viral delivery constructs that can be selected to deliver the nucleic acids described herein to a given cell or subject in need thereof. See, for example, Gomes et al., 2017, Expert Opin Drug Deliv., 2017, 14(3):319-330, the entire contents of which are incorporated herein by reference.

[0255] As described in detail herein, the inventors have now developed methods for treating inflammation, such as lung inflammation, that are derived from and / or based on Isthmin 1 (ISM1), a secreted protein that studies described herein demonstrate plays a role in inhibiting, preventing, and / or resolving inflammation, particularly inflammation of the lungs. In the studies detailed below, supplementation of the lungs with exogenous recombinant ISM1 protein (rISM1) suppressed the lung inflammation phenotype in ISM1-deficient lungs, and the results indicate that administration of ISM1 may help resolve inflammation by inducing apoptosis in alveolar macrophages. The results further suggest that ISM1 may play an important role in inhibiting and / or resolving sterile lung inflammation and / or inflammation triggered by infection and / or injury.

[0256] Thus, in one embodiment, provided herein is a method for modulating GRP78 activity in a subject in need thereof, the method comprising:

[0257] A polypeptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof is administered to the lungs of a subject in need thereof, or an expressible nucleic acid encoding the polypeptide.

[0258] In certain embodiments, regulating GRP78 activity can include administering a polypeptide, nucleic acid or composition as described herein to the subject, thereby triggering GRP78 activity (providing a pro-apoptotic effect) and thus alleviating the subject's inflammation. Typically, regulating GRP78 activity can include, when the pro-apoptotic activity level of GRP78 in the subject is too low to fully suppress, prevent and / or subside the inflammation and particularly lung inflammation of the subject, increasing the GRP78 activity level in the subject (i.e., increasing the pro-apoptotic effect caused by GRP78 binding). In certain embodiments, regulating GRP78 activity can include, when the GRP78 level in the subject is too low to induce sufficient alveolar macrophage apoptosis to alleviate inflammatory conditions, increasing the GRP78 level in the subject. In certain embodiments, when treating a lung disease or illness, mentioning that GRP78 activity level can include, for example, the activity level of GRP78 in the lung tissue or lung cells (such as the alveolar macrophages (AM) of the subject). It will be understood that, in certain embodiments, references herein to GRP78 activity levels or GRP78 activity may be understood as references to the level or activity of GRP78 in transmitting signals into cells following ligand binding, which may provide a pro-apoptotic effect.

[0259] A review of GRP78 can be found in Ni, et al., Biochem J., 2011, 434(2): 181-188, which is incorporated herein by reference in its entirety.

[0260] In certain embodiments, it is expected that ISM1 may favor or more selectively kill or target cells with high csGRP78 levels. Healthy cells under normal conditions should have relatively low levels or no csGRP78. GRP78 expression levels increase in cells under stress, and it is expected that cells containing high levels of csGRP78 may respond to ISM1 and be triggered to apoptosis. Therefore, stressed cells may be the preferred targets of extracellular ISM1 (see also Chen et al., Cell Death & Differentiation, 21(5):797-810, 2014, incorporated herein by reference). Without wishing to be bound by theory, it is expected that cells with increased csGRP78 levels will respond more strongly to ISM1-induced apoptotic signals than healthy cells, which in certain embodiments can reduce side effects.

[0261] In certain embodiments, the polypeptide or nucleic acid can be administered to the subject's lungs by intratracheal administration, intranasal administration or oral inhalation administration. In certain embodiments, the polypeptide or nucleic acid or composition can be administered as an aerosol, inhaler or nebulizer. In certain embodiments, the polypeptide, nucleic acid or composition can be formulated as a dry powder and by aerosolization to administer to the lungs, or can be formulated as a liquid and by atomization to administer to the lungs. In certain embodiments, such as to carry out the administration using a pulmonary drug delivery device as described herein. It has been considered that those skilled in the art who have taught herein will understand the various suitable methods and techniques for administering to the lungs (i.e., for pulmonary delivery). Delivery of medicaments and particularly proteins to the lungs has always been the subject of important research, such as, for example, Bodier-Montagutelli, E., et al., 2018, Designing inhaled protein therapeutics for topical lung delivery: what are the next steps? , Expert Opinion on Drug Delivery, 15(8): 729-736; and Labiris, NR, et al., 2003, Pulmonary Drug Delivery. Part II: The role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications, Br J Clin Pharmacol, 56: 600-612, each of which is incorporated herein by reference in its entirety.

[0262] In yet another embodiment, provided herein is a method for inducing apoptosis of proinflammatory cells or alveolar macrophages (AMs) in a subject in need thereof, or for reducing AM levels in a subject in need thereof, the method comprising:

[0263] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the lungs of the subject in need thereof, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0264] In certain embodiments, inducing apoptosis of alveolar macrophages (AM) may comprise administering to the subject a polypeptide, nucleic acid or composition as described herein, thereby triggering at least some alveolar macrophages (AM) in the lungs of the subject to undergo apoptosis. In certain embodiments, for example, a spirometer may be used to non-invasively measure decreased lung function. High inflammation may indirectly indicate high AM, which may account for approximately 95% of airspace immune cells. High inflammation may damage the lungs and reduce their function. In a clinical setting, when lung condition declines and is accompanied by hypoxemia and dyspnea, etc., clinicians may administer anti-inflammatory drugs. Therefore, in certain embodiments, the polypeptide or nucleic acid may be administered when a decline in lung function is observed, when high inflammation is observed, and / or when it is determined that the level of AM apoptosis is low.

[0265] In certain embodiments, inducing apoptosis in proinflammatory cells can include administering a polypeptide, nucleic acid, or composition as described herein to the subject, thereby triggering at least some proinflammatory cells in the subject's lung to undergo apoptosis. Proinflammatory cells can include AMs, neutrophils, interstitial macrophages, T and B cells, NK cells, and the like. In certain embodiments, ISM1 can be advantageous for selectively targeting proinflammatory cells, such as AMs that carry high levels of csGRP78. For example, in the presence of cigarette smoke, most AMs may be stress-activated and have high levels of csGRP78, and thus may become targets of ISM1.

[0266] In certain embodiments, reducing the level of alveolar macrophages (AM) in a subject can include administering to the subject a polypeptide, nucleic acid, or composition as described herein, thereby triggering a reduction in the level of alveolar macrophages (AM) in the lungs of the subject. In certain embodiments, it is contemplated that administration is performed when the AM level in the subject is determined to be elevated compared to a healthy control or a disease control with milder symptoms. In certain embodiments, it is contemplated that the AM level can be determined from a sample of bronchoalveolar lavage fluid (BALF) that can be collected from the subject, and that the AM can be measured in the BALF. Alternatively or additionally, lung function can be monitored as an indirect measurement of AM levels.

[0267] In some embodiments, the polypeptide or nucleic acid can be administered by intratracheal administration, intranasal administration or oral inhalation administration to experimenter's lung.In some embodiments, the polypeptide or nucleic acid or or composition can be used as aerosol, inhaler or nebulizer administration.In some embodiments, the polypeptide, nucleic acid or composition can be formulated as dry powder and by aerosolization to pulmonary administration, or can be formulated as liquid and by atomization to pulmonary administration.In some embodiments, pulmonary drug delivery device as described herein can be used to carry out the administration.Considering that those skilled in the art of this paper teaching will understand the various applicable methods and techniques for pulmonary administration (i.e., for pulmonary delivery).

[0268] In yet another embodiment, provided herein is a method for treating, ameliorating or preventing lung inflammation in a subject in need thereof, the method comprising:

[0269] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the lungs of the subject in need thereof, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0270] In certain embodiments, lung inflammation can generally include any inflammatory state or condition of the lungs, including but not limited to sterile lung inflammation and / or inflammation caused by infection and / or injury. In certain embodiments, lung inflammation can be lung inflammation associated with COPD, emphysema, bronchitis, ALI, ARDS, IPF, pneumonia, etc. In certain embodiments, lung inflammation can be measured by measuring decreased lung function and / or hypoxemia, and administration is performed when inflammation or elevated inflammation is identified.

[0271] In some embodiments, the polypeptide or nucleic acid can be administered to the lungs of the experimenter by intratracheal administration, intranasal administration or oral inhalation. In some embodiments, the polypeptide or nucleic acid or composition can be used as an aerosol, inhaler or nebulizer administration. In some embodiments, the polypeptide, nucleic acid or composition can be formulated as a dry powder and by aerosolization to the lungs, or can be formulated as a liquid and by atomization to the lungs. In some embodiments, a pulmonary drug delivery device as described herein can be used to carry out the administration. It has been considered that those skilled in the art of this paper will understand the various applicable methods and techniques for pulmonary administration (i.e., for pulmonary delivery).

[0272] In yet another embodiment, provided herein is a method for treating, ameliorating, or preventing a pulmonary disease or disorder associated with pulmonary inflammation in a subject in need thereof, the method comprising:

[0273] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the lungs of the subject in need thereof, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0274] In certain embodiments, a pulmonary disease or condition associated with pulmonary inflammation can generally include any disease, disorder, state, or condition of the lung caused by or accompanied by inflammation. Examples can include, but are not limited to, chronic obstructive pulmonary disease (COPD), chronic obstructive bronchitis, asthma, emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pulmonary fibrosis (such as, for example, idiopathic pulmonary fibrosis), or any combination thereof.

[0275] In some embodiments, the polypeptide or nucleic acid can be administered to the lungs of the experimenter by intratracheal administration, intranasal administration or oral inhalation. In some embodiments, the polypeptide or nucleic acid may be administered as an aerosol, inhaler or nebulizer. In some embodiments, the polypeptide, nucleic acid or compositions can be formulated as a dry powder and administered to the lungs by aerosolization, or can be formulated as a liquid and administered to the lungs by atomization. In some embodiments, a pulmonary drug delivery device as described herein can be used to carry out the administration. It has been considered that those skilled in the art of this paper will understand the various suitable methods and techniques for administering to the lungs (i.e., for pulmonary delivery).

[0276] In another embodiment, provided herein is a method for treating, ameliorating or preventing chronic obstructive pulmonary disease (COPD), chronic obstructive bronchitis, asthma or emphysema in a subject in need thereof, the method comprising:

[0277] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the lungs of the subject in need thereof, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0278] In yet another embodiment, provided herein is a method for treating, ameliorating or preventing acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof, the method comprising:

[0279] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the lungs of the subject in need thereof, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0280] In another embodiment, provided herein is a method for preventing or reducing excessive proliferation of type II (AE2) cells on the surface of alveolar walls in a subject in need thereof, the method comprising:

[0281] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the subject in need thereof, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0282] In yet another embodiment, provided herein is a method for treating, improving or preventing pulmonary fibrosis in a subject in need thereof, the method comprising:

[0283] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the lungs of the subject in need thereof, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0284] In further embodiments of any one or more of the above methods, the polypeptide or nucleic acid may comprise any polypeptide or nucleic acid as described herein, such as those described above. The previous section provides a broad description and examples of suitable polypeptides and nucleic acids. In certain embodiments, the polypeptide and / or nucleic acid may be provided or formulated in a composition, as described in detail above. In certain embodiments, the polypeptide, nucleic acid and / or composition may be provided in a pulmonary drug delivery device, as described in detail above.

[0285] In further embodiments of any one or more of the above methods, the method may further comprise the step of administering to the subject an agent for preventing or reducing lung inflammation in combination with, simultaneously with, or sequentially with the polypeptide or nucleic acid.

[0286] In a further embodiment of any one or more of the above methods, the method may further comprise the step of administering to the subject one or more additional agents for preventing or reducing lung inflammation. In certain embodiments, it is contemplated that the polypeptides, nucleic acids, compositions and / or pulmonary drug delivery devices as described herein may optionally further comprise one or more additional agents for preventing or reducing lung inflammation, or may optionally be combined with one or more additional agents for preventing or reducing lung inflammation, simultaneously or sequentially in a subject in need thereof. Conventional agents for preventing or reducing lung inflammation are well known to those skilled in the art having considered the teachings herein, and may include, for example, steroids. Examples of conventional agents have been described above.

[0287] In certain embodiments of any one or more of the above methods, the method may further comprise the following steps:

[0288] determining the level of ISM1 in the subject; determining the level of GRP78 protein in the subject; determining the level of alveolar macrophages (AMs) in the subject; determining the level of sputum in the subject; determining the level of decreased lung function in the subject; or any combination thereof; and

[0289] The administering step is performed or repeated if: a decreased level of ISM1 is determined in the subject relative to a healthy control level or relative to a low severity disease control level; an increased level of GRP78 protein is determined in the subject relative to a healthy control level or relative to a low severity disease control level; an increased level of alveolar macrophages (AMs) is determined in the subject relative to a healthy control level or relative to a low severity disease control level; an increased level of inflammation is determined in the subject relative to a healthy control level or relative to a low severity disease control level; an increased level of decreased lung function is determined in the subject relative to a healthy control level or relative to a low severity disease control level; or any combination thereof.

[0290] In certain embodiments, the level of ISM1 in a subject can be determined using any suitable technique known to those skilled in the art in light of the teachings herein. For example, in certain embodiments, the level of ISM1 can be determined by ELISA testing of a blood, serum, sputum, or BALF sample from a subject. In certain embodiments, the level of ISM1 can be determined by, for example, mass spectrometry quantification in a blood or sputum sample. In certain embodiments, the level of ISM1 can be determined by immunocytochemistry and / or immunofluorescence staining of an isolated AM sample (e.g., from BALF). In certain embodiments, the level of ISM1 can comprise the level of ISM1 in lung tissue of a subject, the level of ISM1 in lung tissue of a subject excised, or the level of ISM1 in alveolar macrophages (AM) of a subject. In certain embodiments, the determined level of ISM1 for a subject can be compared to a healthy control level (i.e., the level or range of ISM1 observed for a group of healthy control subjects) and / or can be compared to a low severity disease control level (i.e., the level or range of ISM1 observed for a disease control group of subjects with a disease or condition but with a lower degree of severity). If the subject's ISM1 level is determined to be lower than a healthy control level or a low severity disease control level, the subject can be treated or additionally treated with a polypeptide, nucleic acid, or composition as described herein. In certain embodiments, a reduced ISM1 level relative to a control level can be identified as a level that is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% compared to the control level.

[0291] In certain embodiments, subjects with low ISM1 levels can be identified as potentially particularly susceptible to treatment with ISM1, or a fragment or derivative thereof. However, it should be recognized that, in certain embodiments, even in subjects with relatively high levels of endogenous ISM1, if inflammation persists, the ISM1 levels may still not be high enough to overcome the severe inflammation and it may be advisable to administer additional exogenous ISM1, or a fragment or derivative thereof, as described herein.

[0292] In certain embodiments, any suitable technique known to those skilled in the art taking into account the teachings herein can be used to determine the GRP78 protein level of a subject. In certain embodiments, the GRP78 protein level can be total cellular GRP78 level, csGRP78 protein level, or both. In certain embodiments, for example, a fluorescent probe (such as a peptide ligand) can be used in fluorescence imaging, or a radioactive isotope-labeled probe can be used, such as in PET imaging, to determine the GRP78 level. In certain embodiments, such probes can be used in living organisms such as mammals or human subjects. In certain embodiments, the GRP78 protein level can include the GRP78 protein level in the subject's lung tissue, the GRP78 protein level in the excised subject's lung tissue, or the GRP78 protein level in the subject's alveolar macrophages (AM). In certain embodiments, the GRP78 protein level can be determined by ELISA, immunostaining, Western blotting, or other suitable techniques. In certain embodiments, the GRP78 protein level can be determined from a lung tissue sample obtained from a biopsy. In certain embodiments, the determined GRP78 protein level of the subject can be compared to a healthy control level (i.e., a GRP78 protein level or range observed from a healthy control subject group), and / or can be compared to a low severity disease control level (i.e., a GRP78 protein level or range observed from a disease control group with a disease or condition but with a lower severity). If the subject's GRP78 protein level is determined to be higher than the healthy control level or the low severity disease control level, the subject can be treated or additionally treated using a polypeptide, nucleic acid, or composition as described herein. In certain embodiments, an increase in GRP78 protein level relative to a control level can be identified as an increase of at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% compared to the control level. In certain embodiments, the GRP78 protein level can be the GRP78 protein level in the subject's alveolar macrophages. However, it will be appreciated that in cases where a subject has inflammation, it is contemplated that, in certain embodiments, treatment with ISM1, or a fragment or derivative thereof, as described herein, may be desirable regardless of whether elevated GRP78 levels are determined. Because cells with elevated csGRP78 levels are targeted by ISM1-induced apoptosis, even if only a subset of a subject's AMs have elevated csGRP78 levels, it is contemplated that eliminating these cells by triggering cell death may still be beneficial.

[0293] In certain embodiments, any suitable technology known to those skilled in the art considering this paper's teachings can be used to determine the alveolar macrophage (AM) level of a subject. For example, in certain embodiments, induced sputum can be used to determine the AM level. In certain embodiments, for example, the induced sputum program known to those skilled in the art considering this paper's teachings can be used to determine the content of sputum, including the cytokine levels of alveolar macrophages and / or sputum. In certain embodiments, for example, alveolar macrophage (AM) levels can include the alveolar macrophage (AM) levels in the subject's lung tissue or the alveolar macrophage (AM) levels in the subject's lung tissue excised. In certain embodiments, AM levels can be determined by collecting BALF and counting cell numbers. In certain embodiments, the alveolar macrophage (AM) levels of the determined subject can be compared with healthy control levels (i.e., alveolar macrophage (AM) levels or scope observed from healthy control subject groups), and / or can be compared with low severity disease control levels (i.e., alveolar macrophage (AM) levels or scope observed from disease control groups suffering from disease or illness but with lower severity). If it is determined that the alveolar macrophage (AM) level of the experimenter is higher than the alveolar macrophage (AM) level of the healthy control level or the alveolar macrophage (AM) level of the low severity disease control level, the experimenter can be treated or additionally treated using a polypeptide, nucleic acid or composition as described herein. In certain embodiments, the alveolar macrophage (AM) level increased relative to the control level can be identified as an increase of at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 100% compared to the control level.

[0294] In certain embodiments, administration may be initiated or repeated if the subject exhibits decreased lung function, dyspnea, hypoxemia, or another symptom of a lung disease or disorder associated with inflammation, such as COPD.

[0295] In certain embodiments, the level of inflammation in a subject can be determined using any suitable technique known to those skilled in the art, taking into account the teachings herein. In certain embodiments, for example, the level of inflammation can include the level of inflammation in the subject's lung tissue, the level of inflammation in the excised lung tissue of the subject. In certain embodiments, inflammation can be determined or indicated by fever (and its severity), the severity of symptoms such as hypoxemia, dyspnea, shortness of breath, cough, sputum production, white blood cell count, or other such inflammation measurements. In certain embodiments, inflammation can be determined or indicated by the number of immune cells, the level of proinflammatory cytokines such as TNF-α, NF-kB signaling levels, the level of proteases such as MMPs, or other such measurements. In certain embodiments, the determined level of inflammation in a subject can be compared to a healthy control level (i.e., the level or range of inflammation observed in a group of healthy control subjects), and / or can be compared to a low severity disease control level (i.e., the level or range of inflammation observed in a control group of subjects with a disease or condition but with a lower severity). If the subject's level of inflammation is determined to be higher than the healthy control level or the low severity disease control level, the subject can be treated or given additional treatment using a polypeptide, nucleic acid, or composition as described herein. In certain embodiments, elevated inflammation relative to a control level can be identified as a level that is increased by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% compared to the control level.

[0296] In certain embodiments, the level of decreased lung function in a subject can be determined using any suitable technique known to those skilled in the art taking into account the teachings herein. For example, spirometry can be used, or it can be determined by symptoms such as dyspnea, hypoxemia, etc. as shown above. In certain embodiments, the determined level of decreased lung function in the subject can be compared with a healthy control level (i.e., a level or range observed from a group of healthy control subjects), and / or can be compared with a low severity disease control level (i.e., a level or range observed from a disease control group with a disease or condition but with a lower severity). If it is determined that the subject's level of decreased lung function is higher than the healthy control level or the low severity disease control level, the subject can be treated or additionally treated using a polypeptide, nucleic acid, or composition as described herein. In certain embodiments, a level of decreased lung function relative to a control level that is elevated can be identified as a level of decreased lung function that is impaired by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 100% compared to the control level. For example, the clinical criterion for COPD is to have FEV1 / FVC<0.7 (Tiffeneau-Pinelli index), so by spirometry, a decrease in lung function is typically at least 30%, thereby determining whether COPD conditions are met under these criteria. However, for example, it is contemplated that early treatment may be the first choice for preventing the progression of COPD.

[0297] In yet another embodiment, provided herein is a method for treating, ameliorating or preventing a disease or disorder associated with macrophage-mediated inflammation in a subject in need thereof, the method comprising:

[0298] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the subject, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0299] In yet another embodiment, provided herein is a method for treating, ameliorating or preventing a pulmonary disease or disorder associated with pulmonary inflammation in a subject in need thereof, the method comprising:

[0300] A GRP78-activator is administered to the lungs of the subject.

[0301] In yet another embodiment, provided herein is a method for maintaining lung homeostasis and / or resolving lung inflammation and / or promoting lung repair with reduced remodeling in a subject in need thereof, the method comprising:

[0302] A polypeptide or an expressible nucleic acid encoding the polypeptide is administered to the lungs of the subject, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof.

[0303] In certain embodiments, the subject referred to herein can include any suitable subject in need of treatment. In certain embodiments, the subject can include a mammal. In certain embodiments, the subject can include a human.

[0304] In another embodiment, provided herein is a use of a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide, for modulating GRP78 activity in a subject in need thereof; for inducing apoptosis of proinflammatory cells in a subject in need thereof; for inducing apoptosis of alveolar macrophages (AMs) in a subject in need thereof; for reducing AM levels in a subject in need thereof; for treating, ameliorating or preventing lung inflammation in a subject in need thereof; for treating, ameliorating or preventing a lung disease or condition associated with lung inflammation in a subject in need thereof; for treating, ameliorating or preventing chronic obstructive pulmonary disease (COPD) in a subject in need thereof. OPD), chronic obstructive bronchitis or emphysema; for treating, improving or preventing asthma in a subject in need thereof; for treating, improving or preventing acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in a subject in need thereof; for preventing or reducing excessive proliferation of type II (AE2) cells on the surface of alveolar walls in a subject in need thereof; for treating, improving or preventing pulmonary fibrosis in a subject in need thereof; for treating, improving or preventing a disease or condition associated with macrophage-mediated inflammation in a subject in need thereof; for maintaining lung homeostasis and / or resolving lung inflammation and / or promoting lung repair with reduced remodeling in a subject in need thereof; or any combination thereof.

[0305] In some embodiments, the polypeptide or nucleic acid can be used for pulmonary administration to the experimenter. In some embodiments, the polypeptide or nucleic acid can be administered by intratracheal administration, intranasal administration or oral inhalation administration to the experimenter's pulmonary administration. In some embodiments, the polypeptide or nucleic acid or composition can be used as an aerosol, inhaler or nebulizer administration. In some embodiments, the polypeptide, nucleic acid or composition can be formulated as a dry powder and by aerosolization to pulmonary administration, or can be formulated as a liquid and by atomization to pulmonary administration. In some embodiments, pulmonary drug delivery device as described herein can be used to carry out the administration. It is considered that those skilled in the art of this paper teaching will understand the various suitable methods and techniques for pulmonary administration (i.e., for pulmonary delivery).

[0306] In some embodiments, the polypeptide or nucleic acid can be used in combination with an additional agent for preventing or reducing lung inflammation. In some embodiments, it is contemplated that polypeptide, nucleic acid, compositions and / or pulmonary drug delivery devices as described herein may optionally further comprise one or more additional agents for preventing or reducing lung inflammation, or may optionally be combined with one or more additional agents for preventing or reducing lung inflammation, simultaneously or sequentially in a subject in need thereof. Conventional agents for preventing or reducing lung inflammation are known to those skilled in the art who have considered this paper's teachings, and may include, for example, steroids. The example of conventional agents has been described above.

[0307] In yet another embodiment, provided herein is a use of a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an Isthmin 1 (ISM1) protein or a GRP78-activating fragment thereof, or an expressible nucleic acid encoding the polypeptide, in the manufacture of a medicament for modulating GRP78 activity in a subject in need thereof; for inducing apoptosis of alveolar macrophages (AMs) in a subject in need thereof; for treating, improving or preventing lung inflammation in a subject in need thereof; for treating, improving or preventing a lung disease or condition associated with lung inflammation in a subject in need thereof; for treating, improving or preventing chronic obstructive pulmonary disease in a subject in need thereof for treating, improving or preventing a disease or condition associated with macrophage-mediated inflammation in a subject in need thereof; for maintaining lung homeostasis and / or resolving lung inflammation and / or promoting lung repair with reduced remodeling in a subject in need thereof; or any combination thereof.

[0308] In certain embodiments, the polypeptides, nucleic acids, and / or compositions described herein can be administered to a subject in need thereof once, twice, or more times per day; once every 1, 2, 3, or more days; or as needed, for example, based on symptoms. In certain embodiments, for example, the polypeptides, nucleic acids, and / or compositions described herein can be administered via a nebulizer. In the following studies using a mouse model, intratracheal administration of 5 μg / dose once every 2 days was effective.

[0309] Methods for diagnosing and / or identifying subjects susceptible to treatment

[0310] In yet another embodiment, provided herein is a method of identifying a subject having or at risk of developing a pulmonary disease or condition associated with pulmonary inflammation, the method comprising:

[0311] determining the level of ISM1 in the subject; determining the level of GRP78 protein in the subject;

[0312] determining the level of alveolar macrophages (AMs) in a subject; determining the level of inflammation in a subject; or a combination thereof; and

[0313] The subject is identified as having, or being at risk for developing, a lung disease or condition associated with lung inflammation if: the level of ISM1 in the subject is determined to be reduced relative to the level of a healthy control, or relative to the level of a low severity disease control; the level of GRP78 protein in the subject is determined to be increased relative to the level of a healthy control, or relative to the level of a low severity disease control; the level of alveolar macrophages (AMs) in the subject is determined to be elevated relative to the level of a healthy control, or relative to the level of a low severity disease control; the level of inflammation in the subject is determined to be elevated relative to the level of a healthy control, or relative to the level of a low severity disease control; or any combination thereof.

[0314] In some embodiments of the above-mentioned method, the method may further include performing one or more conventional determinations or techniques to further confirm or support identifying the subject as suffering from a pulmonary disease or illness associated with pulmonary inflammation or having a risk of developing a pulmonary disease or illness associated with pulmonary inflammation. In some embodiments, for example, a total white blood cell count and / or differential count of blood may be used. In some embodiments, for example, conventional determinations or techniques may include measuring the main outcomes of pulmonary function (FEV1 / FVC, FRC, PEFR, etc.) and / or the St. George's Respiratory Questionnaire for COPD patients (SGRQ-C) for quality of life (cough, sputum, shortness of breath, wheezing, etc.) and / or exacerbation times.

[0315] In yet another embodiment, provided herein is a method of identifying a candidate subject for treatment using the treatment methods detailed herein, the method comprising:

[0316] determining the level of ISM1 in the subject; determining the level of GRP78 protein in the subject; determining the level of alveolar macrophages (AMs) in the subject; determining the level of inflammation in the subject; or any combination thereof; and

[0317] The subject is identified as a candidate for treatment if: the level of ISM1 in the subject is determined to be decreased relative to the healthy control level, or relative to the low severity disease control level; the level of GRP78 protein in the subject is determined to be increased relative to the healthy control level, or relative to the low severity disease control level; the level of alveolar macrophages (AMs) in the subject is determined to be elevated relative to the healthy control level, or relative to the low severity disease control level; the level of inflammation in the subject is elevated relative to the healthy control level, or relative to the low severity disease control level; or any combination thereof.

[0318] In certain embodiments, the method of identifying a subject as having a pulmonary disease or condition associated with pulmonary inflammation as described herein or as being at risk of developing a pulmonary disease or condition associated with pulmonary inflammation as described herein can further comprise the step of treating the pulmonary disease or condition associated with pulmonary inflammation in the identified subject. In certain embodiments, the step of treating the identified subject can comprise treating the subject using any of the therapeutic methods detailed herein.

[0319] In certain embodiments, the method of identifying a candidate subject for treatment using the methods detailed herein can further comprise the step of treating the identified subject. In certain embodiments, the step of treating the identified subject can comprise treating the subject using any of the methods detailed herein.

[0320] Example

[0321] In the following examples, the secreted protein ISM1 was removed from mice by a gene targeting approach, resulting in spontaneous lung inflammation and progressive emphysema with characteristics similar to those of human COPD. In addition, removal of ISM1 resulted in an enhanced inflammatory response to lipopolysaccharide (LPS)-induced acute lung injury (ALI). Furthermore, intratracheal supplementation (i.e., local administration to the lungs) of exogenous recombinant ISM1 protein (rISM1) suppressed the lung inflammation phenotype in ISM1-deficient lungs and cigarette smoke-induced COPD in mice. These studies suggest that ISM1 may contribute to the resolution of inflammation by inducing apoptosis of alveolar macrophages. These results suggest that ISM1 may be an inhibitor and / or pro-resolution mediator of lung inflammation and may play a role in suppressing and / or resolving sterile lung inflammation and / or inflammation triggered by environmental insults (such as cigarette smoke, infection, and / or injury). Therefore, ISM1 may represent a therapeutic agent and / or target for inflammatory lung diseases such as ALI and COPD.

[0322] Example 1: Isthmin 1-Protecting lung homeostasis and therapeutic effects on COPD

[0323] Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death. COPD is characterized by progressive and largely irreversible airway obstruction due to emphysema (destruction of the alveolar walls and enlargement of the alveoli) and chronic obstructive bronchitis of the small airways. 1 A significant increase in alveolar macrophages (AMs) has been widely implicated in the pathogenesis of COPD. 2 However, the molecular mechanisms and pathophysiology of COPD are poorly understood, and there has been a lack of drugs that can block or reduce the progression of COPD.

[0324] Pulmonary inflammation is integral to COPD pathogenesis, and alveolar macrophages (AMs), the most abundant lung-resident immune cells, are key effector cells of the disease. Disease severity in COPD patients is directly correlated with the accumulation of AMs, particularly in inflamed peripheral airways and alveolar spaces (Finkelstein, Fraser et al., 1995). These observations are supported by studies in mice, which exhibit complete protection against experimental COPD following targeted AM depletion (Ueno, Maeno et al., 2015) or knockout of the potent macrophage elastase MMP-12 (Hautamaki, Kobayashi et al., 1997). However, many COPD patients are resistant to corticosteroid treatment, and several anti-inflammatory agents are being explored for COPD treatment development (Vogelmeier, Criner et al., 2017).

[0325] In this study, the extracellular pro-apoptotic protein ISTHMIN 1 (ISM1) was developed and investigated as an anti-inflammatory agent and in maintaining lung homeostasis. rISM1 was designated as a therapeutic agent for COPD. In these studies, Ism1 knockout (Ism1 Δ / Δ ) mice exhibit increased AM and develop spontaneous chronic lung inflammation with progressive emphysema. Cell surface GRP78 (csGRP78), a high-affinity receptor for ISM1, is primarily present on AM and its level is significantly increased in Ism1 mice. Δ / Δ Ism1 is highly upregulated in mice with COPD induced by CS and in human COPD lungs. Δ / Δ Intratracheal delivery of recombinant ISM1 (rISM1)-depleted AMs blocked emphysema progression and restored lung function in COPD lungs induced by CS. Consistently, high ISM1 expression in COPD patients correlates with high AM apoptosis levels and low AM numbers. These results support the idea that ISM1 may be a protective agent of lung homeostasis and could provide therapeutic benefit in halting COPD progression. The data presented here suggest that Isthmin 1 may protect against COPD.

[0326] The following studies have shown that the secreted protein Isthmin 1 (ISM1) is an anti-inflammatory protein that can induce AM apoptosis through the cell surface GRP78 (csGRP78) receptor. Δ / Δ AM accumulation and spontaneous emphysema in mice. Our results indicate that csGRP78 is involved in the regulation of Ism1 Δ / Δ Ism1 is highly upregulated in AM of COPD lungs from mice, cigarette smoke-induced COPD mice, and humans. Δ / Δ Intratracheal delivery of recombinant ISM1-depleted AMs blocked emphysema and lung function decline in COPD mice. Consistently, ISM1 expression in human COPD lungs correlates with increased AM apoptosis. Thus, our results suggest that ISM1 is a protective agent of lung homeostasis and support the therapeutic use of targeting csGRP78 on AMs for COPD.

[0327] The lungs are constantly exposed to the external environment, and homeostasis is important for limiting immune responses and inflammation. CS is a major risk factor for COPD, currently the third leading cause of death, with an estimated cumulative lifetime risk of 25%. 3 Although bronchodilator medications can relieve patients' symptoms, they do not reduce COPD progression or mortality.

[0328] ISM1 has been previously identified as a secreted protein that inhibits angiogenesis and experimental cancer in mice by acting through csGRP78 and αvβ5 integrin on certain cells, such as some cancer cells and activated endothelial cells. 4-7 .

[0329] Recombinant ISM1 (rISM1) binds to αvβ5 integrin and activates caspase-8, or binds to csGRP78, where it is internalized and transported to mitochondria, inhibiting ATP production and triggering apoptosis. The Ism1 gene is present in vertebrates from fish to humans (Osório, Wu et al., 2014, Xiang et al., 2011); however, the physiological function of Ism1 remains unknown.

[0330] The studies described here demonstrate that ISM1 plays a key role in maintaining lung homeostasis in mice by regulating AM numbers through csGRP78-mediated apoptosis. The results indicate that pulmonary delivery of rISM1 effectively quenches lung inflammation by depleting AMs through apoptosis induction, leading to emphysematous obstruction and recovery of lung function in the CS-induced COPD mice studied. Correspondingly, ISM1 expression in the lungs of human COPD patients correlates with increased AM apoptosis. These results suggest that rISM1 may offer therapeutic approaches for COPD, for example, by targeting csGRP78 on AMs to suppress inflammation by inducing AM apoptosis and blocking lung tissue damage in emphysema.

[0331] Ism1 Δ / Δ Mice develop spontaneous emphysema in ambient air

[0332] In mice, Ism1 is expressed at the highest level in both fetal and adult lungs, nearly 30-fold higher than the second-highest-expressing organ (brain) and much higher than in other organs (Osório et al., 2014). To further investigate the physiological functions and roles of Ism1, CRISPR / Cas9 was used to generate Ism1 knockout (Ism1) in two different mouse strains, FVB / Ntac and C57BL / 6J. Δ / Δ ) mice( Figure 22 Ism1 in two genetic backgrounds. Δ / Δ The mice were viable, with no obvious morphological abnormalities or behavioral phenotypes, despite the presence of Ism1 + / Δ Compared with wild-type (WT) FVB / N mice, the litter size was smaller (data not shown). Histopathological examination of all major organs showed that Ism1 Δ / Δ Mouse lungs developed spontaneous emphysema in both genetic backgrounds ( Figure 1 A and Figure 5A–C) and progressive enlargement of alveolar spaces with age quantified by mean linear intercept (MLI) ( Figure 1 B and Figure 5 D). In Ism1 up to 9 months of age Δ / Δ No obvious pathology was observed in other major organs of the mice. These results indicate that ISM1 is important for murine lung homeostasis, consistent with its highest expression in the lung. For subsequent studies in this article, FVB / NTac Ism1 Δ / Δ Mouse. Whole-mount stereomicroscopy and fluorescent dye labeling of collagen and elastin showed that Ism1 Δ / Δ Significant air entrapment in the lungs of mice ( Figure 1 C) and deterioration of the overall alveolar network ( Figure 1 D). Ism1 was also visualized using a modified van Hoof-van Gieson (VVG) stain and detection of ruptured septa. Δ / Δ Loss of alveolar elastin fibers in mouse lungs ( Figure 5 E). In addition, Ism1 + / Δ Mice developed mild emphysema, which was observed in wild-type (WT) and Ism1 Δ / Δ There was only moderate MLI between mice ( Figure 1 E and F), indicating that Ism1 is a haploinsufficient gene in mice.

[0333] To determine whether ISM1 deficiency impairs lung physiology, a forced lung maneuver system (Buxco) was used in 2-month-old Ism1 Δ / Δ A series of lung function tests were performed on mice. Δ / Δ Mice exhibited increased total lung capacity (TLC) ( Figure 1 G), which is synonymous with the highly swollen lungs observed in COPD patients (Gagnon, Guenette et al., 2014). The increased volume compartment also shows Ism1 Δ / Δ The mouse lung is highly swollen, such as with functional residual capacity (FRC) ( Figure 1 H) and residual volume (RV) ( Figure 1 These changes are also reflected in pressure-volume measurements, whereby static and dynamic compliance (Cchord and Cdyn) are expressed in Ism1 Δ / Δ Mice increased ( Figure 1 J and K). Importantly, Ism1 Δ / Δ Mice showed lower forced expiratory volume (FEV 100 )( Figure 1 L) and having a FEV less than 0.7 100 / FVC mean (equivalent to the FEV1 / FVC index in humans with COPD) Figure 1 M,Ism1 Δ / Δ :0.63±0.05), 0.7 is the standard commonly used for COPD diagnosis in patients (Singh et al., 2019). Ism1 Δ / Δ Increased airway resistance (RI) in mice may be attributed to mucus hypersecretion and inflammatory changes in the airway wall, including hyperplasia and thickening of the airway epithelium ( Figure 1 N, Figure 5 F and G) (Barnes, 2016). Overall, these data suggest that Ism1 Δ / Δ The mice exhibited lung lesions similar to those seen in experimental emphysema / COPD in mouse models and in human COPD patients.

[0334] Figure 5 L and M show that Ism1 Δ / Δ The increased AMs in mouse lungs are not the result of abnormal embryonic lung development. AMs are formed by fetal liver mononuclear cells during embryonic development, which migrate to the lungs and differentiate into AMs during the perinatal and postnatal periods. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine for AM formation and differentiation. The results showed that when GM-CSF was at its highest level in the developing lungs, there was no significant difference in GM-CSF levels between P1 and P7. Therefore, the higher number of AMs in adult lungs (8 weeks old) is not the result of changes in AM formation during lung development. Figure 5 L and M show P1 FVB / NTac WT and Ism1 Δ / Δ Representative immunofluorescence staining of GM-CSF (red) and nuclei (DAPI; blue) in mouse lungs (left) (n=4 mice / group. P1 FVB / NTac WT and Ism1 Δ / Δ Western blot of GM-CSF in mouse lungs with β-actin as a loading control ( Figure 5 M) and fold change (AU, arbitrary units). n = 8-9 mice / group. Data are mean ± sem and analyzed by unpaired two-tailed Student's t-test).

[0335] In adult mouse lungs (8-weeks old), Ism1 Δ / Δ Using a multiplex ELISA assay, we found that Ism1 was expressed in 8-week-old adult mice compared to WT lungs. Δ / Δ The lungs have an altered cytokine microenvironment. Multiple cytokines are upregulated by more than 1.5-fold, including GM-CSF, G-CSF, IL-1α, Rantes, MIP-1α, IL-2, IP-10, and MCP-2 (see Figure 5 of K). Figure 5 K shows 2-month-old FVB / NTac WT and Ism1 Δ / Δ Heat map of relative cytokine expression between mouse lungs (n = 3 WT mice and 6 Ism1 Δ / Δ mice).

[0336] Indeed, Western blot confirmed higher levels of GM-CSF compared to WT mice. This higher GM-CSF level may be due to Ism1 Δ / Δ Sterile lung inflammation in mice is induced. Higher GM-CSF levels may stimulate AM proliferation, thereby promoting Ism1 Δ / Δ Higher numbers of AM in the lungs. Figure 2 H shows the expression of WT and Ism1 in 2-month-old FVB / N cells. Δ / Δ Western blot (left) and fold change (right; AU, arbitrary units) of GM-CSF in mouse lungs, with β-actin as a loading control (n = 4 mice / group. Data are mean ± sem and analyzed by unpaired two-tailed Student's t-test. ***P < 0.001).

[0337] Emphysema gradually worsens as mice age ( Figure 1 These symptoms are similar to those seen in human COPD patients. Strikingly, the severity of emphysema was also ISM1 dose-dependent. Δ / Δ Mice showed a higher expression of Ism1 Δ / + The mice had a more severe emphysema phenotype. It appears that the presence of ISM1 in the lung microenvironment is important for lung homeostasis and preventing sterile inflammation (inflammation without infection or injury). Consistently, ISM1 expression levels were highest in the adult mouse lung among all organs examined, supporting the important role of this protein in lung function.

[0338] Alveolar macrophage accumulation drives Ism1 Δ / Δ emphysema in the lungs

[0339] Ism1 Δ / Δ Emphysema in mice is accompanied by multifocal aggregates of AM in the alveolar spaces ( Figure 2 A), and centrifugation of bronchoalveolar lavage fluid (BALF) cells and flow cytometric analysis confirmed that Ism1 Δ / Δ Increased AM in mouse lungs ( Figure 2 B–D). Notably, from Ism1 Δ / ΔAMs from mice displayed distinct morphologies, resembling macrophage subsets described in COPD patients (Dewhurst, Lea et al., 2017). Western blot analysis of whole lung lysates for known COPD-associated proteases and mediators revealed that Ism1 Δ / Δ Increased levels of MMP-12, MMP-9, and NF-κB p65 in the lungs ( Figure 2 E). Immunohistochemistry (IHC) staining of lung tissue sections showed Ism1 Δ / Δ Increased expression of MMP-12 and MMP-9 in AM of mice ( Figure 2 F), consistent with other mouse models of emphysema and human COPD pathology (Woodruff, Koth et al., 2005). Δ / Δ Primary AM isolated from mice showed increased nuclear translocation of NF-κB p65, indicating NF-κB activation in these cells ( Figure 2 In addition, TGF-β1 and VEGF-A were expressed in Ism1 Δ / Δ Moderately upregulated in the lung and AM of mice ( Figure 5 Ism1 is expressed in H and I, consistent with the AM accumulation and gene expression patterns in COPD patients (de Boer, van Schadewijk et al., 1998, Kranenburg, de Boer et al., 2005). Δ / Δ The lungs also produced higher levels of reactive oxygen species (ROS) ( Figure 5 J). In contrast, Ism1 Δ / Δ The levels of neutrophil elastase and α-1-antitrypsin in the lungs of mice did not show any changes ( Figure 5 H). Microarray analysis showed that Ism1 Δ / Δ Inflammatory cytokines were upregulated in the lungs of mice, including IL-1a, G-CSF, GM-CSF, MIP-1a, RANTES, IP-10, and MCP-2 ( Figure 5 Since GM-CSF drives AM development (Guilliams, De Kleer et al., 2013) and GM-CSF-overexpressing mice develop emphysema and AM accumulation (Suzuki, McCarthy et al., 2020), we sought to elucidate the role of GM-CSF in Ism1. Δ / Δ Ism1 is constitutively upregulated in mice. Immunostaining and Western blotting of P1 mouse lungs did not show Ism1 Δ / Δ The difference in GM-CSF expression between WT and WT mice ( Figure 5 L and M). In addition, 1-month-old Ism1Δ / Δ Increase in MMP-12 in mouse lungs precedes GM-CSF upregulation ( Figure 5 Therefore, it can be inferred that Ism1 Δ / Δ Increased GM-CSF in mouse lungs ( Figure 2 H) is instead linked to emphysema exacerbations and inflammation primarily caused by excessive AM accumulation and activation.

[0340] Figure 2 Figures B and C show the results of the Ism1 knockout mouse model. Δ / Δ Based on bronchoalveolar lavage fluid (BALF) analysis, the lack of ISM1 in the lungs of mice resulted in airway inflammation and increased alveolar macrophages (AMs). Analysis of cells from bronchoalveolar lavage fluid (BALF) confirmed that Ism1 was expressed in the lungs of WT mice compared with WT mice. Δ / Δ Increased alveolar macrophages (AMs) in mouse lungs ( Figure 2 In contrast, no neutrophil infiltration was found in the airways. Δ / Δ AMs in mice displayed distinct morphologies, resembling macrophage subsets described in COPD patients (Dewhurst, Lea et al., 2017). Significantly increased lymphocytes were also observed in BALF, similar to whole lung analysis. Figure 2 As shown in B and C, BALF analysis demonstrated that Ism1 Δ / Δ Airway immune cells are upregulated in mice. Shown are WT and Ism1 cells from 2-month-old mice. Δ / Δ Liu-stained cytospin preparations of bronchoalveolar lavage fluid (BALF) cells from mouse lung ( Figure 2 B) and quantitative ( Figure 2 C) (n=4 mice / group. Data are mean±sem and analyzed by unpaired two-tailed Student's t-test. *P<0.05, ***P<0.001).

[0341] ISM1 expression in mouse bronchial and alveolar epithelium has been previously reported (Osório et al., 2014, Venugopal, Chen et al., 2015). Here, we have shown that AM is a novel source of ISM1 ( Figure 2 I, Figure 6 (A and B), although it is clear that not all AMs constitutively express ISM1 at similar levels in healthy lungs. Notably, AMs also stained strongly for GRP78, and compared to WT mice, Ism1 Δ / Δ Mouse lungs showed more AM with different periplasmic GRP78 ( Figure 2J). Cell surface GRP78 (csGRP78) has been previously detected on mouse peritoneal macrophages (Misra, Gonzalez-Gronow et al., 2005) and human monocytes (Lu, Lai et al., 2010). Primary AMs were treated with recombinant ISM1 (rISM1) to determine whether csGRP78 is present and whether it functions as an ISM1 receptor on AMs. It was observed that rISM1 binds to csGRP78 on non-permeabilized AMs ( Figure 6 C) and colocalized with GRP78 in cells ( Figure 6 D), leading to AM apoptosis ( Figure 2 K and Figure 6 Similarly, rISM1 induced apoptosis in immortalized mouse AM cells (MH-S) after thapsigargin (TG) pretreatment ( Figure 6 F), thapsigargin (TG) is an ER stress inducer known to promote GRP78 translocation to the cell surface (Li, Ni et al., 2008). In addition, anti-GRP78 antibody neutralization effectively blocked rISM1-induced apoptosis ( Figure 6 G). These results demonstrate that rISM1-induced apoptosis is mediated by csGRP78 on AM. Δ / Δ Primary AM isolated from mice showed reduced apoptosis and no change in proliferation ( Figure 2 L and M), indicating that the lack of endogenous ISM1-mediated autocrine / paracrine apoptosis may be caused by Ism1 Δ / Δ Basis of AM accumulation in the lung. Ism1 Δ / Δ Increased AM in the lung contributes to the protease-antiprotease imbalance, thereby leading to Ism1 in ambient air. Δ / Δ Spontaneous COPD in mice.

[0342] Figure 6 The results in Figure B indicate that Ism1 mRNA is expressed in bronchial epithelial cells and alveolar macrophages. Indeed, in situ hybridization using mouse lung tissue sections revealed that Ism1 is expressed in bronchial epithelial cells and some AMs, consistent with the IHC data. This also suggests that the IHC data showing ISM1 expression in AMs are reliable.

[0343] Figure 6 Figures C, F, and G show that rISM1 induces AM apoptosis by targeting csGRP78. rISM1 interacts with csGRP78 on the cell surface of freshly isolated primary AM, as demonstrated by confocal microscopy ( Figure 6C). This data supports that rISM1 targets csGRP78 on the surface of AMs to trigger their apoptosis. Figure 6 In C, representative confocal images of primary AMs showing co-localization of rISM1 (red) and GRP78 (green) after 1 μM rISM1 treatment for 1 hour are shown (nuclei were stained with DAPI (blue)). In addition, when cells were pretreated with thapsigargin (TG), which induces ER stress and upregulates csGRP78, the mouse AM cell line MH-S cells underwent ISM1-induced apoptosis. Denatured ISM1 (cooked) lost this pro-apoptotic activity, indicating that this pro-apoptotic activity is a function of the ISM1 protein. Anti-GRP78 antibodies interfered with rISM1-induced apoptosis, supporting that ISM1 targets csGRP78 on the surface of MH-SAM cells to trigger apoptosis. It should be noted that higher concentrations of anti-GRP78 antibodies themselves can cause cell death, so only concentrations that do not have any effect on cells themselves are used to block csGRP78 and ISM1-induced apoptosis. Figure 6 Figures F and G show quantification of apoptosis in the mouse AM cell line MH-S after 24 hours of pretreatment with 50 nM thapsigargin (TG) and 16 hours of treatment with 1 μM rISM1 (left) and neutralization with GRP78 antibody (right). Treatment conditions are as indicated. Analysis was performed in triplicate wells, and four images per well were captured for quantification using the IncuCyte Live Cell Analysis System.

[0344] rISM1 rescues Ism1 Δ / Δ and cigarette smoke-induced emphysema in COPD mice

[0345] Emphysema due to AM in mice / CO Plays a key role in the pathogenesis of PD 11 ,18 ,19 We evaluated whether exogenously supplied rISM1 could block Ism1 by inducing / promoting AM apoptosis and / or suppressing inflammation. Δ / Δ Development / progression of emphysema in mice. rISM1 was delivered intratracheally twice weekly to 1-month-old Ism1 mice. Δ / Δ Mice were treated with phosphate-buffered saline (PBS) or liposome-clodronate (an established AM-depleting agent) for 4 weeks and compared with mice treated with phosphate-buffered saline (PBS) or liposome-clodronate (an established AM-depleting agent). Immunostaining showed that rISM1 was internalized by AM and induced apoptosis ( Figure 7 A and B), significantly reduced the number of AMs in a dose-dependent manner, similar to clodronate ( Figure 3 A) rISM1 and clodronate-treated Ism1 Δ / Δ The lungs of mice showed a significant decrease in emphysema ( Figure 3Depletion of AMs by rISM1 or clodronate may contribute to alveolar regeneration after resolution of inflammation, as demonstrated by increased proliferating type 2 alveolar epithelial cells in both treatment groups ( Figure 7 C and D). More importantly, lung function tests showed that rISM1 and clodronate-treated Ism1 Δ / Δ There was considerable airflow recovery in mice ( Figure 3 D). Taken together, these results suggest that excess AM is the primary driver of Ism1 Δ / Δ Figure 1: Spontaneous emphysema and decreased lung function in mice. As shown, lung-delivered rISM1 can rescue Ism1 by AM depletion. Δ / Δ Emphysematous phenotype.

[0346] Next, because chronic AM inflammation is associated with lung tissue damage, we evaluated whether rISM1 could alleviate cigarette smoke (CS)-induced COPD in mice. WT BALB / cAnNTac mice were subjected to 2 and 8 weeks of room air (sham) or CS exposure and treated intratracheally with PBS or rISM1 ( Figure 3 E and F). Cytospin analysis of BALF cells from 2-week CS-exposed mice showed that rISM1 effectively suppressed inflammation and reduced the number of AMs and neutrophils ( Figure 3 G). Histological analysis of 8-week CS-exposed mice showed emphysema near the terminal bronchioles, with a large accumulation of immune cells mainly containing AMs, similar to those in smoking COPD patients ( Figure 3 H and Figure 7 Mice treated with rISM1 produced more apoptotic AMs ( Figure 7 F), significantly reduced AM ( Figure 3 I) and MMP-12 levels ( Figure 3 The neutrophil count was also significantly reduced after rISM1 treatment ( Figure 7 of G), which may be the result of reduced chemotaxis after AM depletion (Murugan & Peck, 2009). Therefore, lung-delivered rISM1 effectively blocked CS-induced emphysema in COPD mice by AM depletion ( Figure 3 K) and preserved lung function ( Figure 3 L and Figure 7 H to J).

[0347] like Figure 3As shown in the results, rISM1 inhibited cigarette smoke (CS)-induced acute lung inflammation. CS is known to induce acute inflammatory responses in the lungs by inducing neutrophils and AMs. Using a 2-week CS model in mice, intratracheally delivered rISM1 effectively inhibited CS-induced lung inflammatory responses, as shown by a decrease in total BALF cells. AMs and neutrophils were both suppressed, while lymphocytes ( Figure 3 This result is consistent with data from an 8-week chronic CS-induced emphysema (COPD) model in mice, in which intratracheally delivered rISM1 effectively quenched lung inflammation and reduced AM number by triggering AM apoptosis. Figure 3 Figure E shows the experimental design of a 2-week cigarette smoke-induced COPD model in WT BALB / c mice. Room air-exposed (Sham), cigarette smoke-exposed (CS) (n=5 mice / group) were treated with vehicle (CS+PBS) or rISM1 (CS+10 μg rISM1) at the indicated frequencies and intervals. Figure 3 G shows quantification of bronchoalveolar lavage fluid (BALF) cells from experimental groups in 2-week cigarette smoke-induced COPD mice (n=4-5 mice / group).

[0348] Human ISM1 expression is associated with apoptosis in alveolar macrophages

[0349] Since Ism1 Δ / Δ Since mice developed spontaneous emphysema and exogenously supplied rISM1 protected mice from CS-induced COPD pathogenesis, we hypothesized that changes in endogenous ISM1 levels in human lungs could also affect COPD development. We first validated the specificity of our antibody for human ISM1 (hISM1) using hISM1-overexpressing cells ( Figure 8 A), and then examined hISM1 expression in lung tissue sections from 60 COPD and 18 non-COPD patients (Table A).

[0350] Table A: Patient demographics (forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC). Data are mean ± sem)

[0351]

[0352]

[0353] Similar to mice, hISM1 is also mainly expressed in AM ( Figure 4 However, although ISM1 is also expressed in the bronchial epithelium of mice, especially after CS exposure ( Figure 8B), but hISM1 was not detected in the bronchial epithelium of either COPD or non-COPD human lungs ( Figure 8 C). hISM1 expression was then graded by scoring the IHC staining intensity and hISM1 expression frequency in AM ( Figure 4 A positive correlation was found between hISM1 expression and smoking ( Figure 4 D and E), and higher hISM1 expression was observed in current smokers than in ex-smokers ( Figure 8 These findings are consistent with the fact that ISM1 is significantly upregulated in mouse AM, particularly after CS exposure ( Figure 8 E), while ISM1 staining still could not detect other immune cells such as polymorphonuclear leukocytes and lymphocytes ( Figure 8 Importantly, hISM 1 expression was significantly positively correlated with AM apoptosis, regardless of smoking status ( Figure 4 F, Figure 23 Notably, csGRP78 was upregulated on AMs of COPD patients compared with non-COPD patients ( Figure 4 In fact, more apoptotic AMs were observed in COPD patients compared with non-COPD patients with similar hISM1 expression ( Figure 4 H), and only csGRP78-positive AM apoptosis occurred in COPD patients and CS-exposed mice ( Figure 23 C and D).

[0354] Based on these results, we hypothesized that physiological ISM1 may be important for maintaining adult lung homeostasis by regulating AM apoptosis through csGRP78. Loss of ISM1 could lead to AM accumulation from attenuated apoptosis, thereby ensuring Ism1 expression even under ambient air. Δ / Δ Lung inflammation and emphysema in mice ( Figure 24 ).

[0355] Figure 4 Figures E, F, and H show that ISM1 expression in human COPD and non-COPD lungs is associated with cigarette smoke and AM apoptosis. ISM1 expression analysis using immunohistochemistry (IHC) from 60 COPD and 18 non-COPD human lung tissue samples showed that the expression level of human ISM1 (hISM1) protein was associated with cigarette smoke, with smokers showing higher ISM1 expression ( Figure 4 Furthermore, the level of AM apoptosis was correlated with the level of hISM1 in lung tissue, with higher hISM1 levels in the lungs indicating higher AM apoptosis ( Figure 4F). Higher hISM1 levels showed higher AM apoptosis in COPD and non-COPD lungs ( Figure 4 H). Indeed, Figure 4 Figures E, F, and H show hISM1 expression in COPD and non-COPD lungs. Figure 4 E) smoking status and hISM1 and ( Figure 4 F) Correlation between hISM1 expression and AM apoptosis. The patient sample size is depicted on the graph. Figure 4 E) and Pearson correlation ( Figure 4 F) Data were analyzed. Percentage of apoptotic AM in non-COPD and COPD patients stratified by COPD status and hISM1 expression. The graph depicts the patient sample size (data are mean ± sem and analyzed by one-way ANOVA with Tukey's post hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001).

[0356] I don’t want to be bound by theory. Figure 24 A proposed mechanism by which ISM1 regulates AM apoptosis and lung homeostasis is provided. Figure 24 (Left) Autocrine / paracrine ISM1 specifically targets AMs with high csGRP78 and induces apoptosis. AM numbers remain controlled, inflammation is regulated, and lung homeostasis is maintained. Figure 24 (Right) Middle, absence / low ISM1 leads to AM accumulation in the alveolar space and the onset of emphysema, with a gradual decline in lung function.

[0357] As discussed, chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide, with smoking, long-term exposure to environmental pollution, and aging being the main risk factors. Its main characteristics are irreversible obstruction of air flow due to emphysema (destruction of the alveolar walls) and chronic obstructive bronchitis (airway inflammation) of the small airways. Patients experience recurring respiratory symptoms such as coughing and dyspnea. In addition, exercise intolerance leads to muscle weakness and sarcopenia in COPD patients. Current treatments are mainly bronchodilators, which can only relieve symptoms, and there are no drugs that can block disease progression. In the studies described herein, using genetic and pathological mouse models, the results show that the pro-apoptotic ISTHMIN 1 (ISM1) protein is a protector of lung tissue homeostasis. Loss of ISM1 leads to spontaneous COPD caused by accumulation of alveolar macrophages (AMs). In mouse and human lungs, AMs simultaneously express ISM1 and its high-affinity receptor csGRP78, thereby enabling self-regulation of apoptosis. In cigarette smoke-induced COPD mice, local lung delivery of recombinant ISM1 (rISM1) reduced AM number, suppressed inflammation, and preserved lung function. Furthermore, human ISM1 expression in the lung was significantly positively correlated with AM apoptosis.

[0358] The results described herein identify ISM1 as an anti-inflammatory protein that protects normal lung function and blocks CS-induced COPD progression. Without wishing to be bound by theory, this work supports the use of rISM1 as a protein therapeutic to quench lung inflammation by targeting csGRP78 on pathological AM. The results suggest that ISM1-csGRP78-mediated AM targeting could provide a therapeutic strategy in a wide range of inflammatory lung diseases, for example, where AM plays a pathological role.

[0359] Macrophage clearance has been shown to be mediated primarily through local apoptosis during resolution of inflammation 26 , and AM apoptosis is important in the resolution of infection-associated acute lung inflammation 27-29 AM is the basis of COPD pathogenesis and lung exacerbations 2,20,25 , and are notoriously resistant to apoptosis 23 and corticosteroid treatment 30 Our findings suggest that an autocrine / paracrine mechanism controls AM number and maintains lung homeostasis through the pro-apoptotic protein ISM1. ISM1 targets csGRP78 on AM to induce apoptosis and limit lung inflammation, whereas loss of ISM1 results in the loss of Ism1. Δ / Δ AM accumulation and spontaneous emphysema in mice ( Figure 24 B). Incidentally, AM accumulation is associated with the development of emphysema in smokers 2Intratracheal instillation of rISM1 prevented AM accumulation in CS-exposed mice and effectively blocked COPD progression, consistent with previous mouse studies demonstrating the efficacy of AM depletion. 18,19 Consistently, high hISM1-expressing patients presented with more AM apoptosis, lower AM numbers, and lower frequency of severe COPD. The heterogeneity of hISM1 expression in COPD patients may provide better insights for early intervention in COPD patients. The current findings highlight the important role of regulated AM apoptosis in maintaining lung homeostasis and identify a protective role of ISM1 as an inflammation suppressor in lung homeostasis. For example, rISM1 may prevent COPD progression by quenching AM-driven lung inflammation.

[0360] COPD is a growing global epidemic with a substantial socioeconomic burden, yet no effective drugs exist to interrupt disease progression and / or restore lung function. The studies described here demonstrate that the secreted 50-kDa protein ISM1, an anti-inflammatory protein, effectively quenches lung inflammation and prevents CS-induced COPD progression in mice. Local pulmonary delivery of rISM1 specifically targets AMs via their high-affinity receptor, csGRP78, leading to their apoptosis.

[0361] AMs constitute more than 95% of lung immune cells and are the main inflammatory coordinators of COPD. They are also resistant to apoptosis and thus contribute to chronic lung inflammation even after smoking cessation (Barnes, 2016, Maskey-Warzechowska et al., 2003, Kojima, Araya et al., 2013). Intratracheally delivered rISM1 effectively blocked AM accumulation and prevented lung function decline in CS-induced COPD mice, consistent with previous mouse studies demonstrating the role of AM depletion in emphysema prevention. Thus, for example, the results described herein support rISM1 as a therapeutic agent for COPD to block disease progression and / or maintain lung function. By inducing AM apoptosis, it is expected that rISM1 can not only hinder direct proteolytic damage caused by AM-secreted proteases such as MMP-12, but also prevent MMP-12-driven tumor necrosis factor-α (TNF-α)-related endothelial cell activation, neutrophil chemotaxis, and further macrophage activation, a process estimated to account for up to 70% of CS-induced lung injury (Churg, Wang et al., 2004). Therefore, AMs are and should continue to be an important target for novel anti-inflammatory COPD therapies. However, AM are known to be unresponsive to steroids (Barnes, 2013a) and the poor translatability between mouse models and human clinical trials remains a major challenge in COPD drug development in terms of targeting discrete proinflammatory molecules (Barnes, 2013b). In this context, rISM1 has a clear advantage because it can specifically target csGRP78 on AM. Incidentally, pathological AM in COPD contain high levels of csGRP78 ( Figure 4 G), making them ideal candidates for rISM1-mediated apoptosis. Thus, rISM1 can effectively inhibit AM inflammation and simultaneously kill multiple proinflammatory factors. Without wishing to be bound by theory, it is anticipated that rISM1 can induce apoptosis in proinflammatory AM without damaging immunosuppressive interstitial macrophages that do not express csGRP78 (Quesada Calvo, Fillet et al., 2011), thereby allowing them to perform homeostatic functions in the lung.

[0362] In this work, the results reveal a novel physiological function of ISM1 in maintaining lung homeostasis, consistent with its highest expression level in the mouse lung compared to other organs. The results show that loss of ISM1 in mice leads to the development of spontaneous emphysema under ambient air, accompanied by excessive AM accumulation in the alveolar space. The results indicate that AM are a new source of ISM1 in the mouse lung, in addition to the previously reported bronchial epithelial cells and endothelial cells. In addition, in normal mouse lung, AM express ISM1 and its high-affinity cell surface receptor GRP78 ( Figure 2ISM1 can specifically target cells with high levels of csGRP78 for apoptosis (Chen et al., 2014). The results described here support a model in which ISM1 selectively targets AMs with high csGRP78 for apoptosis, while leaving AMs without / with low csGRP78 intact, thereby controlling the number of AMs for lung homeostasis ( Figure 24 ). From Ism1 Δ / Δ Freshly isolated AMs from mice showed lower apoptosis levels compared to AMs from WT mice, indicating autocrine / paracrine regulation of AM apoptosis by endogenous ISM1 in the mouse lung. Δ / Δ AM depletion in mice rescued emphysema, similar to clodronate treatment. Δ / Δ These results demonstrate that AM accumulation induced by dysregulated apoptosis is essential and sufficient for the development of emphysema even in the absence of environmental insults, consistent with AM being a major orchestrator of COPD pathogenesis.

[0363] ISM1's critical role in lung homeostasis is unique in mammals, as previous loss-of-function studies of Ism1 in lower vertebrates resulted in contrasting phenotypes, such as craniofacial defects in Xenopus (Lansdon, Darbro et al., 2018) and angiogenesis defects accompanied by altered hematopoiesis in zebrafish (Berrun, Harris et al., 2018, Xiang et al., 2011). The highly divergent and intrinsically disordered N-terminal region of ISM1 likely contributes to distinct biological functions in different species (Babu, 2016). On the other hand, the high sequence similarity (93.5% identity) between mouse and human ISM1 suggests that ISM1 has conserved functions between these species (Joshi & Xu, 2007).

[0364] The results of this work reveal an autocrine / paracrine signaling axis between endogenous ISM1 and csGRP78 in inducing AM apoptosis and maintaining lung homeostasis. Local macrophage apoptosis and clearance contribute to the resolution of inflammation (Hamidzadeh, Christensen et al., 2017) and have been described in early atherosclerosis (Arai, Shelton et al., 2005), experimental peritonitis (Gautier, Ivanov et al., 2013), and infection-related acute lung inflammation (Aberdein, Cole et al., 2013). Upregulation of ISM1 in COPD lungs of mice and humans may be a biological response, similar to the pleiotropic TNF-α and type I interferons that are upregulated in various lung diseases and can induce AM apoptosis via autocrine signaling (Wei, Sun et al., 2006, Xaus, Comalada et al., 2000). Indeed, hISM1 expression is strongly correlated with AM apoptosis and has also been observed to be elevated in COPD patients ( Figure 4 ).

[0365] Although previous genome-wide association studies (GWAS) have not linked the Ism1 locus to COPD, it would be interesting to understand the heterogeneity of hISM1 expression in larger COPD patient populations to reveal potential epigenetic or genetic influences on Ism1 and its regulatory genes. Furthermore, it would be interesting to determine whether hISM1 expression has any association with the multiple comorbidities that contribute to the high mortality rate in COPD.

[0366] It should be noted that the presence of αvβ5 integrin, a low-affinity receptor for ISM1, has been reported on lung endothelial and airway epithelial cells (Teoh, Tan et al., 2015). However, we did not observe any obvious targeting of these cells in mice when rISM1 was delivered intratracheally ( Figure 7 ), which would aggravate emphysema due to the expected apoptosis of these lung structural cells. Δ / Δ It alleviated emphysema and improved lung function in mice.

[0367] The relatively large size of rISM1 (~50 kDa) suggests that ISM1 is not rapidly cleared from the lungs and absorbed into the bloodstream (Labiris & Dolovich, 2003, Patton, Fishburn et al., 2004). Significant progress has been made in the aerosolization of protein therapeutics for local pulmonary delivery in various clinical trials. For example, multiple Phase II / III clinical trials have been conducted using α-1 antitrypsin (52 kDa) as an inhaled therapeutic for α-1 antitrypsin deficiency and cystic fibrosis (Bodier-Montagutelli, Mayor et al., 2018). In certain embodiments, for example, due to its comparable size to α-1 antitrypsin, ISM1 can be used with pulmonary delivery via aerosolization.

[0368] In summary, the results described herein highlight the critical role of AM apoptosis regulation in maintaining lung homeostasis and the important role played by ISM1 in this function under both physiological and pathological conditions. The results support Ism1 as a novel gene associated with the pathogenesis of emphysema / COPD and AM apoptosis, and demonstrate that rISM1 attenuates emphysema, suppresses inflammation, and preserves lung function in chronic CS-induced COPD in mice. The results suggest that rISM1 may be used to treat COPD by specifically targeting csGRP78 on AM, the primary pathological immune cell in COPD. It is anticipated that the findings described herein may also have implications for a wide range of respiratory conditions driven or contributed to by AM, such as pulmonary ischemia-reperfusion injury (Naidu, Krishnadasan et al., 2003), acute lung injury (Dagvadorj, Shimada et al., 2015), pulmonary fibrosis (Misharin, Morales-Nebreda et al., 2017), and asthma (Nabe, Matsuda et al., 2018). In certain embodiments, pathological expression of csGRP78 in other non-cancer diseases, such as rheumatoid arthritis and systemic lupus erythematosus (Lu et al., 2010, Weber, Haslbeck et al., 2010), may also provide therapeutic opportunities for rISM1 to modulate inflammation.

[0369] One or more illustrative embodiments have been described by way of example. It will be apparent to those skilled in the art that numerous changes and modifications may be made without departing from the scope of the invention as defined in the claims.

[0370] method

[0371] Study Design. The primary objective of this study was to determine the physiological function of mammalian Ism1 using in-house generated CRISPR / Cas9-mediated knockout of Ism1 in two genetic backgrounds (FVB / Ntac and C57BL / 6J mice). Sample size for phenotypic characterization and rescue experiments was maintained at a minimum of three animals per group for statistical analysis, and n numbers are indicated in the corresponding figures and legends for each experiment. Age- and sex-matched mice were randomly assigned to experimental groups, and no outliers were excluded from the animal study. Ism1 Δ / Δ Rescue experiments in mice were repeated twice, and lung function parameters and histology were analyzed separately. Rescue experiments in mice with chronic CS-induced COPD were performed once, with lung function parameters measured and the left lung lobe fixed for histological analysis, and the right lung lobe homogenized for biochemical analysis. Immune cell quantification for all mouse experiments was performed in a blinded manner. De-identified human lung samples were used for immune cell quantification, staining, and hISM1 expression grading. No data were excluded from the human cohort study.

[0372] Mice. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the National University of Singapore (IACUC protocols BR15-1100 and R18-0588). Wild-type mice (FVB / Ntac, C57BL / 6J, and BALB / cAnNTac; 6 to 8 weeks old) were purchased from InVivos Pte Ltd, Singapore. Ism1 Δ / Δ Mice (FVB / Ntac and C57BL / 6J) were transfected using pronuclear microinjection of recombinant Cas9 and the 5'-CTGCACATCACGGTTCTGCG targeting exon 1 of Ism1. CGG -3' (gRNA1, SEQ ID NO: 5 underlined PAM sequence) and 5'-GCGGATCCGGAGCCTCCGAC CGG Ism1 was identified by genotyping primer pairs P1 (5'-CAGCTCCTGGGATTGCTCCG-3') (SEQ ID NO: 7) and P2 (5'-CCTTCTGCAATGTACCAAGCTCT-3') (SEQ ID NO: 8) for FVB / NTac and 5'-cgcgcgactcaagaggatgg-3' (SEQ ID NO: 22) and 5'-actgggacccgctgacgttg-3' (SEQ ID NO: 23) for C57BL / 6J and sequencing. Δ / ΔThe offspring of the mice were then selected for subsequent breeding and colony maintenance (IACUC protocol BR15-1100). All mice were housed under a standard 12-hour light-dark cycle with food and water available ad libitum. Mice were anesthetized with isoflurane before all tracheal instillations.

[0373] Cell. MH-S(CRL-2019 TM ) were purchased from ATCC and cultured in RPMI-140 medium supplemented with 10% heat-inactivated FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL). Δ / Δ Primary alveolar macrophages were harvested from FVB / NTac mice (Chavez-Santoscoy, Hunterman et al., 2012) and cultured in RPMI-140 medium supplemented with 10% heat-inactivated FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL). Cells were maintained at 37°C in a 5% CO2 incubator. 2-month-old wild-type and Ism1 mice were isolated as described. Δ / Δ Primary alveolar macrophages harvested from FVB / NTac mice 32 , and cultured in DMEM medium supplemented with 10% heat-inactivated FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL). Primary alveolar macrophages were maintained at 37° C. in a 5% CO 2 incubator.

[0374] Reagents. Primary antibodies used for Western blotting: anti-MMP-12 (ab52897, Abcam), anti-MMP-9 (ab38898, Abcam), anti-p65 (10745-1-AP, Proteintech), anti-β-actin antibody (C4, Santa Cruz Biotechnology), anti-TGF-β1 (V, Santa Cruz Biotechnology), anti-VEGF-A (A-20, Santa Cruz Biotechnology), anti-neutrophil elastase (ab68672, Abcam), anti-A-1-antitrypsin (16382-1-AP, Proteintech). Primary antibodies used for immunohistochemistry: anti-MMP-12 (ab66157, Abcam), anti-MMP-9 (ab38898, Abcam), anti-TGF-β1 (V, Santa Cruz Biotechnology), anti-VEGF-A (A-20, Santa Cruz Biotechnology), anti-ISM1 (for mouse lung: E-20, Santa Cruz Biotechnology; for human lung: custom antibody 3M8, AbMart), anti-GRP78 (A-10, Santa Cruz Biotechnology), anti-His-probe (H-15, Santa Cruz Biotechnology), anti-cleaved caspase-3 (Asp175, Cell Signaling Technology).Primary antibodies used for immunofluorescence: anti-ISM1 (E-20, Santa Cruz Biotechnology), anti-p65 (10745-1-AP, Proteintech), anti-CD68 (M-20, Santa Cruz Biotechnology), anti-His-probe (H-15, Santa Cruz Biotechnology), anti-GRP78 (A-10, Santa Cruz Biotechnology), anti-cleaved caspase-3 (Asp175, Cell Signaling Technology), anti-SP-C (FL-197, Santa Cruz Biotechnology), anti-PCNA (PC10, Santa Cruz Biotechnology), anti-GRP78 (A-10, Santa Cruz Biotechnology), neutrophil marker (NIMP-R14, Santa Cruz Biotechnology) using OxiSelect. TM In vitro ROS / RNS assay (STA-347, CellBiolabs) was used to measure reactive oxygen species according to the manufacturer's protocol. TM Cell proliferation was measured using the EdU proliferation assay (Invitrogen, C10499) according to the manufacturer's protocol. Recombinant ISM1 (rISM1) was produced as previously described (Xiang et al., 2011). Liposome-encapsulated clodronate was purchased from Liposoma.

[0375] Mouse rISM (mature form, without signal peptide) was expressed in E. coli using the vector pET-M and purified as a 6xHis-tagged protein (as described in Xiang et al., 2011, JCMM, which is incorporated herein by reference in its entirety). Mouse rISM1 was used in these experiments. Mature ISM1 (without signal peptide) was biologically active under the test conditions. The ISM1 sequence used comprised NP_001263418.1 and was as follows:

[0376]

[0377] (SEQ ID NO: 9; underline indicates the native ISM1 sequence, bold indicates the vector sequence and His tag, italics indicate the N-terminal M residue)

[0378] Liposomally encapsulated clodronate was purchased from Liposoma.

[0379] Lung Histology and Imaging. Mouse lungs from each experiment were inflated and fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned at 5 μm thickness. Mouse and human lung sections were deparaffinized in detergent (Sigma-Aldrich) and serially diluted with ethanol and PBS. FVB / NTac WT and Ism1 were analyzed by a veterinary pathologist. Δ / Δ Histological and pathological scoring of emphysema in mice. Pathological scoring: 0 = absent; 1 = minimal (>1%); 2 = mild (1–25%); 3 = moderate (26–50%); 4 = moderately severe / high (61–75%); and 5 = severe / high (76–100%). Quantification of the mean linear intercept (MLI) was performed as described. 34 (Knudsen, Weibel et al., 2010). Quantification of bronchial epithelial cell counts and measurements was performed using ImageJ software (NIH). For immunofluorescence and immunohistochemical staining, after dewaxing, lung sections were subjected to antigen retrieval by pressure cooking in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). The slides were cooled to room temperature and rinsed with PBS, then blocked with 3% BSA in PBS for 1 hour. Lung sections for immunohistochemistry were quenched with 3% hydrogen peroxide for an additional 30 minutes. The lung sections were then incubated overnight with the corresponding primary antibodies in a humidified chamber at room temperature, washed three times with 0.1% PBST to remove unbound antibodies, incubated with the corresponding secondary antibodies at room temperature for 1 hour and washed again with 0.1% PBST three times. Before installing coverslip, DAPI is used to counterstain the slides of immunofluorescence staining, while in liquid DAB+ chromogenic substrate (Dako), the slides of immunohistochemistry staining are incubated 5 to 15 minutes, then counterstained with hematoxylin and coverslip is installed. Lung section staining is carried out according to the manufacturer's protocol using periodic acid-Schiff (87007, Thermo Fisher) and elastin staining kit (ab150667, Abcam). Images are collected using Zeiss Axiovert 200 and Zeiss LSM-510Meta confocal microscopes, and brightness and contrast are analyzed and adjusted using ImageJ software (NIH).

[0380] BALF immune cell quantification. Bronchoalveolar lavage fluid (BALF) was collected and cells were analyzed by flow cytometry using standard procedures. Figure 25 The gating strategy used to select AMs is shown.

[0381] Apoptosis determination. Apoptosis was measured using the IncuCyte ZOOM live cell imaging system (Essen Bioscience). MH-S cells or surrogate alveolar macrophages were seeded at a density of 50,000 cells per 96-well, and apoptosis was measured every hour using IncuCyte caspase-3 / 7 green apoptosis detection reagent (Cat.No.4440, Essen Bioscience) according to the manufacturer's instructions. MH-S cells were pretreated for 25 hours using 50nM thapsigargin (Sigma-Aldrich) in RPMI-140 culture medium supplemented with 1% heat-inactivated FBS, and then treated for 16 hours with 1 μM rISM1 with and without anti-GRP78 (A-10, Santa Cruz Biotechnology) antibody neutralization under the same culture conditions. Processed in three parallel wells, and 4 images were taken per well for quantification. Primary alveolar macrophages were treated for 16 hours using 1 μM rISM1 under the same culture conditions. Experimental groups were performed in four parallel wells, and four independent fields of view were collected per well for quantification.

[0382] Pulmonary function tests were performed as previously described in FVB / NTac wild-type and Ism1 Δ / Δ Spirometry was performed on mice and experimental COPD WTBalb / cAnNTac mice 35 (Peh, Tan et al., 2017). Briefly, mice were anesthetized with a mixture of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the trachea was cut open. Mice were intubated and placed in a whole-body plethysmograph connected to a computer-controlled ventilator (forced lung motor system, Buxco Research System). FinePointe TM The data acquisition and analysis software (Buxco) recorded total vital capacity (TLC), functional residual capacity (FRC), residual volume (RV), static compliance (Cchord), dynamic compliance (Cdyn), forced expiratory volume at 100ms (FEV 100 ), Tiffeneau–Pinelli index (FEV 100 The work of breathing was calculated using the area under the pressure-volume plot.

[0383] Whole-mount lung imaging. Thoracotomy was performed to obtain lung tissue from 6-month-old FVB / NTac wild-type and Ism1 Δ / ΔMouse harvest mouse lung, and remain in cold PBS until imaging or processing.Use Olympus MXV10 Macro Zoom to collect the image of peripheral left lung lobe under 1.26x magnification.For the immunofluorescence staining of pulmonary elastin and collagen, in the DMEM supplemented with 1% heat-inactivated FBS, penicillin (100U / mL), streptomycin (100 μg / mL) and 1 μM Col-F fluorescent probe (Immunochemistry Technologies), gently shake the lung incubation of each harvest overnight at 4 ° C.Afterwards, use three times PBS that lung is thoroughly cleaned and use Olympus MXV10 Macro Zoom to shoot the image of peripheral lung lobe under 5x magnification with fluorescence excitation.Use ImageJ software (NIH) to adjust the brightness and contrast of image.

[0384] FVB / NTac Ism1 Δ / Δ Rescue of emphysema in mice. Δ / Δ Mice were intratracheally administered 50 μl PBS, 1 μg or 5 μg rISM1 in 50 μl PBS, or 350 μg liposomally encapsulated clodronate in 50 μl PBS twice a week for 4 weeks. Pulmonary function tests were recorded 24 hours after the last day of treatment, and lungs were fixed for histological analysis.

[0385] Cigarette smoke-induced COPD mouse model. 8-week-old female Balb / cAnNTac mice were subjected to 8-week chronic cigarette smoke exposure as previously described. 35 (Peh et al., 2017). Briefly, mice were whole-body exposed to 4% cigarette smoke at a frequency of three 3R4F reference cigarettes (University of Kentucky, Lexington) every 2 hours for a total of nine cigarettes per day. This smoking regime was continued for 5 consecutive days per week for 8 weeks. Sham mice were rotated in separate ventilated chambers and exposed to the same room air. After the first 4 weeks of cigarette smoke exposure, the corresponding treatment groups were given 50 μl PBS or 10 μg rISM1 in 50 μl PBS for an additional 4 weeks. On days 1, 3, and 5 of each week, after the last round of daily cigarette smoke exposure, PBS and rISM1 treatment were instilled intratracheally. 24 hours after the last day of cigarette smoke exposure, lung function tests were recorded, and the lungs were fixed for histological analysis.

[0386] Mouse model of acute cigarette smoke-induced lung inflammation. Eight-week-old female Balb / cAnNTac mice were exposed to chronic cigarette smoke for two weeks as described above. During the second week of cigarette smoke exposure, PBS and rISM1 were administered intratracheally for five consecutive days following the last bout of daily cigarette smoke exposure, in a manner similar to that described above.

[0387] Human lung tissue. The use of human samples was approved by the Institutional Review Board of the National University of Singapore (NUS-IRB Reference No. N-18-057E). Formalin-fixed and paraffin-embedded de-identified lung sections were provided by the Lung Tissue Research Consortium (LTRC), the National Heart, Lung, and Blood Institute (NHLBI), and the National Institutes of Health (NIH). COPD patients were selected based on clinical diagnosis of emphysema and post-bronchodilator / pre-spirometry criteria of FEV1 / FVC <0.7 and FEV% predicted ≤80. Non-COPD patients were identified based on post-bronchodilator / pre-spirometry criteria of FEV1 / FVC ≥0.7 and FEV% predicted ≥80. Patient smoking history and status were provided. Two independent researchers blindly graded the expression of hISM1 in non-COPD and COPD patients. Six to ten random fields were selected for each human lung section.

[0388] Statistical analysis. Statistical analysis was performed using Prism (Graphpad) software. Comparisons between two groups were performed using an unpaired, two-tailed Student's t-test, and multiple group comparisons were performed using a one-way ANOVA with a Tukey post hoc test. The correlation between hISM1 expression and smoking or AM apoptosis was determined using point bivariate and Pearson correlation, respectively. The results show mean ± sem and the sample size of each experiment is shown in the figures or legends accordingly. P values ​​< 0.05 were considered significant. Additional annotations are shown in the legends accordingly.

[0389] Example 2: Isthmin 1 inhibits lipopolysaccharide-induced acute lung injury and lung inflammation

[0390] Isthmin 1 (ISM1) is highly expressed in the mouse lung in bronchial and alveolar epithelial cells, endothelial cells, alveolar macrophages, and NKT cells. In response to intratracheal lipopolysaccharide (LPS) instillation, ISM1 is upregulated in the lung (Venugopal et al 2015, Cardiovas. Res.). In this study, Ism1 knockout mice (Ism1) were used on FVB / N and C57BL / 6J backgrounds. Δ / Δ ), our results demonstrate that ISM1 deficiency leads to mild sterile inflammation in the lungs of mice. After respiratory LPS challenge, Ism1 expression was significantly upregulated compared with wild-type mice. Δ / Δ Mice exhibited an exaggerated lung inflammatory response characterized by increased recruitment of leukocytes, including neutrophils, macrophages, T cells, and B cells. Although innate immune cells regressed to baseline levels by day 7 after LPS insult, Ism1 Δ / Δ Mice exhibited elevated lung fibrosis on day 9, accompanied by increased myofibroblasts, excessive collagen accumulation, and upregulation of TGF-β. Intratracheal instillation of recombinant ISM1 (rISM1) suppressed LPS-induced inflammation in the lungs. These results suggest a therapeutic benefit of ISM1 in protecting the lungs from excessive inflammatory responses and promoting the restoration of homeostasis in injured lungs after LPS-triggered ALI.

[0391] Following infectious or non-infectious respiratory insults, the host mounts an acute inflammatory response in the lungs to protect itself. Healthy hosts also have ways to limit this lung inflammation, which ultimately resolves to prevent collateral damage to surrounding tissues. Pervasive acute lung inflammation is observed in a variety of lung diseases, including acute lung injury (ALI) and its more severe manifestation, acute respiratory distress syndrome (ARDS). These are severe clinical syndromes with a mortality rate of up to 50% in the absence of effective medical treatment. 36 ALI is characterized by increased vascular permeability, infiltration of inflammatory cells (primarily neutrophils), and release of proinflammatory mediators by infiltrating leukocytes and lung parenchymal cells. 37 .

[0392] LPS is a glycolipid component of the cell wall of Gram-negative bacteria that can induce severe inflammatory effects in mice and humans. Short-term intranasal LPS challenge in mice typically stimulates a mixed inflammatory response in the airways and lungs. This includes disruption of the lung endothelial and epithelial barriers, increased inflammatory cell infiltration, and the release of pro-inflammatory and cytotoxic mediators. 38-40 These phenotypes are clinically relevant to ALI and ARDS. Multiple intracellular signaling events are initiated upon LPS challenge. Most LPS binds to the Toll-like receptor 4 (TLR-4) complex and signals to activate nuclear factor kappa B (NF-κB). 41-43Activated NF-κB translocates into the nucleus and stimulates the transcription of many proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), by directly binding to consensus target sequences in their enhancer / promoter regions44. 45 Importantly, NF-κB is active in alveolar macrophages from patients with ARDS. 46 , suggesting that NF-κB signaling is involved in the development and progression of ALI and ARDS.

[0393] ISM1 was first identified as a secreted anti-angiogenic and pro-apoptotic protein and has also been studied in the induction of vascular permeability 47 ,48 ,49 Systemic infusion of an antibody against glucose-regulated protein 78 kDa (GRP78), a high-affinity receptor for ISM1, attenuated LPS-induced pulmonary hyperpermeability. 49 .

[0394] In this study, Ism1 knockout mice (Ism1 Δ / Δ ) investigated the role of ISM1 in LPS-induced acute lung inflammation. The data support that ISM1 is an inflammatory suppressor, protecting the lung from excessive inflammatory responses in the sterile lung and during LPS-induced ALI. The presence of ISM1 in the lung also promotes the return of lung homeostasis after acute injury.

[0395] ISM1 deficiency leads to increased leukocyte infiltration in the lung under sterile conditions

[0396] Using CRISPR / Cas9 gene editing, an Ism1 knockout was generated (Ism1 Δ / Δ )C57BL / 6J mice ( Figure 17 ). In ambient air, Ism1 Δ / Δ Mice exhibit spontaneous inflammation in the lungs. Histological examination of coronal lung sections from 8-week-old knockout mice revealed multifocal, nondemarcated clusters of inflammatory cells, including alveolar macrophages, polymorphonuclear cells, and lymphocytes ( Figure 9 A). Differential immune cell counts and immunohistochemistry (IHC) staining of whole lung single cell homogenates showed a significant increase in total leukocytes, macrophages, and neutrophils ( Figure 9 Focal areas of alveolar wall hyperplasia and emphysema were also observed in the knockout mice ( Figure 9 A and C). Meanwhile, Ism1 Δ / Δ Peripheral blood analysis of mice also showed a significant increase in the total number of white blood cells compared to wild-type mice. Δ / ΔLymphocyte and neutrophil numbers were significantly higher in the knockout and wild-type mice, whereas other cell types remained low ( Figure 9 F).

[0397] Figure 17 Ism1 is shown - / - (Ism1 Δ / Δ ) Details of mouse generation. Figure 17 A shows a schematic diagram of CRISPR / Cas9 targeting Ism1 via guide RNA pairs gRNA1 and gRNA3. P1 and P2 represent primers used for T7E1 assay and genotyping. Figure 17 B shows Ism1 Δ / Δ The DNA sequence of the knockout line revealed a 23 bp deletion that resulted in a premature stop codon and no production of ISM1 protein. Figure 17 C shows C56BL / 6J WT, Ism1 + / Δ and Ism1 Δ / Δ RT-PCR gel image. Figure 17 D, shows C57BL / 6J WT and Ism1 Δ / Δ Representative immunohistochemical staining of ISM1 (brown) and nuclei (hematoxylin, blue) in mouse lung sections. Br, ductules; Al, alveoli. Scale bar, 20 μm.

[0398] ISM1 deficiency leads to an enhanced acute immune response to LPS in the lung

[0399] To examine the role of ISM1 in acute lung inflammation, we expressed Δ / Δ 2 mg / kg LPS was instilled intratracheally into the lungs of WT and WT mice. Both groups of mice survived and developed an acute inflammatory response to LPS. Δ / Δ Mice showed a significant increase in total lung leukocytes during the 7-day acute response period ( Figure 10 A). In Ism1 Δ / Δ In mice, higher numbers of neutrophils ( Figure 10 B), T-cells ( Figure 10 D) and B-cells ( Figure 10 E), while increased macrophage recruitment was observed from day 3 ( Figure 10 C). By day 7, wild type and Ism1 Δ / Δ Neutrophils in mice decreased to basal levels, but in Ism1 Δ / Δ Higher numbers of alveolar macrophages, T and B cells were still observed in the lungs. Δ / ΔMuch higher total bronchoalveolar lavage (BAL) protein was observed in mice, reflecting hyperpermeability associated with excessive lung inflammation ( Figure 10 F). Consistently, histological analysis of lungs harvested 1 day after LPS challenge showed that Ism1 Δ / Δ The immune cells in the alveolar cavity of the lung increased significantly, accompanied by a significant increase in neutrophils at this time point ( Figure 10 G).

[0400] Thus, ISM1 deficiency in mice resulted in a more severe inflammatory response to respiratory LPS challenge, supporting a role for ISM1 in regulating lung inflammation.

[0401] Exogenous rISM1 inhibits LPS-induced inflammatory responses in the lung

[0402] Based on the above results, we hypothesized that ISM1 might inhibit LPS-induced inflammation. To test this hypothesis, we pretreated wild-type mice with 50 μg of rISM1 intratracheally one day before LPS instillation. rISM1 treatment was continued on the day of LPS instillation and for three more days thereafter ( Figure 11 BAL fluid was then collected and rISM1-treated mice indeed showed a significant reduction in total BAL protein ( Figure 11 B). Leukocyte infiltration into the alveolar space was reduced to almost basal levels (without LPS challenge) ( Figure 11 C). Neutrophils ( Figure 11 D) and alveolar macrophages ( Figure 11 The E) of rISM1 was significantly reduced under rISM1 treatment. Although T and B cells tended to decrease under rISM1 treatment ( Figure 11 These changes were not statistically significant due to high variability in the PBS-treated mouse group. Taken together, these findings support that ISM1 can function as a suppressor of lung inflammation and that locally delivered rISM1 quenches LPS-induced lung inflammation in mice.

[0403] ISM1 deficiency leads to defective lung repair and remodeling after LPS-induced acute lung injury

[0404] Inflammation is an important response to external insults, but it can also cause damage to tissues. Affected tissues attempt to repair the damage triggered by inflammation and restore tissue homeostasis. Excessive inflammatory responses can overwhelm repair mechanisms, leading to tissue remodeling. Δ / Δ Mice showed an enhanced immune response to LPS challenge, and we sought to determine whether this increased inflammatory response would affect lung repair and return to homeostasis. Lung tissue histology was examined on day 9 after LPS challenge. Figure 12As shown in A, Ism1 Δ / Δ The lungs showed severe distortion of lung architecture with extensive thickening of the alveolar arms. A significant increase in collagen deposition was also observed by Picro-Sirius red staining ( Figure 12 In addition, an increased abundance of myofibroblasts (α-smooth muscle actin positive) was noted, particularly in the small fiber clusters ( Figure 12 Excessive accumulation of extracellular matrix (ECM) and myofibroblasts is a hallmark of pulmonary fibrosis. 50 .

[0405] Following LPS challenge, the inflammatory response typically results in damage / injury to the epithelium. There are two types of alveolar epithelial cells: type I (AE1) cells are terminally differentiated, flat, squamous, and cover 90% of the alveolar wall surface; type II (AE2) cells are less numerous but have stem cell-like properties. 51 AE2 cells are important for lung repair and regeneration after injury. To maintain homeostasis and the integrity of the alveolar epithelium, AE2 cells proliferate and differentiate into AE1 cells to re-epithelialize the alveolar wall. 52,53 Abnormal replacement of AE1 cells by proliferative AE2 cells is a contributing factor to fibrosis 54 On day 9 after LPS challenge, Ism1 Δ / Δ Lungs of wild-type mice showed significantly more proliferating AE2 cells than wild-type lungs, as shown by double immunofluorescence staining of PCNA (proliferation marker) and SP-C (AE2 marker) ( Figure 13 This result suggests that AE2 over-proliferation may lead to the inhibition of Ism1 Δ / Δ Increased fibrosis in the lungs.

[0406] TGF-β is the most potent profibrotic mediator characterized to date 55 On day 9 after LPS challenge, Ism1 Δ / Δ TGF-β levels in the lungs were significantly increased ( Figure 14 ).

[0407] Together, these results demonstrate that ISM1 deficiency triggers an enhanced immune response to LPS challenge in mice, leading to aberrant lung repair and fibrosis.

[0408] ISM1 deficiency alters the acute inflammatory cytokine / chemokine profile in the lung after LPS challenge

[0409] To decipher whether ISM1 deficiency leads to altered inflammatory cytokines / chemokines in response to LPS, cytokine and chemokine profiles were examined using a cytokine antibody array.Δ / Δ Seven chemokines and cytokines in the lungs were significantly elevated ( Figure 15 All upregulated cytokines / chemokines were known proinflammatory mediators, such as IL-1α, IL-1β; leukocyte chemoattractants, such as monokine induced by gamma interferon (MIG), CXC chemokines such as CXCL10 / IP-10, MIP-1a, and MIP-2; and soluble ICAM-1. In addition, Ism1 Δ / Δ TNF-α in the lungs also increased significantly ( Figure 15 These results suggest that ISM1 deficiency in the lung leads to an increase in multiple proinflammatory cytokines, which may be responsible for the enhanced lung inflammatory response to LPS challenge.

[0410] ISM1 deficiency activates NF-κB signaling in the lung

[0411] Previous studies have shown that LPS activates NF-κB signaling in the murine lung by inducing nuclear translocation of NF-κB. 45,56 To determine whether ISM1 plays a role in regulating LPS-induced NF-κB translocation in vivo, Ism1 was expressed in LPS-treated cells against NF-κB (p65 subunit). Δ / Δ Lung tissues of WT and WT mice were fixed and stained. Compared with WT mice, Ism1 Δ / Δ Nuclear p65 NF-κB (red) was significantly increased in lung sections of mice ( Figure 16 In addition, Ism1 Δ / Δ p65NF-κB levels were also higher in the lungs ( Figure 16 These data indicate that Ism1 is expressed in response to LPS. Δ / Δ NF-κB is increased in the lung, a possible mechanism for the activation of multiple proinflammatory cytokines / chemokines and enhanced inflammation.

[0412] Using an LPS-induced ALI model, this study demonstrated that ISM1 deficiency leads to excessive inflammation in the mouse lungs. Furthermore, when administered intratracheally to mice, rISM1 can quench LPS-induced lung inflammation. These findings support ISM1 as an anti-inflammatory protein.

[0413] Some angiogenesis inhibitors have been previously reported to inhibit lung inflammation in addition to their anti-angiogenic capacity. For example, thrombospondin-1 (TSP-1) has been reported to be important in physiological inflammation and homeostasis in the lung. 57 As early as 1 month of age, Tsp1 - / -The mice initially displayed patchy sites of inflammation in their lung parenchyma. Neutrophil infiltration was observed in the alveoli and perivascular connective tissue. Furthermore, TSP-1-deficient mice were more susceptible to LPS-induced lung injury. 58 TSP-1 inhibits the inflammatory response by regulating the production of IL-10, a key anti-inflammatory cytokine in the resolution phase of lung injury. Angiostatin is another angiogenesis inhibitor that can inhibit LPS-induced acute lung injury in mice. 59 Treatment with angiostatin effectively reduced protein accumulation in BAL fluid and leukocyte infiltration into the lungs. In this study, ISM1 was shown to function in a manner somewhat related to TSP-1 function, as knockout mice exhibited enhanced inflammatory responses in the lungs under nonpathological conditions and were hyperresponsive to LPS-induced acute lung injury. Concurrently, both exogenous angiostatin and ISM1 suppressed acute lung inflammation after LPS challenge.

[0414] Although Ism1 Δ / Δ The lungs showed a more severe inflammatory response to LPS, but the inflammation peaked on day 3 and then began to subside and reached basal levels by day 7, similar to those of wild-type mice. Δ / Δ The lungs exhibited a widespread fibrotic phenotype, with increased collagen deposition, myofibroblast accumulation, and higher levels of TGF-β expression. In addition, an increase in the number of proliferating alveolar epithelial type II (AE2) cells was observed. These data point to a "hyperresponsive" lung that responds to LPS with excessive inflammation, a condition that normally induces tissue remodeling. 60 ISM1 may play a role in suppressing excessive inflammation induced by LPS in the lungs, thereby protecting the lungs from excessive damage and injury. Δ / Δ Excessive inflammation in the lungs may overwhelm repair mechanisms, leading to structural changes and fibrosis.

[0415] The NF-κB pathway plays a role in LPS-induced inflammation 45 ,56 ,61 Activation of NF-κB leads to the translocation of its active form p65 into the nucleus. LPS can enhance the translocation of NF-κB p65 from the cytoplasm to the nucleus. 61,62 LPS-induced NF-κB activation is known to increase the expression of pro-inflammatory cytokines such as IL-1, MIP-2, and TNF-α, leading to an excessive inflammatory response. 56 ,63 ,64 The increased abundance and nuclear translocation of the active form of NF-κB under ISM1-deficient conditions suggest that the NF-κB signaling pathway is involved in the upregulation of multiple proinflammatory cytokines.

[0416] ALI / ARDS is characterized by the presence of large numbers of activated neutrophils and fluid accumulation in the lungs, leading to impaired lung function and high mortality. 65 ALI and ARDS are common lung diseases in the human population. 66 Unfortunately, no major breakthroughs or discoveries of new treatments for these two diseases have been reported in this field. 60 The findings described herein demonstrate that intratracheally delivered rISM1 is sufficient to effectively reduce leukocyte infiltration in LPS-challenged mice. Importantly, it has been demonstrated that ISM1 treatment also inhibits LPS-induced lung hyperpermeability ( Figure 11 ).

[0417] The results support the idea that ISM1 can provide an inhibitory / pro-resolution mediator against pulmonary inflammasomes. It inhibits the excessive accumulation of alveolar macrophages by inducing apoptosis. Locally delivered rISM1 rescued the emphysematous phenotype in mice and helped preserve lung homeostasis. Therefore, these results support the use of ISM1-based therapeutic approaches for treating, ameliorating, and / or preventing inflammatory lung diseases such as ALI and COPD.

[0418] Materials and methods

[0419] Adult (7- to 8-week-old) female ISM1-deficient (Ism1 Δ / Δ ) mice. Age- and sex-matched wild-type C57BL / 6J mice were obtained from the Jackson Laboratory. Animal care and experimental procedures were performed in accordance with institutional guidelines approved by the NUS Institutional Animal Care and Use Committee (IACUC; protocols 066 / 12 and R16 / 0632; breeding agreement BR15 / 1100). The references used for T7E1 assays and genotyping are as follows:

[0420] PCR primers used for T7E1 assay to screen mutant mice:

[0421] forward 5'cagctcctgggattgctccg 3' (SEQ ID NO: 16) and reverse 5'taagacttcttcctggtgccaaa 3' (SEQ ID NO: 17);

[0422] PCR primers for mouse genotyping:

[0423] forward 5'gacagctcctgggattgctcc 3' (SEQ ID NO: 18) and reverse 5'ttctgcaatgtaccaagctctct 3' (SEQ ID NO: 19);

[0424] (See Figure 17 ).

[0425] Recombinant protein. Recombinant ISM was expressed in Escherichia coli and purified using Ni-NTA affinity chromatography, followed by reverse phase HPLC. The recombinant protein was confirmed to be endotoxin-free. In the rescue experiment, rISM1 was dissolved in filtered PBS and 50 μg of rISM1 per mouse was administered intratracheally into the lungs. Mouse rISM (mature form, without signal peptide) was expressed in Escherichia coli using the vector pET-M (as described in Xiang et al., 2011, JCMM, incorporated herein by reference in its entirety) and purified as a His-tagged protein. Mouse rISM1 was used in these experiments. Mature ISM1 (without signal peptide) may have biological significance under the test conditions. The ISM1 sequence used contained NP_001263418.1 and is as follows:

[0426]

[0427] (SEQ ID NO: 9; underline indicates the native ISM1 sequence, bold indicates the vector sequence and His-tag, italics indicate the N-terminal M residue)

[0428] Intratracheal instillation. As Liao et al. 67 As previously described, mice were anesthetized using 5% isoflurane (Baxter) followed by intratracheal delivery of LPS (2 mg / kg LPS) from E. coli O111:B4 (L2630; Sigma Aldrich) or saline. Control animals received saline alone. Mice were allowed to recover until bronchoalveolar lavage (BAL) collection time on days 1, 3, 5, and 7 after LPS instillation for subsequent analysis.

[0429] Bronchoalveolar lavage (BAL) collection. Freshly euthanized mice were dissected to expose the lungs and heart. The trachea was intubated and the lungs were lavaged twice with 1 ml of ice-cold PBS. BAL samples were centrifuged at 500 x g for 5 minutes at 4°C. The supernatant was collected and stored at -80°C until use. BAL protein was quantified using Bradford reagent. 1 mL of red blood cell lysis buffer was added to the cell pellet to lyse all red blood cells, followed by centrifugation. Viable cells were recovered in FACS buffer and counted using Nucleocounter NC-100 (Chemometec, Denmark), followed by differential immune cell counting.

[0430] Differential immune cell counts. Differential immune cell counts were studied using a NovoCyte flow cytometer and analyzed using NovoExpress software (Acea Biosciences, USA). Differential immune cell counts were performed as previously described. 68 Immune cells are recognized by CD45 + , alveolar macrophages are CD11c + Siglec-F + eosinophils are CD11c - Siglec-F + , neutrophils are GR-1 + CD11b + , B cells are CD3 - / CD19 + and T cells are CD3 + / CD19 - cell.

[0431] Peripheral blood leukocyte counts: Blood was collected from the submandibular vein of anesthetized mice. Fresh blood samples were analyzed using a Hemavet H950FS hematology analyzer (Drew Scientific Group, USA).

[0432] Histology. Fully inflated lungs were fixed in 10% neutral buffered formalin, then paraffin-embedded, sectioned, and stained with hematoxylin and eosin or Picro-Sirius Red stain (ab150681, Abcam, USA). Sections (5 μm) were dewaxed in histoclear and then slowly rehydrated in a series of alcohol isocratic solutions starting with 100% ethanol. After hydration in water, sections were placed in PBS for subsequent staining.

[0433] Immunohistochemistry (IHC) and immunofluorescence (IF) were performed using anti-CD68 (sc-7084, Santa Cruz Biotechnology), anti-NIMP-R14 (sc-59338, Santa Cruz Biotechnology), anti-α-SMA (Santa Cruz Biotechnology), anti-TGF-β (sc-146, Santa Cruz Biotechnology), anti-SP-C (sc-13979, Santa Cruz Biotechnology), anti-p65NF KTissue sections were stained with anti-B (107450-1-AP, Proteintech) and anti-PCNA (sc-56, Santa Cruz Biotechnology) overnight at 4°C. Tissue sections were stained with hematoxylin and eosin (DAKO). All images were acquired using a Zeiss Axiovert.

[0434] Immunoblotting and protein arrays. Fresh tissue was homogenized and centrifuged, and the soluble supernatant was used as a whole tissue lysate. Standard western blots were performed using β-actin as a loading control. The antibodies used were anti-TGF-β (sc-146, Santa Cruz Biotechnology), anti-TNF-α (107590-1-AP, Proteintech), and anti-p65 NF-κB (10745-1-AP, Proteintech). The relative abundance of 40 cytokines was examined using the Mouse Cytokine Proteome Analyzer Array (ARY028, R&D Systems, USA). Whole tissue lysates from 4 mice in each group were used. 200 μg of total protein per sample was hybridized to the array and relative expression was compared. Relative expression was quantified by measuring dot blot intensity using Image J software.

[0435] Statistical analysis. Data are expressed as mean ± standard error (± SEM). Statistical significance was determined using Student's t-test. *P < 0.05; **P < 0.01, n ≥ 3.

[0436] Example 3 - The C-terminal AMOP domain alone is sufficient to mediate the pro-apoptotic activity of ISM1.

[0437] In this example, the structure-function relationship of ISM1 was investigated. Methods were first developed for the expression and purification of bacterial and mammalian recombinant ISM1 (rISM1) proteins. The 15 kDa mass difference between the two rISM1s prompted us to investigate the glycosylation profile of mammalian rISM1, which revealed a dense and highly heterogeneous glycan deposition. Next, by performing co-immunoprecipitation assays and apoptosis assays using various rISM1 truncated proteins containing individual domains, we further demonstrated that the adhesion-associated domain in MUC4 and the other protein (AMOP) domain of ISM1 mediated two receptor interactions (i.e., integrin αvβ5 and cell surface GRP78 (csGRP78)). Accordingly, the AMOP domain of ISM1 exhibited the complete pro-apoptotic activity of the full-length protein.

[0438] Expression and purification of recombinant ISM1 protein.

[0439] To elucidate the structure-function relationship of ISM1, we first set out to generate and purify recombinant ISM1 (rISM1). Because ISM1 is a secreted protein, we first utilized a mammalian expression host. To create a mammalian expression construct, the native signal peptide of mouse ISM1 was replaced with the mouse Igκ1 leader sequence to improve secretion efficiency ( Figure 31 The expression construct was then transfected into Expi293F cells, and the recombinant protein in the conditioned medium was purified via one-step IMAC (immobilized metal affinity chromatography) ( Figure 31 Purified mammalian rISM1 migrates at approximately 70 kDa on denaturing SDS-PAGE ( Figure 31 C).

[0440] To express soluble rISM1 from bacterial hosts, a SUMO (small ubiquitin-like modifier) ​​tag was fused to the N-terminus of mouse ISM1 without a signal peptide sequence ( Figure 31 The cell expression construct was transformed into Shuffle T7 cells and the soluble protein was further purified by IMAC and SEC (size exclusion chromatography) ( Figure 31 E). After cleavage of the SUMO tag, bacterial rISM1 migrated at approximately 55 kDa on denaturing SDS-PAGE ( Figure 31 F).

[0441] Mammalian rISM1 is densely deposited with heterogeneous glycans.

[0442] Surprisingly, there is a size difference of approximately 15 kDa between mammalian and bacterial rISM1, suggesting the presence of post-translational modifications (PTMs) on mammalian rISM1. We then focused on protein glycosylation, as it can significantly increase protein mass. Sequence analysis revealed two potential N-linked glycosylation sites on mouse ISM1, namely Asn39 and Asn282 ( Figure 32 A). Incubation of mammalian rISM1 with PNGaseF (peptide-N-glycosidase F) reduced the protein mass by approximately 10 kDa, confirming the presence of N-glycans ( Figure 32 B). N-glycosylation mutant constructs (N39Q, N282Q, N39 / 282Q) were further generated to disrupt one or two N-glycan sites, and the effects were evaluated in three cell lines (HEK293T, HEK293FT, HeLa) ( Figure 32C). Each single mutant exhibited a reduced protein size (~5 kDa) in the whole cell lysate (WCL) fraction, while the double mutant exhibited a total size reduction of ~10 kDa, confirming that both sites were modified by N-glycans. Interestingly, WT / N282Q exhibited a doublet in the WCL fraction, while N39Q and N39 / 282Q exhibited only a singlet ( Figure 32 C). Similar observations from a previous report suggest that this is due to inefficient core glycosylation at Asn39 (8). In HEK293T cells, disruption of the N-glycan site abolished protein secretion in conditioned medium ( Figure 32 C); however, protein secretion was abolished only in the Asn282 mutant in HEK293FT and HeLa cells. These results suggest that N-glycosylation at Asn282 is critical for regulating ISM1 secretion, while N-glycosylation at Asn39 is crucial in certain cell lines.

[0443] We then used a proteomic approach to further identify the presence of O- and C-linked glycans on mammalian rISM1. Strikingly, 30 additional amino acids were found to be glycosylated ( Figure 32 While some residues are modified with only a single glycan of simple structure, certain residues such as Ser184 and Ser188 are modified with multiple glycans of different structures, indicating microheterogeneity in glycan deposition at these positions. Focusing on the distribution of glycosylation sites, 21 glycan sites are located in the N-terminal unstructured region, 2 sites are located in the TSR domain, and 7 sites are located in the AMOP domain ( Figure 32 D). Since most glycan deposition and glycan complexity are present in unstructured regions, this may indicate a stabilizing effect of glycosylation on the folding of unstructured regions. Additionally, two unconventional glycosylation modifications have been reported to be conserved on TSR domains: O-fucosylation (O-fucose-glucose), which recognizes the Cxx(S / T)CG motif, and C-mannosylation (C-mannose), which has the recognition motif WxxW. On the ISM1-TSR domain, the two recognized glycan modifications are an O-linked disaccharide (deoxyhexose-hexose) on Thr229 (226-CSVTCG-231) and a C-linked monosaccharide (hexose) on Trp220 (220-WSLW-223) (Table S1), which may correspond to putative O-fucosylation and C-mannosylation, respectively.

[0444] Of these 30 glycosylation sites, four candidate sites were further selected for mutational analysis to evaluate their effects: Trp220 and Thr229, as they are putative C-mannosylation and O-fucosylation sites on the TSR domain; and Ser184 and Ser188, as they are highly heterogeneous sites with glycan deposition. Preliminary analysis using Western blotting showed that disruption of these glycosylation sites did not affect ISM1 expression or secretion ( Figure 32 In conclusion, our analysis shows that mammalian rISM1 is densely deposited with heterogeneous glycans.

[0445] The AMOP domain of ISM1 mediates its receptor interaction.

[0446] We have previously identified integrin αvβ5 and cell surface GRP78 as cell surface receptors for ISM1. To elucidate which domain of ISM1 mediates receptor interactions, we generated the following mammalian rISM1 proteins: mamISM1, which contains both domains. 26-461 , mamISM1 containing the TSR domain 26-286 and mamISM1 containing the AMOP domain 26-286( Figure 33 A and B). A recombinant GRP78 truncated form containing only the ATPase domain was expressed and purified from cells ( Figure 33 A, B). Mammalian recombinant integrin αvβ5 heterodimers with only the extracellular domain were commercially available. Co-immunoprecipitation assays using these purified recombinant proteins demonstrated that the AMOP domain alone was able to mediate both ISM1-GRP78 and ISM1-αvβ5 interactions ( Figure 33 In contrast, ISM1 truncations without the AMOP domain abolished binding to both receptors ( Figure 33 In conclusion, the ISM1-AMOP domain mediates its interaction with both receptors.

[0447] AMOP domain boundaries influence its pro-apoptotic activity.

[0448] To investigate the pro-apoptotic activity of the AMOP domain, we first generated two AMOP truncations, mamISM1 with different lengths. 271-461 and mamISM1 287-461 , because the exact boundary of the AMOP is not known and is only an estimate ( Figure 34 A, B). Surprisingly, although mamISM1 287-461 Induces EC apoptosis at levels similar to those in starvation states, while mamISM1 271-461 No induction of EC apoptosis ( Figure 34C). It appears that the 16 additional amino acids at the N-terminus completely abolish the pro-apoptotic effect of the AMOP domain.

[0449] To further investigate the impact of these 16 additional amino acids, we generated the following AMOP truncations of varying lengths in this region: bacISM1 271-461 、bacISM1 282-461 、bacISM1 283-461 、bacISM1 287-461 They are produced from bacterial expression systems via fusion to a SUMO-tag ( Figure 34 Interestingly, as the additional sequence becomes shorter, the AMOP truncations become more active ( Figure 34 F). Thus, the structure suggests that the boundaries of AMOP domains can influence their pro-apoptotic activity.

[0450] When the pro-apoptotic activities of bacterial and mammalian recombinant AMOP truncations were closely compared, it was noted that bacAMOP exhibited similar activity to mamAMOP. 271-461 and mamISM1 271-461 No activity was shown, but bacISM1 287-461 and mamISM1 287-461 showed similar levels of pro-apoptotic activity ( Figure 34 Thus, the results indicate that AMOP activity is not affected by post-translational modifications (PTMs).

[0451] Indeed, structure-function relationship studies using truncated mouse ISM1 proteins have shown that the C-terminal AMOP domain from 287-461 retains the full pro-apoptotic properties of the full-length ISM1 protein ( Figure 29 ). In addition, the results showed that mamISM1 produced by mammalian cells 287-461 The fragment was compared to the bacISM1 produced by E. coli 287-461 The fragments (AMOP domain alone) had the same level of pro-apoptotic activity, indicating that the AMOP domain does not require post-translational modification to mediate the pro-apoptotic activity of ISM1. This is also consistent with the co-IP binding assay results, indicating that ISM1 287-461 It is sufficient for its direct binding to the GRP78 receptor ( Figure 30 Thus, both GRP78 receptor binding and pro-apoptotic functions are mediated through the C-terminal AMOP domain from amino acid residues 287-461 (amino acid residues 290-464 in the human ISM1 equivalent).

[0452] ISM1 287-461(C) Not ISM126-277(N) Supports EC adhesion

[0453] We previously showed that surface-coated rISM1 can support EC adhesion and attachment. To investigate the role of ISM1 in supporting EC attachment, we used Cell adhesion assays were performed using a live cell analysis imaging system, which can monitor the dynamic changes of cell attachment.

[0454] Gelatin was used as a positive control because it is an extracellular matrix protein and supports EC adhesion. The gelatin-supported EC adhesion can be seen by the increase in confluence over time ( Figure 21 In contrast, serum protein BSA was used as a negative control. The confluence of EC in BSA-coated wells remained low or even decreased over time, indicating that BSA did not support cell adhesion ( Figure 21 ).

[0455] In the comparison of ISM1 and its truncations, it was observed that ISM1 containing the AMOP domain (287-461) C Can support ISM1 FL Comparable cell adhesion, as there was no significant difference in the ability of the two to support cell attachment ( Figure 19 In contrast, ISM1 lacking the AMOP domain N Shows reduced ability to support cell adhesion ( Figure 19 This result indicates that the AMOP domain in ISM1 is important in mediating EC adhesion. C Mutations in the RKD motif (RKD341RAA, RKD340AAA) resulted in a significant decrease in cell attachment ( Figure 19 B).

[0456] Internalized ISM1 287-461(C) Inducing EC apoptosis

[0457] To further investigate the internalization of ISM1 C Does EC have ISM1 FL The same pro-apoptotic effect, using EC apoptosis assay was performed using a live cell analysis imaging system. Briefly, HUVECs were seeded in 96-well plates (6,000 cells / well) and 1 μM rISM1 protein (including rISM1 N 、rISM1 C and rISM1 FL ) for up to 24 hours. From the corresponding apoptosis curve ( Figure 20 A), found ISM1 C and ISM1 FLIn contrast, ISM1 N This result is consistent with ISM1 C Not ISM1 N The results of internalization assays were consistent with those of EC internalization assays. Therefore, based on these results, the AMOP domain may be responsible for the pro-apoptotic function of ISM1.

[0458] Sequences relevant to these studies (mammalian and bacterial) are as follows:

[0459] SEQ ID NO:28: bacISM1 26-461

[0460] (recombinant mouse ISM1 full length)

[0461] (underlined : native mouse ISM1 sequence; bold: His-tag and vector sequence; italics: SUMO-tag)

[0462] SEQ ID NO:29: bacISM1 287-461

[0463] (Recombinant mouse ISM1 AMOP domain)

[0464] (underlined : native mouse ISM1 sequence; bold: His-tag and vector sequence; italics: SUMO-tag)

[0465] SEQ ID NO:30: mamISM1 26-461

[0466] (recombinant mouse ISM1 full length)

[0467] ( Underlined : native mouse ISM1 sequence; bold: vector sequence and His-tag; italics: Myc-tag)

[0468] SEQ ID NO:31:mamISM1 287-461

[0469]

[0470] (Recombinant mouse ISM1 AMOP domain)

[0471] ( Underlined : native mouse ISM1 sequence; bold: vector sequence and His tag; italics: Myc-tag)

[0472] SEQ ID NO:32: mamISM1 26-464

[0473] (recombinant human ISM1 full length)

[0474] ( Underlined : native human ISM1 sequence; bold: vector sequence and His-tag; italics: Myc-tag)

[0475] SEQ ID NO:33: bacISM1 26-464

[0476]

[0477] (recombinant human ISM1 full length)

[0478] ( Underlined : native human ISM1 sequence; bold: vector sequence and His-tag; italics: FLAG-tag)

[0479] SEQ ID NO:34: bacISM1 290-464

[0480] (recombinant human ISM1 AMOP domain)

[0481] ( Underlined : native human ISM1 sequence; bold: vector sequence and His-tag; italics: FLAG-tag)

[0482] SEQ ID NO:35: mamISM1 290-464

[0483]

[0484] (recombinant human ISM1 AMOP domain)

[0485] ( Underlined : native human ISM1 sequence; bold: vector sequence and His-tag; italics: Myc-tag)

[0486] These results support the idea that ISM1 287-461 (22 kDa) (human ISM1 equivalents are amino acid residues 290-464) may function in a similar manner to the full-length rISM1 protein in inhibiting CS-induced lung inflammation in mice. For example, rISM1 and rISM1 287-461 (The human ISM1 equivalents are amino acid residues 290-464) could provide a therapy for COPD.

[0487] In this example, mammalian and bacterial expression systems were used to produce soluble, functionally active recombinant ISM1 protein. This approach not only facilitates structure-function studies but can also be used to produce ISM1 for various applications.

[0488] The apparent size difference between mammalian and bacterial rISM1 prompted us to investigate the protein glycosylation profiles on ISM1. We unexpectedly discovered that mammalian rISM1 is a highly heterogeneous glycoprotein, and that the extensive deposition of glycans significantly contributes to protein quality. Nevertheless, it should be noted that protein glycosylation is influenced by cell type. Different cells express different types and levels of glycosylation enzymes, thus resulting in variations in glycan profiles. For example, significant differences in glycan profiles were observed between proteins produced in CHO cells and HEK293 cells. HEK293-produced proteins generally contain more complex glycan structures, whereas CHO-derived proteins have higher levels of sialylation. In another example, the O-glycosylation profiles of 12 human cell lines of different tissue origin were analyzed, which revealed a unique O-glycoprotein group within each cell line (Steentoft, C., Vakhrushev, SY, Joshi, HJ, Kong, Y., Vester-Christensen, MB, Schjoldager, KT, Lavrsen, K., Dabelsteen, S., Pedersen, NB, Marcos-Silva, L., Gupta, R., Bennett, EP, Mandel, U., Brunak, S., Wandall, HH, Levery, SB, and Clausen, H. (2013) Precision mapping of the human O-GalNAc glycoproteome through Simple Cell technology. EMBO J 32, 1478-1488). More than half of the identified sugar sites were found in only one cell line, and each cell line also contributed a plethora of unique glycoproteins. Therefore, protein glycosylation analysis performed on mammalian rISM1 produced in Expi293F cells can be used as a reference but may differ in other contexts.

[0489] Unexpectedly, this example shows that ISM1-OP, but not TSR, is mediating receptor binding and pro-apoptotic activity. As different ISM1-AMOP constructs exhibited different activities, it seems that the activity may be regulated at least in part by the border sequence.

[0490] Methods and Materials

[0491] Construct construction

[0492] To generate a mammalian expression construct of ISM1, mouse ISM1 cDNA was amplified by PCR and cloned into the pSECtag-2B vector (Invitrogen) via the BamHI and XhoI restriction enzyme sites. This vector contains an N-terminal signal peptide from the mouse Igκ1 leader sequence to allow efficient protein secretion, as well as a C-terminal hexahistidine tag and a Myc tag to facilitate protein detection and purification.

[0493] To generate a bacterial expression construct of ISM1, SUMO-tagged cDNA was first ligated to the N-terminus of mouse ISM1 cDNA via overlapping PCR and further cloned into the pRSFDuet-1 vector (Novagen) via BamHI and XhoI restriction enzyme sites. This vector contains an N-terminal hexahistidine tag to allow protein detection and purification.

[0494] Cell lines and cell culture

[0495] HUVECs from Merck (SCCE001) were cultured in EndoGROLS complete medium (Merck, SCME001) supplemented with HyClone antimycotic solution (GE Healthcare) to a final concentration of 100 units / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B. HUVECs between passages 4 and 8 were used for the experiments.

[0496] Human embryonic kidney 293T cells (HEK293T) were obtained from the American Type Culture Collection (ATCC); human embryonic kidney 293FT cells (HEK293FT) were a gift from Dr. Adam Yuan's laboratory (Department of Biological Sciences, National University of Singapore); and a human cervical cancer cell line (HeLa CCL-2) was obtained from ATCC. All of these cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) (GE Healthcare) supplemented with 10% fetal bovine serum (GE Healthcare) and HyClone antimycotic solution (GE Healthcare) to a final concentration of 100 units / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B.

[0497] Antibodies and reagents:

[0498] The antibodies used for immunoprecipitation and immunoblotting are as follows: anti-His (A00186, GenScript); anti-Myc (ab9106, Abcam); anti-GRP78 (LS-C165064, LSBio); anti-integrin αvβ5 (P1F76, Santa Cruz); anti-integrin αv (ab179475, Abcam); anti-integrin β5 (ab184312, Abcam).

[0499] The following reagents were used: Caspase-3 / 7 Green Detection Reagent (C10423, Invitrogen); PNGaseF (P0704S, New England Biolabs); SUMO Protease (SAE0067, Sigma).

[0500] transient transfection

[0501] For transient transfection in HEK293T / HEK293FT / HeLa cells, lipofectamine 3000 (Invitrogen) was used according to the manufacturer's instructions. Twenty-four hours after transfection, the culture medium was replaced with fresh serum-free medium. After an additional 24 hours of incubation, conditioned medium and whole-cell lysates were collected and analyzed by Western blotting.

[0502] Mammalian recombinant ISM1 expression and purification

[0503] To express and purify mammalian recombinant ISM1 protein, 50 ml of Expi293F suspension cell culture in Expi293 medium (Gibco) was transiently transfected with 1 μg / mL expression plasmid complexed with Expifectamine 293 (Gibco) transfection reagent according to the manufacturer's protocol. The culture was incubated at 37°C, 8.0% CO2, with a 25 mm orbital diameter and a shaking speed of 120 rpm. After 16 hours, expression enhancer was added. Cell viability was monitored using a Countess II (Invitrogen). When cell viability fell below 70%, spent culture medium was separated from cells by centrifugation at 3000 × g and adjusted with conditioning buffer (5x conditioning buffer: 100 mM HEPES, 150 mM NaCl, 25 mM imidazole, 2.5% (v / v) glycerol, pH 7.5, 1x protease inhibitor cocktail). Two milliliters of Smart Ni-NTA resin (BioBasic) 50% slurry balanced in conditional buffer was added to the conditioned medium and incubated for at least one hour under gentle stirring. The resin was then collected and washed with IMAC buffer (50mM HEPES, 300mM NaCl, 10% (v / v) glycerol, pH 7.5) containing an increasing concentration of imidazole. The protein was eluted with the buffer containing 500mM imidazole. A PD10 desalting column (GE Healthcare) was balanced in a storage buffer (20mM HEPES, 300mM NaCl, 10% (v / v) glycerol, pH 7.5) and used for buffer exchange IMAC elution fractions. Protein was then concentrated using a Vivaspin centrifugal concentrator (Sartorius) with a 30KDa molecular weight cut-off. Final protein concentration was measured using Nanodrop (ThermoFisher). The protein was aliquoted, quick-frozen in liquid nitrogen and stored at -80°C.

[0504] Bacterial recombinant ISM1 expression and purification

[0505] To express and purify the recombinant ISM1 protein, the SUMO-ISM1 bacterial expression construct was first transformed into Shuffle T7 cells (New England Biolabs) following the manufacturer's instructions. A single colony was inoculated into a starter culture (10 ml) and grown overnight at 220 RPM in a 30°C shaker incubator. The next day, all the starter cultures were poured into 1 L of culture medium and the cells were grown at 220 RPM in a 30°C shaker incubator until OD 600 nm reached approximately 0.4-0.6. To induce protein expression, IPTG was then added to a final concentration of 0.25 mM. Induction was performed at 16°C shaker incubator at 180 RPM for 16-20 hours. Finally, the bacterial pellets were collected by centrifugation at 5,000 × g, 4°C for 10 minutes.

[0506] Before initiating bacterial lysis, an osmotic shock was performed to remove periplasmic proteins from E. coli (19). The cell pellet was first resuspended in sucrose buffer (50 mM HEPES, 20% sucrose, 1 mM EDTA, pH 7.4; 10 mL per liter of culture) and then centrifuged at 7000 × g, 4°C for 30 minutes. The pellet was further resuspended in 5 mM MgSO4 (10 mL per liter of culture) and incubated on ice for 10 minutes, followed by further centrifugation at 4500 × g, 4°C for 20 minutes. The supernatant was discarded and the bacterial pellet was kept on ice until lysis.

[0507] To initiate bacterial lysis, lysozyme (final concentration 1 mg / mL) and a protease inhibitor cocktail (Roche) were freshly added to bacterial lysis buffer (50 mM Tris, 250 mM NaCl, 40 mM imidazole, 10% glycerol, pH 7.5). The cell pellet was then resuspended in lysis buffer (10 mL / L culture) and incubated on ice for at least 10 minutes. Afterwards, ultrasonic treatment was performed for 10 minutes at 20% amplitude and 1 second on / off intervals to aid lysis. The lysate was centrifuged at 13,000 × g, 4°C for 20 minutes to clarify. The supernatant containing the soluble recombinant protein was filtered through a 0.45 μm syringe filter unit (Sartorius) and transferred to a new tube.

[0508] For immobilized metal affinity chromatography (IMAC) purification, a HisTrap column (GE Healthcare) was first washed with 5 column volumes of IMAC elution buffer (50 mM Tris, 250 mM NaCl, 250 mM imidazole, 10% glycerol, pH 7.5), followed by 10 column volumes of IMAC binding buffer (50 mM Tris, 250 mM NaCl, 40 mM imidazole, 10% glycerol, pH 7.5). The clarified bacterial lysate was loaded onto the chromatographic column using a pure 25M chromatography system (GE Healthcare). At least 10 column volumes of IMAC binding buffer were used to wash away unbound proteins. The bound proteins were finally eluted using a 20 column volume linear gradient of IMAC elution buffer.

[0509] Size exclusion chromatography (SEC) was performed as the second step of final purification. The previously eluted fractions with relatively high purity from IMAC were merged and loaded onto a Superdex200 10 / 300 column (GE Healthcare) pre-equilibrated with SEC buffer (50 mM Tris, 250 mM NaCl, 10% glycerol, pH 7.5). The peak fractions from SEC were collected, concentrated with an Amicon ultracentrifugal filter (Millipore), and the protein concentration was measured using the Bradford assay. The purified protein was aliquoted, snap-frozen in liquid nitrogen, and stored at -80°C.

[0510] Co-immunoprecipitation

[0511] To perform a Co-IP assay between purified recombinant ISM1 and GRP78 proteins, 2 μg of GRP78 antibody (LS-C165064, LSBio) was first conjugated to protein A / G agarose beads (Santa Cruz Biotechnology) by incubating with 20 μL of beads in PBS at 4°C for 2 hours. The antibody-conjugated beads were then centrifuged at 5,000 x g, 4°C for 3 minutes to remove unbound antibody. Next, 4 μg of rGRP78 and 2 μg of rISM1 were added to the beads in 1 mL of Co-IP binding buffer (PBS, pH 7.4) and incubated overnight at 4°C on a rotator. The next day, the beads were further washed 3 times with Co-IP wash buffer (PBS containing 0.1% Tween 20), with 10 minutes between each wash. The antibody-protein complex was finally eluted from the beads with 20 μL of 2xSDS loading dye and analyzed using Western blotting.

[0512] Apoptosis assay

[0513] To measure endothelial cell apoptosis induced by recombinant ISM1, the assay was performed using the IncuCyte ZOOM live cell imaging system (Essen Bioscience). HUVECs were seeded in 96-well plates at a density of 5500 cells / well overnight. The next day, HUVECs were first starved for 3 hours in 2% FBS medium and then treated with recombinant protein in the presence or absence of VEGF (293-VE-010, R&D Systems). Caspase-3 / 7 green apoptosis detection reagent (C10423, Invitrogen) was also included at a 1:1000 dilution to measure apoptotic events. Apoptosis measurements were performed every hour for a total of 24 hours. Final summary statistics were compiled from at least three separate biological replicates.

[0514] statistics

[0515] Statistical analysis and plotting of results were performed using GraphPad Prism software. Comparisons between two experimental groups were performed using an unpaired, two-tailed Student's t-test. Results are plotted as mean ± sem. P values ​​less than 0.05 were considered significant.

[0516] Example 4 - Recombinant ISM1 inhibits inflammation in house dust mite (HDM)-induced asthma in mice

[0517] Asthma is a disease of the large airways and the asthmatic immune response is primarily mediated by eosinophils, rather than AMs or neutrophils. The HDM-induced asthma model is a widely used asthma model for studying allergic asthma.

[0518] According to Hammad et al. (2009) and Peh et al. (2015) and Figure 26 The mouse asthma model was generated using the protocol described in . Briefly, female C57BL / 6J mice (6-8 weeks) were anesthetized with isoflurane and sensitized with 40 μL of 100 μg HDM extract (house dust mite) via the intratracheal route on days 0, 7, and 14. Two hours after the HDM challenge, on days 15 and 16, a single daily dose of bacterially produced recombinant ISM1 (2 mg / kg, 40 μg / mouse) or an equal volume of vehicle (normal saline) was continuously administered. All mice were euthanized on day 17, and bronchoalveolar lavage fluid (BALF) was collected for immune cell infiltration analysis. A naive group consisting of five healthy mice was used as a control.

[0519] like Figure 27As shown, intratracheal delivery of rISM1 effectively inhibited HDM-induced asthmatic airway inflammation, as shown by a significant decrease in total leukocytes, eosinophils, and lymphocytes in mice treated with rISM1. Consistent with previous reports, intratracheal HDM mainly triggers eosinophil-mediated airway inflammation, with neutrophils rarely involved. Under rISM treatment, eosinophils were suppressed by more than 70%. Alveolar macrophages (AMs) were also increased after HDM challenge, and rISM treatment showed a decrease in AMs under this treatment regimen, although there was no statistical significance. The results show that airway-delivered ISM1 can inhibit HDM-induced airway inflammation in mouse asthma. These results support the use of rISM1 for the treatment of asthma and the preparation of anti-asthma drugs.

[0520] Elevated serum immunoglobulin E (IgE) levels are a hallmark of Th2 immune responses. Serum was collected from mice 24 hours after the last rISM treatment. Compared with the HDM-treated group, rISM1 treatment significantly reduced total blood IgE levels, indicating that ISM1 suppressed Th2 immune responses ( Figure 28 ).

[0521] One or more illustrative embodiments have been described by way of example. It will be apparent to those skilled in the art that numerous changes and modifications may be made without departing from the scope of the invention as defined in the claims.

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Claims

1. Use of Isthmin 1 (ISM1) protein or its GRP78 activation fragment in the manufacture of a medicament for the following diseases: chronic obstructive pulmonary disease (COPD); asthma; acute lung injury (ALI) or acute respiratory distress syndrome (ARDS); in, The ISM1 protein consists of SEQ ID NO: 1, 2, 3 or 4, Wherein, the GRP78 activation fragment consists of the following amino acid sequence: OR and Wherein, the ISM1 protein or the GRP78 activation fragment is to be administered to the lungs of the subject via a nebulizer, a metered dose inhaler (MDI) or a dry powder inhaler (DPI).

2. The use according to claim 1, wherein The chronic obstructive pulmonary disease (COPD) is chronic obstructive bronchitis or emphysema.

3. The use according to claim 1 or 2, wherein The ISM1 protein or the GRP78 activation fragment thereof is used for intratracheal administration, intranasal administration or inhalation administration to the subject.

4. The use according to claim 1 or 2, wherein the ISM1 protein or the GRP78 activating fragment thereof is recombinant.

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