WO2023104199A1

WO2023104199A1 - Use of soluble receptor for advanced glycation end products protein for preventing or treating pulmonary infection diseases

Info

Publication number: WO2023104199A1
Application number: PCT/CN2022/138041
Authority: WO
Inventors: 肖瑞平; 张秀琴; 胡新立; 王珏; 吕凤祥; 温蔚
Original assignee: 南京景瑞康分子医药科技有限公司
Priority date: 2021-12-10
Filing date: 2022-12-09
Publication date: 2023-06-15
Also published as: CN116251171A

Abstract

The present invention relates to a use of a soluble receptor for advanced glycation end products (sRAGE) protein, and a functional variant or fragment thereof for preventing or treating pulmonary infection diseases, preferably viral pulmonary infections, more preferably viral pulmonary infections caused by coronaviruses. The present invention also relates to a pharmaceutical composition comprising the sRAGE protein and the functional variant or fragment thereof. The present invention also relates to a method for preventing or treating pulmonary infections, comprising the usage of the sRAGE protein, the functional variant or fragment thereof, or the composition.

Description

Use of soluble advanced glycation end product receptor protein for preventing or treating pulmonary infection

technical field

The invention relates to the field of using macromolecule drugs to treat diseases related to lung infection of organisms. Specifically, it relates to the use of soluble receptor for advanced glycation end products (sRAGE) protein for preventing or treating lung infection-related diseases, especially related to coronavirus (HCoV) such as new coronavirus (COVID-19) infection of the above-mentioned diseases. It also relates to functional variants and fragments of sRAGE, or pharmaceutical compositions comprising sRAGE or functional variants and fragments thereof, and their use in preventing or treating the above diseases.

Background technique

Regarding coronaviruses, especially the novel coronavirus SARS-CoV-2, in addition to developing vaccines for vaccination for prevention, we are actively looking for safe and effective strategies to treat pneumonia and related symptoms caused by SARS-CoV-2 infection , in order to save the lives of patients and reduce the sequelae of patients with new crown after cure.

To date, several different types of vaccines are available and have proven their effectiveness in reducing the incidence of COVID-19 in mass vaccinations around the world. However, the development of therapeutic drugs for lung infection-related diseases caused by SARS-CoV-2 infection has lagged far behind. Current treatment options all have their limitations.

Currently reported clinical treatment drugs for COVID-19 include: convalescent plasma from convalescent patients, tocilizumab (for patients with extensive lung disease and severe patients, and for those with elevated IL-6, to block IL6 receptors), intravenous Inject COVID-19 human immunoglobulin, as well as remdesivir (antiviral), remdesivir + baricitinib (JAK inhibitor), dexamethasone (corticosteroid), and the combination of coronavirus spike protein Combination of neutralizing monoclonal antibodies bamlanivimab, casirivimab and imdevimab.

According to the clinical results, the existing drugs can only alleviate the disease to a certain extent and reduce the mortality rate, but the overall clinical efficacy is still uncertain. Especially for severe patients with inflammatory storm and sepsis, there is currently no effective treatment. In addition, partially cured patients had variable sequelae.

Like SARS-CoV and MERS, SARS-CoV-2 virus infection may trigger a cytokine storm and cause severe damage to multiple organs. SARS-CoV-2 new coronary pneumonia is a respiratory infectious disease, and the lung is the main organ attacked.

In the chest CT manifestations, small patchy shadows outside the lungs and changes in the lung interstitium can be seen in the early stage of new coronavirus pneumonia, and then develop into ground glass opacities, infiltrates, and lung consolidation in both lungs. Pulmonary consolidation can also occur, and pleural effusion is rare. The consolidation area mainly presents diffuse alveolar damage and exudative alveolitis. Serous fluid, fibrinous exudate, and hyaline membrane formation can be seen in the alveolar cavity, and mucus plug formation can also be seen in the small bronchi and bronchioles. Therefore, it is believed that reducing the organ damage caused by inflammation is of great significance to controlling the course of the disease, reducing sequelae, and ensuring the quality of life of patients after recovery. Existing anti-inflammatory treatments for COVID-19 will play a role, but none are very effective.

For example, treatment of patients with severe COVID-19 infection with the JAK1/2 inhibitor ruxolitinib, although the median time to clinical improvement was shorter, was not statistically different from a control group receiving placebo for the primary efficacy endpoint , it can be seen that the effect is not satisfactory (Y.Cao et al., J Allergy Clin Immunol, July 2020).

In the past period of time, many drugs that are considered to be mechanistically promising for the treatment of new coronary pneumonia have been tried in this field, but none of them have achieved the expected effect. Such examples include oxyquinoline, tocilizumab, and the antiviral drugs ensovibep, Ritonavir (K.-T. Choy, et al. Antiviral Research 178 (2020) 104786). This fact shows that although theoretically there are many candidates that can be used as therapeutic drugs for the new crown, it is difficult to predict whether these candidates can really work before conducting experimental research.

Receptor for advanced glycation end products (RAGE, receptor for advanced glycation end products) and its ligands are involved in the pathological process of many diseases. Studies have shown that it is related to the occurrence and development of related diseases such as aging, inflammation and cell stress.

Specifically, RAGE is an inducible pattern recognition receptor whose endogenous ligands are a series of damage-associated pattern molecules (DAMPs), including advanced glycation end products (AGEs), high-mobility group protein B1 (HMGB1), S100, pathogen DNA and protein, etc. Among them, HMGB1 plays an important role in inflammation-related diseases such as sepsis and autoimmune diseases. AGEs are a kind of glycotoxins. The accumulation of AGEs in the body leads to DNA damage, oxidative stress and inflammation, and thus is closely related to aging and degenerative diseases.

The downstream pathways of RAGE include JAK-STAT, PI3K-Akt, Ras-ERK, etc. The activation of these pathways can lead to enhanced intracellular immune response, promote the release of inflammatory factors and the production of reactive oxygen species (ROS). The RAGE pathway is involved in various pathological processes including inflammation, oxidative stress, and vascular endothelial dysfunction.

sRAGE (soluble receptor for advanced glycation end-products) is the N-terminal extracellular domain of RAGE, derived from the expression of enzyme-cleaved or spliced variants of the full-length receptor. A separate sRAGE protein can also bind to the ligand of RAGE, which prevents the RAGE protein from binding to these ligands by binding to the RAGE ligand, thereby effectively blocking the signal transduction of RAGE and playing an anti-inflammatory role. In addition, it has been reported It is said that sRAGE plays an anti-apoptotic role by inhibiting the JAK2/STAT3 signaling pathway.

Studies have shown that serum sRAGE levels in COVID-19 patients with pulmonary symptoms are higher than those in healthy controls and asymptomatic patients with COVID-19 infection (after age correction) (Kehribar et al., Cancer Gunaydin & Metin Ozgen (2020)). At the same time, it was found that if patients with diabetes were infected with the new coronavirus, the incidence and mortality of respiratory distress syndrome were higher than those of non-diabetic patients. The study showed that the overall level of sRAGE was also significantly increased in these patients, and some subtypes Compared with non-diabetic patients with new coronary pneumonia, the changes of the RAGE pathway are significantly different, suggesting that the RAGE pathway is involved in the occurrence and development of new coronary pneumonia.

However, so far there is no report on the use of sRAGE in the treatment of pneumonia virus infection, especially the disease caused by coronavirus infection, nor has it been studied whether sRAGE is effective for patients with severe new coronary pneumonia.

Therefore, there is still an urgent need to find therapeutic drugs and methods that can effectively inhibit lung inflammation, reduce the incidence of severe pneumonia, and reduce toxic and side effects.

Contents of the invention

By administering sRAGE to the hamster SARS-CoV-2 infection model, the inventors found that it significantly improved the lung lesions of the hamster SARS-CoV-2 infection model. The experimental results show that sRAGE can reduce the excessive activation of inflammatory signals, reduce the infiltration of immune cells and the expression of inflammatory factors in the lungs, and significantly reduce the incidence of severe pneumonia in virus-infected model animals.

Based on these results, it was confirmed that sRAGE can be used as a therapeutic and/or preventive agent for COVID-19-infected pneumonia, thereby completing the present invention. In addition, the control of lung inflammation helps to reduce the systemic inflammatory response, thereby reducing the organ damage caused by inflammation, providing patients with a better chance of recovery and reducing sequelae.

The present invention includes the following contents:

In a first aspect, the present invention relates to the use of an isolated soluble receptor for advanced glycation end products (sRAGE) polypeptide in the preparation of a medicament for the prevention or treatment of viral lung infections, preferably caused by SARS-CoV -2 Viral lung infection caused by infection.

In a second aspect, the present invention provides a method for treating or preventing a disease associated with SARS-CoV-2 infection, comprising administering an isolated sRAGE polypeptide to a subject.

In a third aspect, the present invention provides a pharmaceutical composition comprising an isolated sRAGE polypeptide and a pharmaceutically acceptable carrier, and the pharmaceutical composition is used for treating or preventing diseases associated with SARS-CoV-2 infection.

Preferably, the isolated sRAGE polypeptide described in the first, second and third aspects above has the following characteristics:

(a) comprising or consisting of the amino acid sequence shown in SEQ ID NO:1;

(b) comprising or consisting of an amino acid sequence having at least 80% homology to the amino acid sequence set forth in SEQ ID NO:1, and has sRAGE activity; or

(c) it is a functional fragment of the polypeptide described in (a) or (b), and the functional fragment has sRAGE activity;

Preferably, the amino acid sequence described in (b) having at least 80% homology or the functional fragment described in (c) comprises one or more of the V domain, C1 domain and C2 domain of sRAGE indivual.

In the fourth aspect, the present invention relates to the combined use of the isolated sRAGE polypeptide of the present invention or its functional fragment or a pharmaceutical composition containing it and other drugs for treating diseases related to viral infection pneumonia. The other drugs are, for example but not limited to, convalescent plasma from convalescent patients, tocilizumab (for patients with extensive lung disease and severe patients, and IL-6 elevation, block IL6 receptors), intravenous injection of COVID-19 Human immunoglobulin, and remdesivir (antiviral), remdesivir + baricitinib (JAK inhibitor), dexamethasone (corticosteroid), and neutralizing mAbs that bind to the coronavirus spike protein Combination of bamlanivimab, casiri vimab and imdevimab.

In a fifth aspect, the present invention relates to an isolated nucleic acid molecule comprising a polynucleotide encoding a sRAGE polypeptide of the present invention.

The advantages of the present invention at least lie in the low toxicity and high safety of sRAGE. Compared with chemical drugs that may have strong side effects, sRAGE is a polypeptide that naturally exists in the human body. When it is used as a biological drug, it has no obvious toxic side effects, so it will not cause additional burden to patients who have already been infected by the virus.

Description of drawings

Fig. 1 is a schematic diagram of the administration scheme of the golden hamster animal model.

Fig. 2 is the histological section diagram of HE staining of hamster model lung, including negative control (uninfected control), positive control (human serum albumin (HSA) processing of 20ng/g body weight), and different doses (10ng/g and 20ng/g/ g body weight) of sRAGE treatment.

Figure 3 shows the proportion of experimental animals with different severity of pneumonia in each treatment group in the sRAGE treatment experiment.

Figure 4 shows the scores and statistics of pneumonia grading in the different treatment groups.

Figure 5 shows the mRNA levels of lung tissue inflammatory factors detected by qPCR in the treatment group and the control group.

Figure 6 shows the levels of neutrophils (NEUT) and lymphocytes (LYMPH) in peripheral blood leukocytes in the blood of each treatment group.

Figure 7 shows the mRNA expression levels of CD68 and the type 1 interferon response marker Mx1 in the lung tissues of the control group and each treatment group.

Fig. 8 is a graph showing the results of immunohistochemistry for CD3 (upper panel) and MxA (lower panel) of sections of lung tissues of each group.

Figure 9 shows the percentage of NF-κB activation changes caused by LPS stimulation after the wild-type (WT) and sRAGE with different modification site mutations were administered to lipopolysaccharide (LPS)-stimulated 293T-RAGE KI cells . The sRAGEs used included: commercially available sRAGE (“sRAGE”), plasmid-expressed wild-type sRAGE (“WT”), sRAGE with T5A mutation (“T5A”), sRAGE with S61A mutation (“S61A”), sRAGE with T5A and S61A double mutation ("T5A/S61A"), sRAGE with N3Q mutation ("N3Q"), sRAGE with N59Q mutation ("N59Q"), sRAGE with N3Q and N59Q double mutation ("N3Q/N59Q"), and a negative blank control ("NC") and a positive control ("LPS") stimulated only with LPS without the addition of sRAGE were included.

specific implementation plan

The practice of some of the methods disclosed herein employs, unless otherwise indicated, conventional techniques of biochemistry, chemistry, molecular biology, cell biology, genetics, immunology and recombinant DNA, which are within the skill of the art. See, eg, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012) et al.

sRAGE

The receptor for advanced glycation end products (RAGE) is a multiligand, pro-inflammatory pattern recognition receptor. It is involved in various conditions that cause chronic and sterile inflammation. RAGE expression is attenuated in many adult tissues except lung and skin.

RAGE recognizes ligands not through short peptide motifs, but through 3D structures. Therefore, it can be activated by various ligands, such as advanced glycation end products (AGEs), HMGB1, S100/calgranulin, β-amyloid, and even DNA and RNA molecules, pathogenic proteins, etc. Once activated, it transduces signals through several downstream kinases MAPKs, PI3K/Akt and JAK, which in turn activate the transcription factors NF-κB, AP-1 and Stat3. These transcription factors promote the expression of important cytokines such as TNFα, IL-1 and IL-6. These cytokines play important roles in lung infection-related diseases.

Soluble RAGE (sRAGE), as the name suggests, is the soluble form of RAGE. Structurally, sRAGE is the extracellular domain of RAGE, which consists of three immunoglobulin-like domains, namely V domain, C1 domain and C2 domain. As RAGE without a transmembrane domain and a C-terminal domain, sRAGE can interact with all ligands due to its extracellular structure and does not induce RAGE-based intracellular signaling cascades due to its lack of intracellular structure area. It is speculated that sRAGE or its functional fragments can act as a decoy receptor to alleviate the inflammatory response triggered by full-length RAGE.

Unlike chemical drugs that may have strong side effects, sRAGE is a polypeptide that naturally exists in the human body, and it has no obvious toxic side effects when it is used as a biological drug. Therefore, when used for treatment, the toxicity is small and the safety is high.

It is not intended to limit the mechanism. Based on the fact that the administration of sRAGE and its fragments does not affect the viral load in the hamster model, the inventors deduce that the therapeutic mechanism does not involve the killing of the virus itself, and does not depend on the specific strain Specific recognition and killing, so it is not affected by the mutation of the new coronavirus. In other words, even if a new mutant strain of the new coronavirus appears, the sRAGE of the present invention and the pharmaceutical composition containing it can effectively exert a therapeutic effect.

A further advantage of the present invention is that it is particularly effective for pulmonary infection, especially pulmonary infectious disease in which factor storm of various inflammatory factors occurs. When the sRAGE of the present invention is used to treat the infection caused by the new coronavirus, it can not distinguish and is not limited to the mutant strain type of the new coronavirus, and is not even limited to the lung infection caused by the new coronavirus.

Therefore, in one embodiment, the administration of sRAGE and its functional fragments can be applied to other lung infection-related diseases with related symptoms, not limited to lung infection-related diseases caused by novel coronavirus. In addition, similar to the lung infection caused by the new coronavirus, even if the virus has mutated, the preventive/therapeutic agent of the present invention still has protective and therapeutic effects on the mutant strain.

In one embodiment, sRAGE is preferably in its N-glycoform, particularly preferably in a form with polyvalent sialic acid, especially N-glycosylation at amino acids 3 and 59. Since insect and mammalian cells have different glycosylation pathways, glycoproteins expressed in the two systems contain polysaccharides with different structures. In addition to affecting the efficacy and in vivo half-life of glycoproteins, glycosylation modification is also a potential source of immunogenicity. Currently, biosafety rules established by major regulatory agencies (FDA, EMEA) require the production of human therapeutic glycoproteins from mammalian sources. Therefore, the N-glycosylated form of sRAGE (which may further contain O-glycosylation) would be a better therapeutic candidate as sRAGE produced in mammalian cells.

As the N-glycosylation form of sRAGE, sialylated N-glycosylation can be cited, preferably including N-glycosylation corresponding to amino acid 3 and/or 59 of SEQ ID NO:1. O-glycosylation can also be further included, especially O-glycosylation corresponding to amino acid 5 and/or 61 of SEQ ID NO:1. When the sRAGE polypeptide is a sRAGE variant having homology to the amino acid sequence of SEQ ID NO: 1 or a fragment thereof, "corresponding to the amino acid n position of SEQ ID NO: 1" means that in the variant or fragment it is identical to SEQ ID NO: The position corresponding to the nth amino acid in ID NO:1 does not mean that the position must be the nth position in the variant or fragment. Those skilled in the art can determine the corresponding positions in similar polypeptide sequences, for example, by aligning two or more similar polypeptide sequences to determine the corresponding positions therein.

In one embodiment, sRAGE or a fragment thereof may be used in combination with a JAK inhibitor to treat COVID-19. At present, JAK inhibitors (JAKi) have been used as anti-inflammatory strategies against COVID-19 by some researchers and clinical trials. It is known that SARS-CoV-2 may hyperactivate the local complement system in a JAK1/2-dependent manner. However, when treated with the JAK1/2 inhibitor Ruxolitinib, the inhibitory effect on the NF-κB signaling pathway is limited, and the effect on improving symptoms in severe COVID-19 patients is not satisfactory. sRAGE effectively inhibits the transcription of inflammatory factors downstream of NF-κB, suggesting that sRAGE combined with JAKi may significantly improve the therapeutic effect in the treatment of COVID-19.

In some embodiments, an "isolated sRAGE polypeptide" or "sRAGE protein" of the present invention may be a recombinant protein and encompasses modified forms of the sRAGE polypeptide so long as it still retains the desired function. With respect to the wild-type sRAGE protein, such as the sRAGE protein having the amino acid sequence shown in SEQ ID NO: 1, the modified form comprises one or more amino acid substitutions, deletions or insertions. For example, the isolated sRAGE polypeptide can be a variant thereof, such as a truncated sRAGE, or a sRAGE having one or more amino acid mutations. The amino acid sequence of the variant may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, At least 99.5% sequence identity, and have sRAGE activity, eg, the ability to compete with RAGE for binding to its ligand. In some instances, such variants are referred to as functional variants of sRAGE. In the context of the present invention, percent homology of amino acid sequences has the same meaning as percent identity of amino acid sequences and is calculated as follows: After aligning two homologous amino acid sequences, the number of positions with identical amino acids is divided by The amino acid number of the longer of the two amino acid sequences, then multiplied by 100%.

The "functional fragment" of sRAGE in the present invention refers to a fragment having the desired biological function of sRAGE activity in the present invention, including a fragment with increased half-life. It should be noted that the meanings of "functional variant" and "functional fragment" are not exclusive, and the "fragment" also includes fragments of functional variants of sRAGE. sRAGE activities contemplated by the present invention include, but are not limited to, competition with endogenous RAGE for binding of RAGE ligands.

In a preferred embodiment, said functional variant or functional fragment comprises amino acids in domains that are important for the function of binding RAGE ligand. For example, a functional fragment of sRAGE may comprise at least one, such as one, two or all three functional modules selected from the group consisting of the V domain of sRAGE (corresponding to amino acid residues 1-94 of SEQ ID NO: 1 position), the C1 domain of sRAGE (corresponding to amino acid residues 103-199 of SEQ ID NO: 1) and the C2 domain of sRAGE (corresponding to amino acid 205-295 of SEQ ID NO: 1). In some embodiments, the functional variant or functional fragment of sRAGE comprises at least one of the V domain or the C1 domain of sRAGE, preferably comprises the V domain, more preferably comprises both the V domain and the C1 domain. In some embodiments, the functional fragment of sRAGE includes a V domain and one selected from a C1 domain and a C2 domain, preferably includes both a V domain and a C1 domain, and more preferably includes a V domain and a C1 domain. domain and C2 domain. In one embodiment, the functional variant of sRAGE comprises a V domain, a C1 domain, and a C2 domain, and differs from SEQ ID NO: 1 by less than 40 amino acids or at least at 87.5% of amino acid positions have the same amino acid.

sRAGE coding sequence

The invention also provides coding sequences for sRAGE polypeptides. The sRAGE coding sequence refers to the nucleotide sequence encoding the sRAGE polypeptide of the present invention or its functional fragment or variant.

In one embodiment, the coding sequence of the present invention is the wild-type coding sequence of the sRAGE polypeptide, such as the sequence shown in SEQ ID NO:2. In another embodiment, the coding sequence is a fragment of the wild-type coding sequence, which encodes a fragment of the sRAGE polypeptide, preferably a fragment comprising one of the V domain or the C1 domain, preferably comprising a fragment of the V domain, more preferably A fragment comprising both the V domain and the C1 domain.

In another embodiment, the coding sequence of the present invention is a variant of the wild-type coding sequence of a sRAGE polypeptide, and encodes a wild-type sRAGE polypeptide as shown in SEQ ID NO: 1 or a fragment thereof. In one embodiment, the coding sequence of sRAGE has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, Sequences that are at least 98% or at least 99% identical. For example, the coding sequence is a codon optimized sequence.

In another embodiment, the coding sequence of the invention encodes a variant of a sRAGE polypeptide.

In a preferred embodiment, the coding nucleotide sequence of sRAGE has one or more nucleotide differences relative to the wild-type coding sequence, and has one or more desirable properties, including but not limited to: The expression level is increased, the encoded product has higher efficacy in treating viral pneumonia, requires a smaller dose, has changed glycosylation patterns, and the like.

For example, in the case where the sRAGE coding sequence of the present invention encodes a sRAGE variant or fragment of a polypeptide different from wild-type sRAGE, the variant or fragment has an altered glycosylation pattern, or in the treatment of viral pneumonia such as new coronary pneumonia It has enhanced potency, or requires a smaller effective dose (such as molar amount) in the treatment of viral pneumonia such as new coronary pneumonia.

For example, in order to increase the expression level, the coding sequence of the present invention may be a codon-optimized coding sequence optimized according to the host cell type expressing the sRAGE polypeptide, so as to improve the expression efficiency of the polypeptide in a specific host.

For example, in order to change the glycosylation pattern of the polypeptide product, the codon corresponding to the amino acid of the glycosylation site in the coding sequence can be modified, for example, the modification can be any one of deletion, substitution, and addition.

Preparation of sRAGE

The sRAGE used in the present invention can be prepared by various expression systems commonly used in the art based on the coding sequence of sRAGE or its recombinant protein. For example, by using Invitrogen's

Baculovirus expression system, use pENTR1 vector to construct baculovirus carrying human T7-sRAGE cDNA, use the constructed recombinant virus to infect CHO cells or 293 cells, and obtain the expression of sRAGE, and can also use Escherichia coli prokaryotic expression system, etc. However, it is not limited to such a method. In the preparation process, as the coding sequence of sRAGE, for example, the sequence shown in SEQ ID No: 2, or a sequence having 95%, 96%, 97%, 98% or more identity thereto can be used. Those skilled in the art can understand that due to codon degeneracy, the coding sequence may not be limited to a specific nucleotide sequence, and may be optimized according to the codon bias of the expression host.

Since post-translational modifications affect the efficacy, half-life, and immunogenicity of sRAGE, the preparation of sRAGE is preferably carried out in mammals, and commonly used mammalian cells cultured in vitro, such as CHO cell lines, 293 cell lines, etc. .

Specifically, as a method for obtaining mammalian post-translationally modified sRAGE, it may be enumerated: using human RAGE (NM_001136) cDNA sequence to construct an sRAGE expression vector; PCR amplifying the coding sequence corresponding to RAGE 23-340 amino acids, and subcloning it into The T7sRAGE CHO-CD14 cell line was established by transient or stable transfection on a membrane-targeted expression vector containing a RAGE signal peptide and a T7 epitope tag to express mammalian post-translationally modified sRAGE. The obtained N-glycosylation information of sRAGE protein can be confirmed by analysis based on HPAEC technology.

post-translationally modified sRAGE

"sRAGE with mammalian post-translational modifications" means that the sRAGE protein has post-translational modifications performed in mammalian cells. Post-translational modifications include, but are not limited to, glycosylation, phosphorylation, sulfation, carboxylation, acetylation, and the like.

The inventors found that the 5th and/or 61st amino acid (O-glycosylation site) of sRAGE, or the 3rd and/or 59th amino acid (N-glycosylation site) of sRAGE were replaced with other amino acids Following substitution, the sRAGE inhibitory effects on inflammation (eg, activation of NF-κB signaling induced by lipopolysaccharide (LPS) stimulation) were attenuated. It can be seen that the glycosylation of these sites plays a key role in the anti-inflammatory effect of sRAGE.

Thus, in a preferred embodiment, said sRAGE polypeptide preferably comprises a mammalian N-glycan profile, preferably with sialylated N-glycosylation. In terms of position, N-glycosylation is particularly preferably at amino acid position 3 and/or position 59. Independently of N-glycosylation, the sRAGE polypeptide preferably comprises mammalian O-glycosylation. The mammalian post-translational modification may include O-glycosylation, preferably at amino acid position 5 and/or position 61. In a preferred embodiment, sRAGE has a mammalian post-translational modification such as described in WO2013103688A1.

Purification of sRAGE

The sRAGE protein and its functional fragments can be purified from the culture medium of cells expressing it. The purification and quantification methods used are not particularly limited, and commonly used protein purification and quantification methods can be used. In the case of having a T7 tag, for example, Novagen's T7 Tag Affinity Purification Kit can be used for purification. The purified protein concentration can be determined by, for example, RAGE ELISA kit, and the purified sRAGE protein can be stored at -80°C.

Indications

Since the beginning of this century, there have been three outbreaks of human coronavirus (HCoV) outbreaks in the world, namely severe acute respiratory syndrome (SARS) caused by human severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and severe acute respiratory syndrome (SARS) in 2012. Middle East Respiratory Syndrome (MERS) caused by Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2019 and Novel Coronavirus Pneumonia (COVID-19) caused by Severe Acute Respiratory Syndrome Novel Coronavirus (SARS-CoV-2) -19).

It should be noted that the clinical manifestations and laboratory tests caused by the three coronavirus infections are basically the same, and the treatment options are also roughly the same. Therefore, although the novel coronavirus is used in the embodiments of the present invention, the therapeutic method and sRGAE protein of the present invention, the pharmaceutical composition can also be applied to other coronaviruses such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV - HKU1, SARS-CoV (causing Severe Acute Respiratory Syndrome) and MERS-CoV (causing Middle East Respiratory Syndrome), preferably SARS-CoV, MERS-CoV, SARS-CoV-2. This is because the principle of action of sRGAE lies in the regulation of the immune inflammatory response, rather than targeting the virus itself. Therefore, its therapeutic effect in the new type of coronavirus pneumonia can also be applied to pneumonia caused by other viruses.

Coronavirus disease

The full name of "Sars-CoV-2" is severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2). In some embodiments, sRAGE is used to treat or prevent infection caused by Sars-CoV-2 or its mutant strains (for example, British variant strain B.1.1.7 (WHO named Alpha), South African variant strain B.1.351 (WHO named Beta), Brazil mutant strain P.1 (WHO named Gamma), Indian mutant strain B.1.617.2 (WHO named Delta, Delta), Danish mink mutant strain, etc.) Complications of pneumonia or inflammatory cytokine storm and multi-system injury, as well as lung infection in subjects with changes in immune factors.

The full name of "COVID-19" is "Coronavirus disease 2019 (Coronavirus disease 2019)", which refers to pneumonia infected by a new type of coronavirus, also referred to as new coronary pneumonia in this article.

Clinical Manifestations of Novel Coronavirus Pulmonary Infection

The clinical manifestations of the novel coronavirus pulmonary infection described in the present invention, the clinical classification (common type, severe type, critical type) etc., can utilize for example the new type of coronavirus pneumonia diagnosis and treatment plan ( Trial implementation of the revised version of the eighth edition, National Health Office Medical Letter [2021] No. 191) for judgment.

As clinical classification criteria, the following items can be referred to, for example.

(1) light

The clinical symptoms were mild, and there was no evidence of pneumonia on imaging.

(2) Ordinary type

With fever, respiratory symptoms, etc., imaging can show pneumonia.

(three) heavy

Adults who meet any of the following:

1. Shortness of breath, RR ≥ 30 times/min;

2. In a resting state, when inhaling air, the oxygen saturation is ≤93%;

3. Partial pressure of oxygen in arterial blood (PaO2)/inhaled oxygen concentration (FiO2)≤300mmHg (1mmHg=0.133kPa);

In high-altitude (more than 1000 meters above sea level) areas, PaO2/FiO2 should be corrected according to the following formula: PaO2/FiO2×[760/atmospheric pressure (mmHg)].

4. The clinical symptoms are progressively aggravated, and lung imaging shows that the lesion progresses >50% within 24 to 48 hours.

The child meets any of the following:

1. Sustained high fever for more than 3 days;

2. Shortness of breath (<2 months old, RR≥60 times/min; 2-12 months old, RR≥50 times/min; 1-5 years old, RR≥40 times/min; >5 years old, RR≥30 times/min), except for the influence of fever and crying;

3. In the resting state, the oxygen saturation is ≤93% when inhaling air;

4. Assisted breathing (nostril flapping, three concave signs);

5. Drowsiness and convulsions;

6. Refusal to eat or difficult to feed, with symptoms of dehydration.

(4) Critical type.

Those who meet one of the following conditions:

1. Respiratory failure occurs and mechanical ventilation is required;

2. Shock occurs;

3. Combined with other organ failure requires ICU monitoring and treatment.

The prevention or treatment method using sRAGE of the present invention can be used to treat mild, common, severe or critical novel coronavirus lung infection, and lung infection caused by other coronaviruses.

Complications of lung infection caused by new coronary pneumonia or other coronaviruses may be selected from one or more of the following group: acute respiratory distress syndrome, septic shock, metabolic acidosis, anorexia, nausea, vomiting and Gastrointestinal symptoms such as diarrhea, eyeball inflammation, cardiovascular damage, Guillain-Barré syndrome, etc.

"Treatment" in the present invention includes curing the disease, or at least partially relieving or alleviating one or more symptoms of the disease. For example, in the context of the present invention, the treatment can reduce the development of severe new coronary pneumonia, eliminate or weaken the imaging signs of pneumonia, improve or alleviate the clinical symptoms mentioned in the above "Clinical manifestations of new coronavirus lung infection", etc. .

The "prevention" in the present invention refers to the main measures to prevent or slow down the development of the disease before the onset of the disease, preventing the occurrence of a specific disease or one or more symptoms caused by the disease. The present invention is mainly aimed at preventing or delaying the occurrence or progress of severe pulmonary infection, and preventing complications, sequelae and even disability caused by pulmonary infection such as coronavirus, such as new coronavirus infection.

subjects

"Subject" in the present invention refers to animals, preferably vertebrates, more preferably mammals, such as rodents, such as mice, rats, hamsters; primates, such as monkeys; most preferably humans.

When the subject is a human, it may be an infant, a teenager, or an adult. In some embodiments, the subject is an elderly patient, who is over 60 years old, or over 65 years old. Elderly patients have lower immunity and lower tolerance to drugs due to the decline of body functions, so there is a higher risk of drug use. However, the treatment scheme using sRAGE of the present invention has good safety and tolerance, and is suitable for elderly patients.

In some embodiments, the subject has other underlying diseases, such as underlying diseases that may lead to increased mortality when suffering from a pulmonary viral infection. For example, the subject has diabetes.

pharmaceutical composition

The present invention provides a pharmaceutical composition comprising sRAGE and a pharmaceutically acceptable carrier.

"Pharmaceutically acceptable carrier" refers to a material other than an active ingredient suitable as a pharmaceutical for delivery to a subject, such as a human, which has no unacceptable toxic or other properties. Pharmaceutically acceptable carriers can be solid, such as powder, or liquid. Examples include excipients (such as sterile water, physiological saline), buffers, solvents, surfactants, chelating agents (such as EDTA), fillers and the like.

The pharmaceutical composition may also include other additives for different functions, such as stabilizers, preservatives, disintegrants, binders, coating agents, lubricants, flavoring agents, sweeteners, solubilizers, etc.

The dosage of sRAGE contained in the pharmaceutical composition of the present invention can be determined according to the effective dosage combined with the administration time interval. As the effective dosage of sRAGE, the doctor can determine it according to the condition, weight, age, and sex of the subject. It can be listed that the sRAGE polypeptide isolated from the drug is 0.001-100 ng/g body weight, preferably 0.1-100 ng/g body weight, More preferably a dose of 5-100 ng/g body weight is administered.

The frequency of administration can be single or multiple times per day, preferably 1, 2 or 3 times per day. The duration of administration can be determined and adjusted at any time by an experienced clinician according to changes in the condition, for example, from one week to several weeks, or from one month to several months, and the administration days may not be continuous. There can be gaps in between.

Administration route

The sRAGE of the present invention and its functional fragments, or pharmaceutical compositions and pharmaceutical preparations comprising them can be administered in any suitable manner, including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal, and, if If necessary, intralesional administration may be used for local immunosuppressive therapy. Systemic administration delivery, such as intravenous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, subcutaneous injection and infusion administration to a subject may be exemplified. Other means include, but are not limited to, for oral, sublingual, transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, transmucosal, inhalation, nasal, eye drops, ear drops, or intravaginal administration.

It is conceivable that the sRAGE polypeptide of the present invention can also be administered to a subject by delivering the polynucleotide sequence encoding the polypeptide, and then undergo expression and post-translational modification in the subject, and then play a role. In this case, the present invention also relates to a polynucleotide encoding the sRAGE polypeptide of the present invention, a vector comprising said polynucleotide, comprising said vector and a pharmaceutically acceptable carrier Pharmaceutical compositions, and their use in the treatment of the indications of the present invention.

drug combination

In one embodiment, sRAGE of the present invention can be combined with JAKi or antiviral drugs, so that the chemical drugs can be applied at a lower dose while maintaining or even enhancing the therapeutic effect. The isolated sRAGE polypeptide or its functional fragment or the pharmaceutical composition containing it of the present invention can be used in combination with other medicines for treating diseases related to viral infection and pneumonia.

As "other antiviral, anti-inflammatory or immunotherapy" mentioned in the present invention, for example, include but not limited to JAKi or antiviral drugs, convalescent plasma from convalescent patients, Tocilizumab (for patients with extensive lung disease and severe patients, And those with elevated IL-6, block IL6 receptors), intravenous injection of COVID-19 human immunoglobulin, and remdesivir (anti-virus), remdesivir + baricitinib (JAK inhibitor), Dexamethasone (corticosteroid), and the combination of bamlanivimab, casirivimab, and imdevimab, neutralizing mAbs that bind to the coronavirus spike protein. When used in combination with convalescent plasma from convalescent patients, the usage and dosage of convalescent plasma from convalescent patients can refer to the "Clinical Treatment Plan for Convalescent Plasma in Convalescent Patients with New Coronary Pneumonia (Trial Version 2)".

Some specific embodiments of the present invention are illustrated below, but are not limited thereto.

1. Use of a soluble receptor for advanced glycation end products (sRAGE) polypeptide in the preparation of a medicament for preventing or treating viral lung infection in a subject.

2. The use of embodiment 1, wherein the pulmonary infection is a viral pulmonary infection caused by human coronavirus (HCoV) infection.

3. The purposes of embodiment 2, wherein said human coronavirus (HcoV) is selected from the group consisting of human severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe Acute respiratory syndrome novel coronavirus (SARS-CoV-2); preferably SARS-CoV-2.

4. The use of any one of embodiments 1-3, wherein the isolated sRAGE polypeptide:

(a) comprising or consisting of the amino acid sequence shown in SEQ ID NO:1;

(b) comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 1, 95%, 96%, 97%, 98%, 99%, 99.5% homologous amino acid sequence or having at least 80%, 85%, 86%, 87% homology with the amino acid sequence shown in SEQ ID NO: 1, Amino acid sequence composition of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology, and has sRAGE activity ;or

(c) It is a functional fragment of the polypeptide described in (a) or (b), and the functional fragment has sRAGE activity.

5. The use according to embodiment 4, wherein said functional fragment comprises one, two or three of the V domain, the Cl domain and the C2 domain of RAGE.

6. The use according to embodiment 5, wherein the functional fragment at least comprises the V domain and/or the C1 domain of RAGE, more preferably comprises the V domain, or comprises the V domain and the C1 domain.

7. The use according to any one of embodiments 4-6, wherein the sRAGE activity comprises competition with endogenous RAGE for binding of a RAGE ligand.

8. The use of any one of embodiments 1-7, wherein the isolated sRAGE polypeptide has a mammalian post-translational modification.

9. The use according to embodiment 8, wherein said mammalian post-translational modification comprises mammalian N-glycosylation, preferably said N-glycosylation is a sialylated N-glycosylation.

10. The use of embodiment 8 or 9, wherein said post-translational modification corresponds to N-glycosylation at amino acid positions 3 and/or 59 of SEQ ID NO:1.

11. The use of any one of embodiments 8-10, wherein said mammalian post-translational modification further comprises O-glycosylation.

12. The purposes of embodiment 11, said O-glycosylation is O-glycosylation corresponding to amino acid 5 and/or 61 of SEQ ID NO:1.

13. The use of any one of embodiments 1-11, wherein the isolated sRAGE polypeptide is produced by a mammalian cell.

14. The use of embodiment 13, wherein said isolated sRAGE polypeptide is produced by CHO cells and 293T cells.

15. The use according to any one of embodiments 1-14, wherein the drug is dosed at 0.001-100 ng/g body weight, preferably 0.1-100 ng/g body weight, more preferably 5-100 ng/g body weight, for example 8-20 ng Dosage administration of isolated sRAGE polypeptide/g body weight.

16. The use according to any one of embodiments 1-15, wherein the medicament is administered to the subject once or more, preferably 1-3 times per day.

17. The use according to any one of embodiments 1-16, wherein the medicament is for use in combination with other antiviral, anti-inflammatory or immunotherapeutics.

18. The use of any one of embodiments 1-17, wherein the medicament is delivered to the subject by systemic administration.

19. The use of embodiment 18, wherein the medicament is delivered to the subject by intravenous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, subcutaneous injection or infusion.

20. The use of any one of embodiments 1-19, wherein the subject is a mammal, preferably a rodent or a primate, more preferably a human.

21. The use according to any one of embodiments 1-20, wherein the subject is an elderly patient and/or suffers from diabetes.

22. The use of any one of embodiments 1-21, wherein the subject has at least one indication selected from the group consisting of:

1) Pneumonia-related clinical manifestations such as fever and/or respiratory symptoms;

2) With imaging features of pneumonia;

3) One or more of the following manifestations: increased D-dimer, progressive decrease in peripheral blood lymphocytes, increased inflammatory factors; liver enzymes, lactate dehydrogenase, muscle enzymes, myoglobin, muscle Increased calcium protein and ferritin; or increased C-reactive protein (CRP) and erythrocyte sedimentation rate.

23. The use of embodiment 22, wherein the imaging features of pneumonia include at least one of the indications selected from the group consisting of:

The lungs showed different degrees of consolidation, and the consolidation area showed diffuse alveolar damage and/or exudative alveolitis; serous, fibrinous exudate, and hyaline membrane formation were seen in the alveolar cavity; the exudative cells were mainly mononuclear and Macrophages, multinucleated giant cells can be seen; type II alveolar epithelial cells hyperplasia, some cells are exfoliated; inclusion bodies can be seen in type II alveolar epithelial cells and macrophages; hyperemia, edema, mononuclear and lymphocyte infiltration can be seen in the alveolar septum; a few alveoli Hyperinflation, rupture of alveolar septum or formation of cysts; partial epithelial shedding of the bronchial mucosa at all levels in the lung, exudate and mucus can be seen in the cavity; mucus plugs can be seen in the small bronchi and bronchioles; small patchy shadows outside the lung, Changes in the lung interstitium, ground-glass opacities of the lungs, infiltrates, consolidation of the lungs, pleural effusion, phlegm and cough in the lungs.

24. The use according to any one of embodiments 1-23, wherein the subject is diagnosed as suffering from the novel coronavirus pneumonia clinically classified as common, severe or critical.

25. A method for treating or preventing a disease associated with human coronavirus (HCoV) infection comprising administering to a subject an isolated sRAGE polypeptide.

26. The method of embodiment 25, wherein said isolated sRAGE polypeptide is characterized by:

(a) comprising or consisting of the amino acid sequence shown in SEQ ID NO:1;

27. The method of embodiment 26, wherein said functional fragment comprises one, two or three of the V domain, the Cl domain and the C2 domain of RAGE.

28. The method of embodiment 27, wherein the functional fragment comprises at least the V domain and/or the C1 domain of RAGE, more preferably the V domain, or the V domain and the C1 domain.

29. The method of any one of embodiments 26-28, wherein the sRAGE activity comprises competition with endogenous RAGE for binding of a RAGE ligand.

30. The method of any one of embodiments 25-29, wherein the isolated sRAGE polypeptide has a mammalian post-translational modification.

31. The method of embodiment 30, wherein said mammalian post-translational modification comprises mammalian N-glycosylation, preferably said N-glycosylation is a sialylated N-glycosylation.

32. The method of

embodiment

30 or 31, wherein the post-translational modification corresponds to N-glycosylation at amino acid positions 3 and/or 59 of SEQ ID NO:1.

33. The use of any one of embodiments 30-32, wherein the mammalian post-translational modification further comprises O-glycosylation.

34. The method of embodiment 33, said O-glycosylation being O-glycosylation corresponding to amino acid positions 5 and/or 61 of SEQ ID NO:1.

35. The use of any one of embodiments 1-24 or the method of any one of embodiments 25-34, wherein 0.001-100 ng/g body weight, preferably 0.1-100 ng/g body weight, more preferably 5-100 ng The isolated sRAGE polypeptide is administered at a dosage per g of body weight.

36. The use of any one of embodiments 1-24 or the method of any one of embodiments 25-34, wherein said isolated sRAGE is administered once or more, preferably 1-3 times per day peptide.

37. The use of any one of embodiments 1-24 or the method of any one of embodiments 25-34, wherein the isolated sRAGE polypeptide is delivered by systemic administration, for example by intravenous injection, intramuscular injection, peritoneal injection Administration by intrasternal injection, intrasternal injection, subcutaneous injection and infusion.

38. A pharmaceutical composition comprising an isolated sRAGE polypeptide and a pharmaceutically acceptable carrier for treating or preventing diseases associated with SARS-CoV-2 infection.

39. The pharmaceutical composition of embodiment 38, wherein said isolated sRAGE polypeptide is characterized by:

(a) comprising or consisting of the amino acid sequence shown in SEQ ID NO:1;

40. A composition comprising an isolated sRAGE polypeptide, or a functional fragment or variant thereof, and at least one other diagnostic and/or therapeutic agent.

41. The composition of embodiment 41 for research use based on competition with endogenous RAGE for binding to a RAGE ligand.

42. A kit comprising an isolated sRAGE polypeptide of the invention, or a functional fragment or variant thereof, for use in the prevention or treatment of viral pulmonary infections.

43. The kit of embodiment 42, wherein the viral lung infection is a viral lung infection caused by SARS-CoV-2 infection.

44. An isolated nucleic acid molecule encoding the nucleotide sequence of the isolated sRAGE polypeptide of embodiment 39 or a functional fragment or variant thereof.

45. An expression construct comprising the isolated nucleic acid molecule of embodiment 44.

46. An expression vector comprising the isolated nucleic acid molecule of embodiment 44 or the expression construct of embodiment 45.

47. A host cell comprising the expression vector of embodiment 46.

48. The host cell of embodiment 47, which is a eukaryotic cell, such as a mammalian cell.

49. The host cell according to embodiment 47 or 48, which is capable of stably and efficiently producing the isolated sRAGE polypeptide having the mammalian post-translational modification described above.

Example

In order to understand and apply the present invention more fully, the present invention will be described in detail below with reference to the examples and accompanying drawings, which are only intended to illustrate the present invention and not intended to limit the scope of the present invention. The scope of the invention is specifically defined by the appended claims.

Materials and Methods

SARS-CoV-2 virus

Adopted standard product SARS-CoV-2 virus SARS-CoV-2/WH-09/human/2020/CHN (Institute of Medical Experimental Animals, Chinese Academy of Medical Sciences) in the embodiment. Experiments with this virus were performed in a Biosafety Level 3 (ABSL3) facility using HEPA-filtered isolators.

experimental animals

Male and female golden hamsters (Golden hamster, Mesocricetus auratus) aged 8-10 weeks were purchased from Beijing HFK Bioscience Co., Ltd. Animal studies were performed in Animal Biosafety Level 3 (ABSL3) facilities using HEPA-filtered isolators. All procedures involving animals in the examples of the present invention were reviewed and approved by the Animal Care and Use Committee of the Institute of Experimental Animal Science, Peking Union Medical College Medical College.

animal handling

A total of 35 hamsters were used, male and female randomly. The hamsters were divided into an infection group (30) and a non-infection group (5). The infection group was inoculated nasally with 10 ⁵ TCID ₅₀ of SARS-CoV-2 stock solution and 100 μl PBS; the non-infection group hamsters were intranasally inoculated with PBS.

According to the administration dose of sRAGE, the infection group is divided into the following groups: sRAGE 20ng/g (10), 10ng/g (5), 5ng/g (5), respectively, after

infection

1, 2, 3 , 4, 5, and 6 days (d) intraperitoneally inject human recombinant sRAGE (purchased from Yiqiao Shenzhou Company, the amino acid sequence is shown in SEQ ID NO: 1) at doses of 20ng/g, 10ng/g, and 5ng/g body weight as treatment; and the hamsters injected with human serum albumin (20ng/g body weight) at the same time point were used as the treatment control group (treatment control: 10).

All hamsters were observed daily for body weight, clinical signs and responses to external stimuli. Hamsters were sacrificed at 7 days, heart blood (1ml) was collected for complete blood count, and whole lung was surgically collected for viral load analysis, pathological examination and mechanism research.

Viral load determination

Viral load was detected by qRT-PCR. Whole lung homogenate was prepared with an electric homogenizer, and total lung RNA was extracted with RNeasy Mini Kit (Qiagen). Reverse transcription was performed using the PrimerScript RT kit (TaKaRa) according to the manufacturer's instructions. Using the PowerUp SYBG Green Master Kit (ABI), the reaction was performed under the following conditions: 50°C for 2 minutes, 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds, and finally 95°C for 15 seconds, 60°C 1 minute at 95°C for 45 seconds.

The qRT-PCR primer sequences are as follows:

Forward: 5'-TCGTTTCGGAAGAGACAGGT-3' (SEQ ID NO:3),

Reverse: 5'-GCGCAGTAAGGATGGCTAGT-3' (SEQ ID NO: 4).

Using the recombinant plasmid with known copy number (1.47×10 ⁹ -1.47×10 ¹ copy per μl), construct a standard curve by 10-fold dilution method. All experiments were performed in a Biosafety Level 3 facility.

The Ct value (number of cycles to reach threshold in qPCR) was compared to a standard curve, and the viral load results for each hamster lung were expressed as the log10 transformation number of equivalent copies of the genome per ml. Differences in viral load between groups were analyzed using a one-tailed t-test. A p-value <0.05 was considered significant (*p<0.05, **p<0.01). Viral loads are expressed as mean ± SD.

Statistical Analysis

Statistical analysis was performed using Prism software (GraphPad software). The data are expressed as the mean ± s.e.m., and the comparison between the experimental group and the control group was performed using a two-tailed Student's t test. P value ≤ 0.05 means the difference is statistically significant. *: p<0.05; **: p<0.01; ***: p<0.001.

Western blot

The lung tissues of the above groups were lysed on ice for 30 min with RIPA lysis buffer (Solarbio, R0010) supplemented with protease inhibitors and phosphatase inhibitors. The tissue was centrifuged at 12000rmp/min for 15min at 4°C in the lysate, the supernatant was collected, and the virus was inactivated at 56°C for 30min. The above experiments were all carried out in a level 3 biosafety facility.

Western blot analysis was performed with 30 μg of protein. Western blotting was performed using the following antibodies: RAGE (Abcam, ab216329), β-actin (Yeasen, 30101), recombinant Anti-MX1 antibody [EPR24485-19] (ab284603, Abcam). Use ImageJ software (https://imagej.nih.gov/ij) to calculate western blot bands to obtain quantitative data for further statistical analysis.

Gene expression in lung tissue

Lung tissue gene expression was detected by qRT-PCR: Whole lung homogenate was prepared with an electric homogenizer, and total lung RNA was extracted with RNeasy Mini Kit (Qiagen). cDNA was prepared by cDNA Synthesis SuperMix (TransGen, AE311-04) according to the manufacturer's protocol. Gene expression analysis was performed using SuperReal PreMix Plus SYBR Green (TIANGEN BIOTECH, FP205). SYBR Green uses ACTB as an internal reference gene. Data were analyzed by the ΔCt method. Primers were designed according to the predicted sequence (Mesocricetus auratus) in the NCBI database (see Table 1).

Table 1

Pathological examination

Necropsy was performed at 7 dpi (day(s) postinfection) in an animal biosafety level 3 (ABSL3) laboratory. Lungs were examined visually, fixed with 10% buffered formalin solution, and paraffin sections (3-4 μm thick) were made. H&E staining and immunohistochemical detection of hamster lung histopathological changes. Pathological diagnosis of H&E-stained sections was performed by two pathologists from independent experimental groups, and histological semi-quantitative scoring was as follows:

Attachment: Evaluation method for pulmonary lesions

1. Grading criteria for single lobe lesions:

1. Widening of the alveolar septum:

+, mild lesions, slightly widened alveolar septa.

++, moderate disease, the alveolar septum is obviously widened, and the extent of the disease is more than 1/2.

+++, severe lesions, alveolar septa were significantly widened, and alveolar septa were widened, fused, and alveolar spaces were significantly narrowed, and the lesion range was greater than 1/2.

++++, very severe lesions, alveolar septa widened, fused, alveolar cavities were narrowed and disappeared, local lung tissue was consolidated, and the extent of the lesion was greater than 3/4.

2. Others, lesions (alveolar exudation, perivascular inflammatory cell infiltration, peribronchial inflammatory cell infiltration, etc.)

+, mild lesions, the lesion range is less than 1/4 of the cut surface of lung tissue.

++, moderate lesions, the lesions occupy about 1/4-2/4 of the lung tissue section.

+++, severe lesions, and the lesion range occupies about 2/4 to 3/4 of the lung tissue section.

++++, very severe lesions, the lesion range is larger than 3/4 of the section of lung tissue.

2. Comprehensive Judgment Criteria for Animal Pulmonary Diseases

1. Mild: widened alveolar septum with inflammatory cell infiltration + appearing in more than 1 lung lobe, and widened alveolar septum with inflammatory cell infiltration + no more than 2 lung lobes.

2. Moderate: Widening of alveolar septa and inflammatory cell infiltration +++ appears in one lung lobe, or widening of alveolar septum and inflammatory cell infiltration++ appears in more than 2 lung lobes (including 2), or alveoli appear in more than 2 lung lobes Septal widening inflammatory cell infiltration++ with intraalveolar exudation++～++++ and perivascular inflammatory cell infiltration++～++++.

3. Severe: Widening of alveolar septa and inflammatory cell infiltration++++ appear in more than 1 lung lobe, or widening of alveolar septum and inflammatory cell infiltration++++ appear in more than 2 lung lobes (including 2), or more than 3 lung lobes Widening of alveolar septa and inflammatory cell infiltration++ with intraalveolar exudation++～++++, and perivascular inflammatory cell infiltration++～++++.

Immunohistochemistry

The ready-to-use immunohistochemistry kit of SABC system (Biolab, ZN1830) was used for immunohistochemistry according to the instructions. The procedure for obtaining slices is the same as for H&E stained slices.

Example 1 SARS-CoV-2 intranasal infection-induced COVID-19 hamster model

Golden hamster has been used as an animal model of SARS-CoV-2-induced pneumonia, and its pathological phenotype is highly similar to that of COVID-19 patients.

In this example, a golden hamster model of COVID-19 was established by intranasal inoculation of SARS-CoV-2 virus (a dose of 1×10 ⁵ TCID ₅₀ ). For the specific inoculation process and animal modeling process, see the "In Vivo Animal Modeling" section in "Materials and Methods".

From the first day after inoculation, hamsters were treated with sRAGE with body weights of 20ng/g, 10ng/g, and 5ng/g, respectively, and 20ng/g of human serum albumin (HSA) was used as the treatment control group. The body weight of each hamster was monitored daily for 7 consecutive days. Hamsters were sacrificed at 7 dpi, and blood samples and lung tissues were collected. The determination of viral load in the lung was performed as described in Materials and Methods, and the virus inoculation schedule of the animal model is shown in Figure 1 .

The results showed that: 7dpi after the hamsters were infected with SARS-CoV-2, about ^1x106 virus copies/ml were detected in the lungs, and all the hamsters infected with SARS-CoV-2 gradually lost weight. sRAGE moderately but significantly alleviated weight loss, suggesting that sRAGE may attenuate the overall damage of SARS-CoV-2 to the body. sRAGE treatment did not have a significant effect on viral load, suggesting that the effect was not achieved by altering viral load.

Example 2 Evaluation of lung injury (hamster model lung tissue section H&E staining)

In this example, the lung tissue sections of each group of animals in Example 1 were examined by H&E staining (for the method, see "Pathological Examination" in "Materials and Methods") to evaluate the severity of pneumonia. The HE staining results of the lung tissue of the golden hamster model in each experimental group are shown in Fig. 2 . Sections were scored and counted according to overall lung damage, alveolar wall thickening, intra-alveolar fibrin deposition, and inflammatory cell infiltration.

The results show that, as can be seen from Figure 2, normal lung tissue structure can be seen in the negative control group (non-infected control group), and there is no secretion in the alveoli; widened and fused, the alveolar cavity was narrowed and disappeared, and local lung tissue was consolidated; while the sRAGE treatment group (10ng, 20ng group) showed moderate pneumonia, and the pathological changes such as widening of the alveolar septum, alveolar fusion, and narrowing of the alveolar cavity were relatively obvious. Significant improvement was observed in the treatment control group.

sRAGE treatment 20ng group and sRAGE treatment 10ng group had the same performance trend (Figure 3). Among the SARS-CoV-2-infected hamsters treated with HSA, 90% of the number showed severe interstitial pneumonia, which was specifically manifested as diffuse alveolar septal thickening, extensive hemorrhage, and a large number of inflammatory cell infiltration in the lung. In contrast, fewer hamsters in the sRAGE-treated group developed severe interstitial pneumonia. Specifically, the ratio of the number of critically ill animals to the total number of animals in the HSA treatment control group was 9/10, that in the sRAGE20ng group was 3/10, and that in the sRAGE10ng group was 1/5. It can be seen that in the 10ng/g sRAGE group, only one animal developed severe pneumonia, which greatly reduced the proportion of animals that reached severe disease.

In the pneumonia grading score statistics shown in Fig. 4, the improvement of the score by the administration of sRAGE can also be seen.

Example 3 Expression levels of various inflammatory factors

In this example, the mRNA levels of various inflammatory factors (IL-1β, IL-6, TNFα, IL-18, IL-10, IL-12, etc.) in the lung tissue obtained in Example 1 were detected , to determine the level of inflammatory response in the lungs. The method was the same as that in the "qRT-PCR" section in Materials and Methods, the primers used are shown in Table 1, and the results are shown in Figure 5. The peripheral blood leukocytes and lymphocytes in the blood of each group were also detected, and the results are shown in FIG. 6 .

The results showed that compared with the HSA-treated control group, the mRNA expression levels of IL-1β, IL-10, IL-6, TNFα, IL-18, IL-12, etc. were significantly reduced in the group treated with sRAGE , and in the group given sRAGE treatment, the 20ng group was superior to the 10ng group. In addition, in the sRAGE-treated group, compared with the HSA-treated control group, the peripheral blood leukocyte count showed a trend of decreased neutrophils, while lymphocytes showed a trend of increased in the sRAGE-treated group (Fig. 6 ).

Example 4 Macrophage marker CD68 and type 1 interferon response marker Mx1

The mRNA expression levels of CD68 and type 1 interferon response marker Mx1 in the lung tissues of each group in Example 1 were detected. Methods were as described in Materials and Methods and statistical results are shown in Figure 7.

The Mx1 gene encodes the protein MxA (Myxovirus resistance protein A, myxovirus resistance protein A). Reliable marker of bioavailability and also as a marker for distinguishing between viral and bacterial diseases. CD68 is a marker of macrophages.

RT-qPCR results showed that, compared with the HSA-treated control group, the mRNA expression levels of CD68 and type 1 interferon response marker Mx1 were significantly decreased in the sRAGE-treated group. It shows that sRAGE significantly reduces the accumulation of macrophages in lung tissue and the degree of lung inflammation.

Example 5 sRAGE significantly inhibited pulmonary inflammatory cell infiltration and inflammatory response

Immunohistochemistry for CD3 and MxA was performed on the slices of lung tissues of each group in Example 1, the method was as described in Materials and Methods, and the staining results are shown in FIG. 8 .

The results showed that infection with SARS-CoV-2 induced pulmonary infiltration of CD3-positive T cells, macrophages, and neutrophils, especially in the perivascular and peribronchial regions. After treatment with sRAGE, the infiltration of these inflammatory cells in the lung was significantly reduced, indicating that sRAGE can inhibit the lung inflammatory response by inhibiting the recruitment of inflammatory cells.

In conclusion, treatment of a hamster model of SARS-COV-2 infection with sRAGE can significantly reduce the accumulation of inflammatory cells in lung tissue, including neutrophils and macrophages, and the expression of cytokines is also significantly reduced. The results showed that the overall severity of COVID-19 pneumonia was significantly reduced, and the proportion of hamsters with severe pneumonia symptoms decreased. It shows that sRAGE can be used as a preventive and therapeutic drug for lung infection-related diseases, especially new coronary pneumonia caused by SARS-CoV-2.

Example 6 Effect of glycosylation site on the anti-inflammatory effect of sRAGE protein

In this example, in order to study the effect of post-translational glycosylation modification on the anti-inflammatory effect of sRAGE, the amino acids involved in glycosylation in sRAGE were mutated to compare the inhibitory effect of sRAGE with different glycosylation modifications. Differences in the effect of LPS stimulation on NF-κB activation. Specifically, the sRAGE used included commercially available sRAGE, wild-type sRAGE expressed from a plasmid, sRAGE mutated at amino acid positions 5 and/or 61 involved in O-glycosylation, and sRAGE involved in N-glycosylation sRAGE with mutations at amino acid positions 3 and/or 59. After administering these sRAGE proteins to 293T cells stably expressing RAGE (293T-RAGE KI cells), the NF-κB activation level of the cells after LPS stimulation was measured. The activation level of NF-κB was determined by measuring the transcriptional activity level of NF-κB using the pNFκB-TA-luc reporter gene plasmid detection system (Beiyuntian). The specific test steps are as follows.

The 293T-RAGE KI cell experiments were divided into 10 groups for the following different treatments: (1) blank control group (NC), (2) LPS positive control group (LPS); and the following LPS plus each The sRAGE groups were: (3) commercially available sRAGE group (purchased from Sino Biological Inc., with a final concentration of 0.01ug/ml), (4) wild-type sRAGE group (WT), (5) sRAGE with T5A single amino acid mutation (T5A), (6) sRAGE with S61A single amino acid mutation (S61A), (7) sRAGE with T5A and S61A double amino acid mutation (T5A/S61A), (8) with N3Q single amino acid mutation Mutated sRAGE(N3Q), (9) sRAGE(N59Q) containing N59Q single amino acid mutation, (10) sRAGE(N3Q/N59Q) containing N3Q and N59Q double amino acid mutation.

Add 5×10 ⁵ CHO-K1 cells to each well of a six-well culture plate and culture until the confluence rate is about 80%. For the above groups (3) to (10), Lipofectamine ^TM 3000 reagent was used to transfect the expression plasmids encoding the wild type and the sRAGE mutant containing amino acid site mutations, and the cells and supernatant were collected 48 hours later for use.

A 293T cell line stably expressing RAGE was constructed by lentiviral transfection, which was named "293T-RAGE KI". 293T-RAGE KI cells were transfected with luciferase expression plasmids (3ug in total), and seeded into 96-well plates (10,000-15,000/well) after 24 hours, and cultured overnight. In each group except the NC group and the LPS group, the corresponding CHO cell supernatant was added to the wells at a ratio of 20% of the liquid volume in the wells.

After adding the cell supernatant for 30 minutes, in all groups except the blank control group, LPS (100ug/ml) was added for 2 hours, and then the cells were lysed. Luciferase activity in cell lysates was detected using the Promega E1910 kit. The results are shown as percent change relative to the blank control group stimulated without the addition of LPS and are shown in FIG. 9 .

As shown in Figure 9, in the LPS group, the activation of the inflammatory factor NF-κB was significantly increased after LPS stimulated the cells. In the sRAGE group and the sRAGE wild-type (WT) group, the administration of sRAGE protein and the expression supernatant of wild-type sRAGE effectively inhibited the activation of NF-κB induced by LPS stimulation, which proved that sRAGE has anti-inflammatory effects. On the other hand, this inhibitory effect of sRAGE was suppressed to varying degrees after amino acids 5 and/or 61 involved in O-glycosylation, or amino acids 3 and/or 59 involved in N-glycosylation were mutated. weakened, especially the effects of N3Q mutation and S61A mutation. The above results indicate that the glycosylation at the relevant sites plays a key role in the anti-inflammatory effect of sRAGE.

Industrial Applicability

The inventors provide the use of sRAGE in significantly attenuating the pro-inflammatory response in the lung after SARS-CoV-2 infection, which can inhibit lung inflammation through multiple mechanisms. sRAGE is a molecule produced by the organism itself, with no obvious side effects and high safety. In addition, the combined treatment of sRAGE with JAKi or antiviral drugs is expected to enable the application of chemical drugs at lower doses while retaining or even enhancing the therapeutic effect. The present invention provides strong evidence to support the application of sRAGE in the real clinical environment, mechanism research, animal model, and drug development for the treatment of lung virus infection.

sequence information

The amino acid sequence of SEQ ID NO:1-sRAGE

The coding sequence-DNA of SEQ ID NO:2-sRAGE

SEQ ID NO:3 - qRT-PCR Primer for Determination of Viral Load - Forward

SEQ ID NO:4 - qRT-PCR Primer for Determination of Viral Load - Reverse

Claims

Use of the isolated soluble receptor for advanced glycation end products (sRAGE) polypeptide in the preparation of a medicament for preventing or treating viral pulmonary infection in a subject, preferably caused by human coronavirus (HCoV ) viral lung infection caused by infection, more preferably a viral lung infection caused by SARS-CoV, MERS-CoV or SARS-CoV-2 infection.
The use according to claim 1, wherein the isolated sRAGE polypeptide:

(a) comprising or consisting of the amino acid sequence shown in SEQ ID NO:1;

(b) comprising or consisting of an amino acid sequence having at least 80% homology to the amino acid sequence set forth in SEQ ID NO:1, and has sRAGE activity; or

(c) it is a functional fragment of the polypeptide described in (a) or (b), and the functional fragment has sRAGE activity;

Preferably, the amino acid sequence described in (b) having at least 80% homology or the functional fragment described in (c) comprises one or more of the V domain, C1 domain and C2 domain of sRAGE indivual.
Use according to any one of claims 1-2, wherein the isolated sRAGE polypeptide has a mammalian post-translational modification, and the mammalian post-translational modification preferably includes mammalian N-glycosylation, so Said N-glycosylation is preferably sialylated N-glycosylation, particularly preferably N-glycosylation corresponding to amino acid 3 and/or 59 of SEQ ID NO:1,

Optionally, said mammalian post-translational modification further comprises O-glycosylation, said O-glycosylation is preferably an O-glycosyl corresponding to amino acid 5 and/or 61 of SEQ ID NO:1 change.
The use according to any one of claims 1-3, wherein the drug is used in combination with other antiviral, anti-inflammatory or immunotherapy.
The use according to any one of claims 1-4, wherein the subject has at least one of the indications selected from the group consisting of:

1) Pneumonia-related clinical manifestations such as fever and/or respiratory symptoms;

2) With imaging features of pneumonia;

3) One or more of the following manifestations appear: increased D-dimer, progressive decrease in peripheral blood lymphocytes, increased inflammatory factors; liver enzymes, lactate dehydrogenase, muscle enzymes, myoglobin, troponin Increased protein and ferritin; or increased C-reactive protein (CRP) and erythrocyte sedimentation rate.
According to the use according to claim 5, the imaging features of pneumonia include at least one of the indications selected from the following group:

The lungs showed different degrees of consolidation, and the consolidation area showed diffuse alveolar damage and/or exudative alveolitis; serous, fibrinous exudate, and hyaline membrane formation were seen in the alveolar cavity; the exudative cells were mainly mononuclear and Macrophages, multinucleated giant cells can be seen; type II alveolar epithelial cells hyperplasia, some cells are exfoliated; inclusion bodies can be seen in type II alveolar epithelial cells and macrophages; hyperemia, edema, mononuclear and lymphocyte infiltration can be seen in the alveolar septum; a few alveoli Hyperinflation, rupture of alveolar septum or formation of cysts; partial epithelial shedding of the bronchial mucosa at all levels in the lung, exudate and mucus can be seen in the cavity; mucus plugs can be seen in the small bronchi and bronchioles; small patchy shadows outside the lung, Changes in the lung interstitium, ground-glass opacities of the lungs, infiltrates, pulmonary consolidation, pleural effusion, and phlegm and cough in the lungs.
The use according to any one of claims 1-6, wherein the subject is diagnosed by a physician as having clinically classified as common type, severe type or critical type of new coronavirus pneumonia.
The use according to any one of claims 1-7, wherein the dose of the sRAGE polypeptide in the drug is 0.001-100 ng/g body weight, preferably 0.1-100 ng/g body weight, more preferably 5-100 ng/g body weight , and/or administered to the subject once or more times per day, preferably 1-3 times.
A pharmaceutical composition comprising an isolated sRAGE polypeptide and a pharmaceutically acceptable carrier, the pharmaceutical composition is used to treat or prevent diseases associated with SARS-CoV-2 infection, and the isolated sRAGE polypeptide has the following characteristics:

(a) comprising or consisting of the amino acid sequence shown in SEQ ID NO:1;

(b) comprising or consisting of an amino acid sequence having at least 80% homology to the amino acid sequence set forth in SEQ ID NO:1, and has sRAGE activity; or

(c) it is a functional fragment of the polypeptide described in (a) or (b), and the functional fragment has sRAGE activity;

Preferably, the amino acid sequence described in (b) having at least 80% homology or the functional fragment described in (c) comprises one or more of the V domain, C1 domain and C2 domain of sRAGE indivual.
The pharmaceutical composition according to claim 9, wherein the dose of sRAGE polypeptide is 0.001-100ng/g body weight, preferably 0.1-100ng/g body weight, more preferably 5-100ng/g body weight, and/or once a day or multiple times, preferably 1-3 times, to the subject.