Proteins that suppress excessive inflammatory responses and their applications

Abstract

The present invention provides a protein that suppresses excessive inflammatory responses and its applications. The protein comprises a soluble CD4 molecule (sCD4), and the applications include treating or preventing excessive inflammatory responses in sepsis or macrophages. The present invention also provides that the target of sCD4's suppression of excessive inflammatory responses in macrophages is cell membrane-type MHC II, and that sCD4 acts by regulating the MHCII / STING / SHP2 complex.

Description

[Technical Field]

[0001] The present invention belongs to the field of biopharmaceuticals; more specifically, the present invention relates to a recombinant protein that suppresses excessive inflammatory responses and its application in the treatment or prevention of sepsis. [Background technology]

[0002] Uncontrolled inflammation due to excessive activation of innate immune cells leads to systematic pathological changes and is often associated with high mortality rates from severe infections. Therefore, inflammatory responses must be tightly controlled at multiple levels by a series of negative regulators to maintain immune stability. Naive T cells have been demonstrated to play an important role in suppressing acute inflammatory responses in beta-coronavirus and Gram-negative bacterial infections. Direct contact with macrophages suppresses the expression of proinflammatory cytokines, such as TNF and IL-6, produced by Toll-like receptor (TLR) activation, without the need for antigen-specific T cell receptors (TCRs). More importantly, effective control of TLR-mediated inflammatory pathological responses on antigen-presenting cells (APCs), such as macrophages, dendritic cells, or B cells, depends on the number of naive T cells, rather than their quality. Therefore, the severity and / or mortality of infections in immunocompromised individuals is directly correlated with low numbers of naive T cells.

[0003] In the early stages of bacterial and viral sepsis, T cells often die rapidly (T lymphopenia) due to mass death, which is associated with multiple organ dysfunction due to early excessive inflammatory responses and later immune paralysis. Over the past 30 years, more than 200 clinical trials aimed at treating sepsis, including increasing T cell numbers, resolving T cell immune exhaustion, and suppressing the exaggerated inflammatory responses of the natural immune system (including neutralizing various inflammatory cytokines), have failed, highlighting the urgent need for research into the mechanisms of sepsis pathogenesis and treatment strategies.

[0004] The CD4 molecule is a transmembrane protein whose extracellular domain (ectodomain) contains four Ig-fold-like functional domains (D1-D4). The D1-D2 functional domain at the distal membrane end binds to the β2 functional domain of MHC-II molecules, enhancing the antigen presentation function of MHC-II and the activation of CD4+ T cells by APCs as a co-receptor for TCR. The CD4 molecule also functions as a receptor for the HIV membrane protein gp120, mediating HIV entry and infection of CD4+ T cells.

[0005] Upon activation of CD4+ T cells, the extracellular domain of the CD4 molecule can be enzymatically shed to form soluble CD4 (sCD4). Early clinical studies have detected sCD4 in the sera of patients with infectious diseases, autoimmune diseases, and cancer, but its physiological and pathological mechanisms have not yet been reported. Since the early 1990s, numerous studies have been conducted to utilize the sCD4 protein and its various derivatives to suppress HIV infection (Chen, Feng, et al., 2014). However, it has not yet been reported whether the sCD4 protein has a regulatory effect on diseases associated with macrophage hyperinflammatory responses. Summary of the Invention

[0006] The objective of the present invention is to provide a recombinant protein sCD4 that suppresses the hyperinflammatory response of macrophages and its application in preparing a pharmaceutical composition for preventing or treating the hyperinflammatory response of sepsis.

[0007] In a first aspect, the present invention provides the use of a soluble CD4 recombinant protein or a construct expressing soluble CD4 (e.g., an expression vector, more specifically, an adenoviral vector, a lentiviral vector, etc.) in the preparation of a pharmaceutical composition for treating or preventing an inflammatory response, wherein the inflammatory response is sepsis hyperinflammation or a macrophage inflammatory response.

[0008] In one or more preferred embodiments, the macrophage inflammatory response comprises macrophage TLR4-mediated inflammation mediated by overexpression of inflammatory cytokines at the very early stage of sepsis onset.

[0009] In one or more preferred embodiments, the macrophage TLR4-mediated inflammation associated with overexpression of inflammatory cytokines includes, but is not limited to, inflammation associated with overexpression of TNF and / or inflammation associated with overexpression of IL-6.

[0010] In one or more preferred embodiments, the soluble CD4 recombinant protein comprises the extracellular domain of the CD4 molecule.

[0011] In one or more preferred embodiments, the extracellular domain of the CD4 molecule comprises the D1, D2, D3 and D4 domains.

[0012] In one or more preferred embodiments, the soluble CD4 recombinant protein is encoded by a codon-optimized coding sequence of human or murine origin.

[0013] In one or more preferred embodiments, the soluble CD4 recombinant protein comprises a protein having the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a conservative variant thereof (a variant that retains the sequence and function of the protein set forth in SEQ ID NO:1 or SEQ ID NO:3).

[0014] In one or more preferred embodiments, the conservative variants include those selected from the following: (1) a derived protein formed by substituting, deleting, or inserting one or more (e.g., 1-20; preferably 1-10; preferably 1-5, 1-3, or 1-2) amino acid residues from the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and retaining the protein function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3; (2) a derived protein having an amino acid sequence that is 80% or more (preferably 85% or more, 90% or more, or 95% or more; e.g., 98% or more or 99% or more) identical to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and retaining the protein function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3; or (3) a protein formed by adding a tag sequence or a signal peptide sequence to the protein (e.g., at the N- or C-terminus) of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0015] In one or more preferred embodiments, the conservative variant is selected from, but is not limited to, a variant comprising an oligopeptide tag that contributes to the detection of the protein shown in SEQ ID NO:1 or SEQ ID NO:3, and / or an Fc-fused recombinant variant that increases the stability of the protein shown in SEQ ID NO:1 or SEQ ID NO:3.

[0016] In one or more preferred embodiments, the soluble CD4 expressing construct includes, but is not limited to, an expression vector.

[0017] In one or more preferred embodiments, the expression vector includes a viral expression vector, a non-viral expression vector.

[0018] In one or more preferred embodiments, the viral expression vector includes, but is not limited to, an adeno-associated virus, a lentiviral vector, or an adenoviral vector.

[0019] In one or more preferred embodiments, the soluble CD4 cross-inhibits the inflammatory response by binding to macrophage MHC II; preferably, the inflammatory response comprises sepsis hyperinflammation or a macrophage inflammatory response; preferably, the macrophage inflammatory response is a macrophage TLR4 inflammatory response.

[0020] In one or more preferred embodiments, the signaling pathway utilized in the TLR4 inflammatory response is the macrophage MHC II-TLR4 inflammatory response complex; preferably, the soluble CD4 recombinant protein or soluble CD4 expressing construct upregulates MHC II, recruits and activates SHP-2, inhibits TRAF6, and cross-inhibits the TLR4 / NF-κB-mediated inflammatory response.

[0021] In one or more preferred embodiments, the soluble CD4 recombinant protein or soluble CD4 expressing construct is included in a pharmaceutically acceptable carrier to form a pharmaceutical composition.

[0022] In another aspect of the present invention, there is provided an application of an artificially established or intracellular signaling pathway, or a system containing such a signaling pathway, in screening for substances (including compounds, compositions, drugs, etc.) that suppress macrophage inflammatory responses; the signaling pathway is selected from macrophage MHC II-SHP2 and / or STING-TRAF6-TLR4; macrophage MHC II-TLR4; macrophage SHP2-TRAF6-TLR4; and macrophage STING-TLR4.

[0023] In another aspect of the present invention, there is provided a method for screening a substance that suppresses macrophage inflammatory response, the method comprising: (1) A candidate substance is contacted with a system containing a signaling pathway selected from the following group: macrophage MHC II-SHP2 and / or STING-TRAF6-TLR4; macrophage MHC II-TLR4; macrophage SHP2-TRAF6-TLR4; macrophage STING-TLR4; (2) The system of (1) is observed, and a substance that modulates the signal transduction pathway of (1) is screened, the substance being a substance (including a potential substance) that is useful for suppressing macrophage inflammatory responses; the modulation includes upregulating (e.g., activating or increasing expression) MHC II, upregulating SHP2, upregulating STING, and downregulating (e.g., suppressing or decreasing expression) TRAF6.

[0024] In one or more embodiments, the signaling pathway in (1) is macrophage MHC II-SHP2-TRAF6-TLR4; and the modulation in (2) includes upregulating MHC II, upregulating SHP2, and the SHP2 downregulating TRAF6, thereby suppressing the TLR4 inflammatory response.

[0025] In one or more embodiments, the signal transduction pathway in (1) is macrophage MHC II-TLR4; and the modulation in (2) includes suppressing TLR4 inflammatory responses by upregulating MHC II.

[0026] In one or more embodiments, the signal transduction pathway in (1) is SHP2-TRAF6-TLR4; and the modulation in (2) includes upregulating SHP2, which then downregulates TRAF6, thereby suppressing the TLR4 inflammatory response.

[0027] In one or more embodiments, the signal transduction pathway in (1) is STING-TLR4; and the modulation in (2) includes suppressing TLR4 inflammatory responses by upregulating STING.

[0028] In one or more embodiments, the method includes establishing a control group, which is a system to which the candidate substance is not added and which contains a signaling pathway selected from the group consisting of macrophage MHC II-SHP2 and / or STING-TRAF6-TLR4; macrophage MHC II-TLR4; macrophage SHP2-TRAF6-TLR4; macrophage STING-TLR4.

[0029] In one or more embodiments, the system further includes MyD88 / IRAK1 / TRAF6, an inflammatory signaling molecule downstream of TLR4; the method also includes monitoring the activation status of MyD88 / IRAK1 / TRAF6, a signaling molecule downstream of TLR4, and if the activation is inhibited, indicating that the candidate substance is useful for inhibiting macrophage inflammatory responses.

[0030] In one or more embodiments, the system further includes NF-B, which drives expression of inflammatory cytokine genes such as TNF / IL-6; and the method includes observing that NF-B drives expression of inflammatory cytokines (e.g., TNF / IL-6), and a decrease in expression indicates that the candidate substance is useful for suppressing macrophage inflammatory responses.

[0031] In one or more embodiments, the suppression of the TLR4 inflammatory response is evaluated by the expression or presence of inflammatory cytokines; preferably, the inflammatory cytokines include TNF and / or IL-6; if the expression or presence of TNF and / or IL-6 does not change significantly (e.g., if there is no significant downregulation compared to a control), the TLR4 inflammatory response is not suppressed, and if the expression of TNF and / or IL-6 is significantly reduced, the TLR4 inflammatory response is suppressed.

[0032] In one or more embodiments, the system containing the signaling pathway is selected from a cell (culture) system, a subcellular (culture) system, a tissue (culture) system, or an animal system.

[0033] In one or more embodiments, the candidate substances include, but are not limited to, regulatory molecules (e.g., upregulators, interference molecules, nucleic acid inhibitors, binding molecules (e.g., antibodies or ligands)) designed to target the signaling pathway or its pathway proteins, or its upstream or downstream proteins or genes, CRISPR constructs, small molecule compounds, and compounds derived from chemical libraries.

[0034] In one or more embodiments, the improvement or enhancement is a statistical improvement or enhancement, for example, an improvement or enhancement of 10% or 20% or more, preferably an improvement or enhancement of 40% or 50% or more, preferably an improvement or enhancement of 80% or 100% or more, compared to a control or baseline.

[0035] In one or more embodiments, the inhibition is one of downregulation, and is a statistical inhibition or downregulation, for example, an inhibition or downregulation of 10% or 20% or more, preferably an inhibition or downregulation of 40% or 50% or more, preferably an inhibition or downregulation of 80% or 100% or more, compared to a control or baseline.

[0036] Other aspects of the present invention will be apparent to those skilled in the art based on the disclosure herein. [Brief explanation of the drawings]

[0037] [Figure 1] We demonstrate that sCD4 suppresses TLR-mediated inflammatory responses in APCs (macrophages and dendritic cells). (A) Mouse BMDMs were cocultured with different concentrations of mouse-derived sCD4 protein (25 nM, 50 nM, 250 nM), and the concentrations of the inflammatory cytokines TNF and IL-6 in the cell supernatant were measured by Luminex analysis. (B) BMDMs were cocultured with mouse-derived sCD4 recombinant protein, and then TLR3, TLR7, and TLR9 signaling pathways were stimulated with polyI:C, CpG, and VSV virus, respectively. (C) Mouse bone marrow-derived dendritic cells (BMDCs) were cocultured with different concentrations of mouse-derived sCD4 protein (25 nM, 50 nM), and the inhibitory effects of sCD4 on different TLRs or cellular inflammatory responses were measured using the same method as above. [Figure 2] Human sCD4 suppresses macrophage TLR4 inflammatory responses. Human sCD4 protein was co-cultured with human peripheral blood mononuclear cell (PBMC)-derived macrophages at different concentrations (1.39 nM, 4.17 nM, 12.5 nM, 37.5 nM, and 112.5 nM), and then stimulated with LPS for the indicated time periods. The concentrations of inflammatory cytokines in the supernatant were detected using the Luminex assay. [Figure 3] This shows that sCD4 protein effectively reduces the mortality rate of LPS-induced sepsis. (A) Mice were intraperitoneally injected with sCD4 protein, followed by intraperitoneal injection of LPS, and the survival rate of the mice was observed. (B) Luminex assay was used to detect TNF and IL-6 concentrations in mouse serum. [Figure 4]This shows that sCD4 protein effectively reduces mortality in CLP sepsis. (A) Mice were intraperitoneally injected with sCD4 protein and then underwent CLP surgery. (A) Luminex analysis was used to detect the suppression of inflammatory cytokine responses in mice. (B) Flow cytometry was used to analyze the suppression of sCD4 activation in splenic macrophages. [Figure 5] This shows that sCD4 suppression of the TLR 4 inflammatory response is dependent on MHC II. (A) sCD4 was co-cultured with WT or MHC II knockout (MHC II- / -) mouse BMDMs, and then the cells were stimulated with LPS. sCD4 failed to inhibit the TLR 4 inflammatory response of MHCII- / - BMDM cells. (B) sCD4 protein was intraperitoneally injected into wt or MHC II- / - mice, and the mice were stimulated with LPS. sCD4 protein failed to reduce mortality or the inflammatory cytokine storm in MHCII- / - mice. [Figure 6] We demonstrate that sCD4 suppression of TLR4-mediated inflammatory responses is dependent on macrophage MHC II molecules. Mice were depleted of macrophages with liposomes and then reinfused with MHC II+ / + (A) or MHC II- / - (B) macrophages. sCD4 protein was then intraperitoneally injected, followed by intraperitoneal injection of LPS. Mice were then monitored for survival and serum inflammatory cytokine levels. [Figure 7] This shows that sCD4 suppression of macrophage TLR4 inflammatory responses is independent of the CD40-mediated anti-inflammatory signaling pathway. (A) sCD40 ligand protein (sCD40L) was co-incubated with wt or MHC II- / - BMDMs, respectively, and (B) sCD4 was co-incubated with wt or CD40 knockout (CD4O- / -) BMDMs, respectively. Furthermore, after stimulation with LPS, the levels of inflammatory cytokines in the supernatant were detected by Luminex. [Figure 8]We demonstrate that sCD4 suppresses TRAF6-mediated regulation of TLR4 inflammatory responses by activating SHP2. sCD4 protein was co-incubated with BMDMs and then stimulated with LPS for different time periods. (A) Protein blotting was used to measure the phosphorylation level of SHP2 in cell lysates. (B) Co-immunoprecipitation (co-IP) of SHP2 and TRAF6 was performed to detect their interaction. (C) Wild-type (SHP2fl / fl) or macrophage-specific knockout SHP2 gene (SHP2- / -) mice were intraperitoneally injected with sCD4 protein and then intraperitoneally injected with LPS. Survival and inflammatory response capacity of the mice were measured. (D) Changes in TLR4 inflammatory responses after pretreatment of wild-type (SHP2fl / fl) or (SHP2- / -) BMDM cells with sCD4 protein were also measured, including detection of IRAK1 and IkBa phosphorylation levels. [Figure 9] We demonstrate that sCD4 suppression of TLR4-mediated inflammatory responses is also STING-dependent. We intraperitoneally injected sCD4 protein into wild-type (STING+ / +) or STING gene knockout (STING- / -) mice, followed by intraperitoneal injection of LPS, and then measured the survival rate and inflammatory response capacity of the mice. [Figure 10] sCD4 disrupts the lipid raft domain of the MHCII / TLR4 complex and suppresses the pro-inflammatory TLR4 plasma membrane distribution. BMDMs were pre-incubated with sCD4, followed by the addition of LPS. (A) Quantitative analysis of the interactions between the indicated protein molecules (red dot structures) in BMDMs using the Duolink method. (B) Colocalization of STING and SHP2 heterodimers (green) stained with Duolink and MHC II molecules (red) immunofluorescently stained in BMDMs (yellow). Cell nuclei were stained with DAPI, and Pearson's coefficients were used to measure the degree of colocalization. Scale bar: 5 μm. [Figure 11]We demonstrate that sCD4 specifically interferes with TLR4 activation but does not affect TNF inflammatory signaling. sCD4 protein was co-incubated with BMDMs and then stimulated with TNF for different periods of time. Protein blotting was used to demonstrate changes in the inflammatory response in cell lysates, including the phosphorylation levels of JnK, p38, and IkBa. DETAILED DESCRIPTION OF THE INVENTION

[0038] As a result of extensive research, the present inventors have demonstrated that soluble CD4 (sCD4) suppresses excessive inflammatory responses in macrophages, and have also demonstrated that MHCII, the target of sCD4's suppression of excessive inflammatory responses in macrophages, and its crosstalk, suppress the TLR / NF-κB inflammatory signal chain response.

[0039] sCD4 sCD4 is derived from the extracellular domain of the CD4 molecule. The amino acid sequence of human-derived sCD4 is shown in SEQ ID NO:3, and its nucleotide sequence is shown in SEQ ID NO:4; the amino acid sequence of mouse-derived sCD4 is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:2. The present invention also encompasses sCD4 homologues from other species and their applications.

[0040] The sCD4 of the present invention may be naturally occurring, for example, isolated or purified from a human or non-human mammal. Alternatively, the sCD4 may be artificially prepared, for example, recombinant sCD4 may be produced using conventional recombinant genetic engineering techniques for experimental or clinical use. Recombinant sCD4 may be used in the present invention. The sCD4 may include full-length sCD4 or a biologically active fragment thereof. Preferably, the amino acid sequence of the sCD4 may be essentially the same as the sequence shown in SEQ ID NO:3 or SEQ ID NO:1. The corresponding nucleotide coding sequence can be easily obtained from the amino acid sequence of sCD4.

[0041] The present invention also encompasses sCD4 amino acid sequences in which one or more amino acid residues have been substituted, deleted, or added. sCD4 or biologically active fragments thereof contain some sequences with conservative amino acid substitutions, which do not affect or retain some of its activity. Appropriate amino acid substitutions are well-known in the art, are easily performed, and ensure that the biological activity of the resulting molecule is not altered. These techniques have led those skilled in the art to recognize that, in general, changing a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity.

[0042] Any biologically active fragment of sCD4 can be used in the present invention. Here, a biologically active fragment of sCD4 refers to a polypeptide that can retain all or part of the function of full-length sCD4. Typically, the biologically active fragment retains at least 50% of the activity of full-length sCD4. Under more preferred conditions, the active fragment can retain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length sCD4.

[0043] Modified or improved sCD4s can also be used in the present invention, for example, to enhance their half-life, potency, metabolism, and / or protein effects. The modified or improved sCD4 can be a complex of sCD4 or can contain substituted or artificial amino acids. The modified or improved sCD4 can have relatively little in common with naturally occurring sCD4, but can also suppress macrophage inflammatory responses (e.g., for treating sepsis). In other words, any altered form that does not affect the biological activity of sCD4 can be used in the present invention.

[0044] The present invention provides a method for applying soluble CD4 protein (sCD4) that can suppress excessive inflammatory responses. sCD4 recognizes and acts on MHC II receptors on the surface of macrophages, thereby suppressing the excessive expression of inflammatory cytokine genes mediated by macrophage TLR4, and exhibits excellent anti-inflammatory function against sepsis caused by gram-negative bacterial lipopolysaccharide (LPS) and cecal ligation and puncture (CLP).

[0045] The inventors' research has revealed for the first time technical solutions including, but not limited to, the following: (1) Mouse and / or human sCD4 proteins suppress the production and secretion of the proinflammatory cytokines TNF and IL-6 in bone marrow-derived macrophages (BMDMs) and peripheral blood mononuclear cell (PBMC)-derived macrophages, respectively, in a dose-dependent manner. sCD4 can also suppress macrophage inflammatory responses induced by TLR3, TLR7, and TLR9 agonists. (2) sCD4 can suppress the progression of inflammation in LPS and CLP sepsis animal models. (3) sCD4 suppresses macrophage TLR4-mediated inflammatory responses independently of CD4 T cell co-inhibitory molecules (e.g., CD40 ligand, CD40L); (4) sCD4 specifically recognizes and utilizes MHC II, exerting a negative regulatory effect on macrophage inflammatory responses, and macrophage MHC II is sufficient to mediate sCD4 regulation of excessive inflammatory responses, such as those in sepsis; (5) sCD4 specifically regulates the inflammatory response of TLR4 in an MHC II-dependent manner, without affecting the chain reaction of other inflammatory cytokines (e.g., TNFR); (6) sCD4 binds MHC II, and the intracellular domain of the latter recruits and activates SHP2 and STING, suppressing the activation of inflammatory signaling molecules MyD88 / IRAK1 / TRAF6 downstream of TLR4 and inhibiting the expression of inflammatory cytokine genes such as TNF / IL-6, which are driven by NF-κB.

[0046] In a specific embodiment of the present invention, human-derived and mouse-derived sCD4 that can be efficiently expressed in human HEK293 cells was obtained, and the effective drug concentrations for suppressing TLR inflammatory responses and the development of sepsis were determined in cultured cells and different animal models of sepsis, demonstrating clear therapeutic and clinical value for the inflammatory responses associated with CD4 T lymphocyte deficiency syndrome.

[0047] Based on the inventors' new findings, the present invention provides the application of sCD4, its upregulators (including constructs expressing it), in the preparation of recombinant proteins that suppress macrophage inflammatory responses.

[0048] As used herein, the terms "macrophage inflammatory response / symptom (macrophage inflammation)" and "macrophage hyperinflammatory response / symptom)" can be used interchangeably, and both refer to clinical indications / diseases caused by an excessive inflammatory response of macrophages (e.g., showing an increase in inflammatory cytokine levels via TLR4; more specifically, showing an increase in TNF or IL-6 levels).

[0049] As used herein, the term "sepsis hyperinflammatory response / symptoms" refers to clinical indications / diseases caused by the hyperinflammatory response of sepsis.

[0050] As used herein, the sCD4 upregulator includes a promoter, an agonist, etc. Any substance that can improve the activity of sCD4, maintain the stability of sCD4, promote the expression of sCD4, promote the secretion of sCD4, prolong the effective duration of sCD4, or promote the transcription and translation of sCD4 can be used in the present invention as an effective substance with upregulatory function.

[0051] As used herein, the sCD4 upregulator includes, but is not limited to, an expression vector or expression construct capable of expressing (preferably overexpressing) sCD4 after being introduced into a cell. Typically, the expression vector contains a gene cDNA subcloning sequence cassette, which contains a gene encoding sCD4 and an expression control sequence operably linked thereto. "Operably linked" or "operably linked" refers to a situation in which one portion of a linear DNA sequence can regulate or control the activity of another portion of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

[0052] In the present invention, the sCD4 polynucleotide sequence may be inserted into a recombinant expression vector, which can then be introduced into cells and overexpressed to produce sCD4. Any plasmid or vector can be used in the present invention as long as it is replicable and stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and a translation control element. The expression vector includes viral vectors and non-viral vectors, and preferably includes, but is not limited to, adeno-associated virus vectors, lentivirus vectors, and adenovirus vectors.

[0053] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence containing sCD4 and appropriate transcriptional / translational control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.

[0054] The present invention also provides a composition comprising an effective amount (for example, 0.000001 to 20% by weight, preferably 0.00001 to 10% by weight) of the sCD4, or an upregulator thereof (for example, an expression vector that overexpresses the sCD4), or an analog thereof, and a pharmaceutically acceptable carrier.

[0055] The composition of the present invention can be used directly to suppress macrophage inflammatory responses, and can also be used in combination with other therapeutic agents or adjuvants.

[0056] In general, these substances can be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably about 6-8.

[0057] As used herein, the term "comprises" indicates that each component may be applied together in the mixture or composition of the present invention. The terms "consisting essentially of" and "consisting of" are included in the term "comprises." As used herein, the term "effective amount" or "effective dosage" refers to an amount that is functional or active in humans and / or animals and tolerated by humans and / or animals.

[0058] As used herein, a "pharmaceutically acceptable" ingredient is one that can be administered to humans and / or mammals without undue adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used in administering a therapeutic agent, and includes various excipients and diluents.

[0059] The compositions of the present invention contain a safe and effective amount of sCD4, or its upregulator (e.g., an expression vector overexpressing the sCD4), or an analog thereof, and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Typically, pharmaceutical formulations are required depending on the dosage form. The pharmaceutical compositions of the present invention are prepared as injections and are prepared by conventional methods using, for example, saline or an aqueous solution containing glucose or other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical formulations of the present invention can also be sustained-release formulations.

[0060] The effective amount of sCD4 or its upregulator according to the present invention may vary depending on the administration method and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by one skilled in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of sCD4 or its upregulator, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated in the patient; the patient's body weight; the patient's immune status; and the route of administration. Generally, sCD4 or its upregulator according to the present invention can be effectively administered at a daily dose of about 0.00001-10 mg / kg of animal body weight. For example, depending on the exigencies of the therapeutic situation, the daily dose may be administered in divided doses or the dose may be proportionally reduced.

[0061] The present invention also provides a method for suppressing inflammatory responses of macrophages, comprising administering to a subject an effective amount of sCD4, or an upregulator thereof (e.g., an expression vector that overexpresses the sCD4), or an analog thereof.

[0062] The administration route of the sCD4 or its upregulator, or its analogue of the present invention is not particularly limited and may be systemic or local. For example, the sCD4 or its upregulator of the present invention can be administered to animals by intraperitoneal injection, intravenous injection, oral injection, subcutaneous injection, intrathecal injection, intradermal injection, etc.

[0063] After understanding the application of sCD4, the sCD4, its encoding gene, or pharmaceutical composition thereof can be administered to a mammal using various methods known in the art. Preferably, gene therapy can be used. For example, sCD4 can be directly administered to a subject by injection or other methods; alternatively, an expression unit (e.g., an expression vector or virus) carrying the sCD4 gene can be delivered to a target site via a specific route to express active sCD4.

[0064] In one embodiment of the present invention, sCD4 can be directly administered to a mammal (e.g., a human). Alternatively, the gene encoding sCD4 can be cloned into an appropriate vector (e.g., a conventional prokaryotic or eukaryotic expression vector, or a viral vector such as a herpesvirus vector or an adenovirus vector) by standard methods, and the vector can be introduced into cells capable of expressing sCD4, thereby allowing the cells to express sCD4. Expression of sCD4 can be achieved by introducing an appropriate amount of cells into an appropriate part of the mammal's body.

[0065] The method of administration of an upregulator or analog of sCD4 will depend primarily on the type and properties of said upregulator, which can be assessed by one skilled in the art.

[0066] In the specific examples of the present invention, several administration schemes for animals such as mice are provided. The conversion of the dosage for animals such as mice to a dosage suitable for humans can be easily performed by those skilled in the art, and can be calculated, for example, based on the Meeh-Rubner formula: A = k × (W 2 / 3) / 10,000, where A is the body surface area and m 2 where W is body weight in grams; K is a constant that varies by animal species, but is generally 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that dosage conversions may vary depending on the drug and clinical situation, as assessed by an empirical pharmacist.

[0067] The present invention can provide a new paradigm for the prevention or treatment of sepsis. Targeting inflammatory cells with sCD4 protein is a fundamental treatment whose mechanism and efficacy are clearly superior to current corticosteroid therapy or symptomatic treatments targeting inflammatory cytokines (TNF, IL-6, etc.) or their homologous receptor antibodies. When used in combination, it can clearly reduce the drug concentration and / or frequency of use of corticosteroids or inflammatory cytokine antibodies.

[0068] Macrophage-related signaling pathways and their regulation As used herein, the term "(signal) transduction pathway" refers to a signaling system formed by the mutual regulation or interaction between a series of genes or proteins or their metabolic products (synthetic or processed products), including interactions between the transduction pathway proteins and other intracellular elements or cellular organelles, and possibly the cooperative participation of upstream or downstream genes or proteins, which generally results in the occurrence of several cellular events. The macrophage-associated signaling pathway primarily involves the following: macrophage MHC II, TRAF6, Toll-like receptor (TLR), SHP2, or STING. Here, the macrophage-associated signaling pathway involves the involvement of macrophages and lysosomes.

[0069] As used herein, the terms "(signal) transduction pathway" and "(signal) pathway" are used interchangeably.

[0070] The amino acid sequences of the MHC II proteins are shown, for example, in GenBank: NP_996988.2, mouse-derived MHC II beta chain; NP_034508.2, mouse-derived MHC II A / Eα chain. The corresponding nucleotide sequences are A / Eα (NM_010378.3) and Aβ (NM_207105.3), respectively.

[0071] The protein amino acid sequence of SHP2 is shown in, for example, GenBank_Q06124 (derived from mouse).

[0072] The amino acid sequence of the STING protein is shown, for example, in GenBank_Q86WV6 (derived from mouse).

[0073] The amino acid sequence of the TRAF6 protein is shown in, for example, GenBank_Q9Y4K3 (derived from mouse).

[0074] The protein amino acid sequence of the TLR4 is shown in, for example, GenBank_O00206 (derived from mouse).

[0075] In the present invention, unless otherwise specified, the signal transduction pathway from downstream of TLR4 to the onset of inflammation is known to those skilled in the art, and information on upstream and downstream proteins / genes is also known to those skilled in the art.

[0076] In the present invention, the pathway may include one selected from macrophage MHC II-SHP2 and / or STING-TRAF6-TLR4; macrophage MHC II-TLR4; macrophage SHP2-TRAF6-TLR4; and macrophage STING-TLR4. The sCD4 suppresses the occurrence of macrophage inflammatory responses (inflammation) by regulating the pathway. The upstream, middle, or downstream of the pathway may include other regulatory genes or upstream / downstream pathways.

[0077] After understanding the function of the macrophage-associated signaling pathway (preferably including its upstream and downstream proteins or genes), it should be understood that the macrophage-associated signaling pathway can be regulated using various methods well known to those skilled in the art. For example, the expression level or activity level of pathway proteins can be upregulated to achieve the purpose of suppressing inflammation.

[0078] A preferred embodiment of the present invention is to provide a substance (e.g., sCD4 or a construct that overexpresses MHC II) that activates / upregulates macrophage MHC II based on the macrophage MHC II-SHP2 and / or STING-TRAF6-TLR4 signaling pathway, which activates MHC II, causing MHC II to recruit and activate SHP2 and STING in the intracellular space, suppressing the activation of inflammatory signaling molecules MyD88 / IRAK1 / TRAF6 downstream of TLR4, and causing the inhibition of the expression of inflammatory cytokines such as TNF / IL-6, which are driven by NF-κB.

[0079] In a preferred embodiment of the present invention, based on macrophage MHC II-TLR4, a substance that activates / upregulates macrophage MHC II (e.g., sCD4 or a construct that overexpresses MHC II) is provided, which activates MHC II and thereby suppresses TLR4 inflammatory responses.

[0080] In a preferred embodiment of the present invention, based on macrophage SHP2-TRAF6-TLR4, a substance that activates / upregulates SHP2 (e.g., a construct that overexpresses SHP2) is provided, which activates / upregulates macrophage SHP2, thereby suppressing TRAF6 and TLR4 inflammatory responses.

[0081] A preferred embodiment of the present invention is to provide a substance that activates / upregulates STING (e.g., a construct that overexpresses STING) based on macrophage STING-TLR4, which activates / upregulates macrophage STING and thereby suppresses TLR4 inflammatory responses.

[0082] In a preferred embodiment of the present invention, sCD4 plays a role by regulating the MHCII / STING / SHP2 complex.

[0083] Substances that regulate the pathway can be used in the present invention as potentially useful substances for suppressing macrophage hyperinflammatory responses. They may be compounds, small chemical molecules, or biomolecules. The biomolecules may be at the nucleic acid level, including DNA and RNA, or at the protein level.

[0084] When used as targets for artificial regulation or to artificially construct screening systems, the above proteins or encoding genes can be naturally occurring, for example, purified and isolated from mammals, or can be recombinantly prepared, for example, recombinant proteins can be produced based on conventional genetic engineering techniques. Furthermore, any modified forms that do not affect the biological activity of these proteins are also useful, for example, derivatives or mutants whose functions are unchanged.

[0085] Drug screening by macrophage-related signaling pathways Based on the inventors' new findings, the study of macrophage-related signaling pathways has many applications, including screening for substances that modulate the signaling pathways to suppress macrophage hyperinflammatory responses, including upregulating (e.g., activating or increasing expression) MHC II, upregulating SHP2, upregulating STING, and downregulating (e.g., suppressing or decreasing expression) TRAF6.

[0086] The present invention provides a method for screening for a substance that suppresses macrophage inflammatory responses, comprising the steps of: (1) contacting a candidate substance with a system containing a signaling pathway selected from the group consisting of macrophage MHC II-SHP2 and / or STING-TRAF6-TLR4; macrophage MHC II-TLR4; macrophage SHP2-TRAF6-TLR4; and macrophage STING-TLR4; (2) observing the system in (1) and screening for a substance that modulates the signaling pathway of (1), wherein the substance is a substance (including a potential substance) that is useful for suppressing macrophage inflammatory responses.

[0087] As used herein, the terms "upregulate," "downregulate," "improvement," "inhibit," "enhance," "weaken," "promote," "reduce," and the like have a statistical meaning. That is, they refer to a significant "upregulation," "downregulation," "improvement," "inhibit," "enhancement," "weaken," "promotion," or "reduction." For example, they refer to a significant "upregulation," "downregulation," "improvement," "inhibit," "enhancement," "weaken," "promotion," or "reduction" of 10%, 20%, 30%, 40%, 50% or more, and more preferably 60%, 70%, 80%, 100% or more, compared to the protein activity, protein expression, protein binding, or level of methylation in a control group.

[0088] The system containing the macrophage-associated signaling pathway is selected from a cell system (or cell culture system), an intracellular system (or intracellular culture system), a solution system, an animal system, or a tissue system (or tissue culture system).

[0089] In a preferred embodiment of the present invention, the above-mentioned method further comprises conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances useful for suppressing excessive inflammatory responses of macrophages from the candidate substances.

[0090] In conducting the screening, the alteration and interaction status of the protein or its encoding gene can be determined using a variety of techniques well known in the art.

[0091] A variety of conventional techniques can be used to assess the transcription or expression status of genes in a system. These techniques include, but are not limited to, oligonucleotide hybridization techniques (e.g., probes), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, etc. Many techniques well known to those skilled in the art, such as co-immunoprecipitation, GST precipitation, phage display, or yeast double hybridization, can be used to detect protein-protein interactions and the strength of those interactions. Protein nuclear localization is also a well-known technique in the art.

[0092] Substances initially screened by the above method can constitute a screening library, from which substances that are truly useful for suppressing excessive inflammatory responses of macrophages can ultimately be screened.

[0093] The present invention also provides potential substances that can be used to suppress excessive inflammatory responses of macrophages, which are obtained by the screening method.

[0094] The present invention provides a method for preparing a drug that suppresses macrophage inflammatory responses (particularly, suppresses macrophage excessive inflammatory responses), which method includes the following steps: synthesizing and / or purifying a substance useful for suppressing macrophage excessive inflammatory responses obtained by the screening, and preparing it as a drug that suppresses macrophage excessive inflammatory responses.

[0095] As described below in the present invention, substances useful for suppressing macrophage hyperinflammatory responses can be used in preparing pharmaceutical compositions.

[0096] Methods for screening substances that act on a protein or gene, or a specific region thereof, as a target are well known to those skilled in the art, and any of these methods can be used in the present invention. The candidate substance may be selected from peptides, polymeric peptides, peptoids, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences. Depending on the type of substance to be screened, those skilled in the art will know how to select an appropriate screening method.

[0097] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely illustrative of the present invention and do not limit the scope of the present invention. Experimental methods in the following examples that do not specify specific conditions are usually carried out according to the standard conditions described in J. Sambrook et al., Guide to Molecular Cloning, Third Edition, Science Press, or according to the conditions recommended by the manufacturer. [Example]

[0098] Materials and Methods Pre-incubation of BMDM cells with sCD4 protein BMDMs were differentiated and matured with 25 nM sCD4 protein in a 24-well flat-bottom cell culture plate overnight (8-12 hours). The cells were then stimulated with 100 ng / mL LPS, 100 μg / mL poly(I:C), 0.3 μM CpG ODN, or VSV (MOI = 5). After 6-12 hours, the cell supernatant was collected and used for cytokine detection. BMDC cells can also be used for specific experiments.

[0099] Detection of the effect of sCD4 protein on the TLR pathway by protein blotting The treated cells were lysed using cell lysis solution, and total cellular protein was obtained. Protein electrophoresis was performed on 10% SDS-PAGE, and the protein was transferred to a PVDF membrane and blocked with 5% nonfat milk. The membrane was then incubated with the primary antibody (PBST + 5% nonfat dry milk for general antibodies, or 5% BSA for phosphorylated antibodies) and incubated overnight at 4°C on a shaker at low speed. The corresponding secondary antibody (PBST + 5% nonfat dry milk) was then incubated for 1 hour at room temperature, followed by exposure and development using the chemiluminescence method.

[0100] Method for administering sCD4 protein to mice Mice were intraperitoneally injected with sCD4 protein at 15 μg / kg body weight, and 12 hours later, LPS was intraperitoneally injected.

[0101] Establishment of a mouse sepsis model LPS model: Eight-week-old male mice were intraperitoneally injected with 8 mg / kg LPS depending on their body weight, and the mortality was observed every six hours.

[0102] CLP model: Eight-week-old male mice were used. After anesthesia, the animals were placed supine on a surgical board. The abdominal surgical area was routinely disinfected and depilated. Under aseptic conditions, a 2-cm incision was made in the abdominal wall with a scalpel. The abdomen was entered through the incision and separated at the distal end of the ileocecal valve. One-third of the cecum was ligated with a No. 3 suture. An No. 18 needle was used to puncture the ligated end, and a small amount of feces was pushed out. The peritoneum and skin were then intermittently sutured with a No. 4 suture.

[0103] Detection of cellular inflammatory cytokines by Luminex First, the standard sample was prepared. The standard powder was centrifuged at 10,000 rpm for 10 seconds to precipitate the protein. 200 μL of heavy suspension buffer was added, and the mixture was thoroughly mixed by shaking on an oscillator. The mixture was then left at room temperature for 10 minutes to fully dissolve the protein. The standard was then serially diluted in eight 4-fold gradients. The antibody magnetic beads were thoroughly mixed using an oscillator for 30 seconds to thoroughly mix the beads. 50 μL of antibody magnetic beads were added per well to a light-shielded 96-well detection plate. The 96-well plate was placed on a magnetic base and allowed to adsorb for 2 minutes, after which the supernatant was discarded. 150 μL of wash buffer was added to the sample plate, allowed to stand for 30 seconds, then placed on a magnetic base and allowed to adsorb for 2 minutes, after which the supernatant was discarded. 25 μL of standard or sample was added per well. The detection plate was sealed with a sealing membrane and incubated at 500 rpm for 1 hour at room temperature. The sample plate was placed on a magnetic base and allowed to stand for 2 minutes, after which the liquid was discarded. Next, wash buffer was added to the sample plate, and after 30 seconds, the liquid was discarded. This was repeated three times. 25 μL of detection antibody solution was added per well and incubated at 500 rpm for 30 minutes at room temperature. The sample plate was placed on a magnetic base and after 2 minutes, the liquid was discarded. 150 μL of wash buffer was added to the sample plate and incubated for 30 seconds. The sample plate was then placed on a magnetic base and allowed to adsorb for 2 minutes. The supernatant was discarded. This was repeated three times. 25 μL of PE detection solution was added per well and incubated at 500 rpm for 30 minutes at room temperature. The sample plate was placed on a magnetic base and after 2 minutes, the liquid was discarded. 150 μL of wash buffer was added to the sample plate and incubated for 30 seconds. This was repeated three times. 120 μL of loading buffer was added per well and detected using a Bio-Rad detector.

[0104] Sequence information Amino acid sequence of sCD4 protein from mouse (SEQ ID NO:1): [ka]

[0105] Nucleotide coding sequence of sCD4 protein from mouse (SEQ ID NO:2; codon-optimized sequence): [ka]

[0106] Amino acid sequence of human sCD4 protein (SEQ ID NO:3): [ka]

[0107] Nucleotide coding sequence of sCD4 protein from human (SEQ ID NO:4; codon-optimized sequence): [ka]

[0108] Example 1 sCD4 suppresses inflammatory responses mediated by TLRs in APCs (macrophages, dendritic cells) 1. sCD4 protein reduces inflammatory cytokine levels The inventors injected different concentrations of mouse-derived sCD4 protein (0, 25 nM, 50 nM, 250 nM) into mouse bone marrow-derived macrophages (BMDM, 5 × 10 4 After 6 hours of stimulation with 100 ng / mL LPS, the concentrations of inflammatory cytokines TNF and IL-6 in the cell supernatant were detected using the Luminex method.

[0109] As shown in Figure 1A, the results showed that the inflammatory cytokines produced by the cells were significantly reduced with increasing sCD4 protein concentration.

[0110] Thus, sCD4 protein reduces inflammatory cytokine levels in a dose-dependent manner.

[0111] 2. Inhibitory effects of sCD4 on different TLRs or cellular inflammatory responses The inventors co-cultured mouse bone marrow-derived BMDMs with mouse-derived sCD4 protein, and then stimulated the TLR3, TLR7, and TLR9 signaling pathways using 100 μg / mL polyI:C, 0.3 μM CpG, or VSV virus (MOI = 5), respectively. Using the PBS group as a control, the concentrations of the inflammatory cytokines TNF and IL-6 in the cell supernatant were detected by the Luminex method.

[0112] As shown in Figure 1B, compared to the PBS control group, sCD4 protein significantly suppressed the intensity of TLR 3, TLR 7, and TLR 9 signaling pathways induced by polyI:C, CpG, or VSV virus. The most notable inhibitory effect was on the inflammatory cytokine IL-6, which was generated by the signaling pathway induced by polyI:C or VSV virus.

[0113] Therefore, sCD4 protein suppresses inflammatory responses mediated by TLR3, TLR7, and TLR9 signaling pathways stimulated by polyI:C, CpG, or VSV virus.

[0114] 3. Inhibitory effects of sCD4 on different Toll-like receptors (TLRs) or cellular inflammatory responses The inventors injected different concentrations of mouse-derived sCD4 protein (0, 25 nM, 50 nM) into mouse bone marrow-derived dendritic cells (BMDCs, 5 × 10 4 After co-culture with 100 μg / well of 1000 cells and stimulation with LPS for the times indicated in the figure, the concentrations of inflammatory cytokines TNF and IL-6 in the cell supernatant were detected using the Luminex method.

[0115] As a result, as shown in Figure 1C, sCD4 was found to have a significant inhibitory effect on different TLRs or cellular inflammatory responses.

[0116] Example 2 Human-derived sCD4 suppresses macrophage TLR4 inflammatory responses The inventors co-cultured human peripheral blood mononuclear cell (PBMC)-derived macrophages with different concentrations of human sCD4 protein (0, 1.39 nM, 4.17 nM, 12.5 nM, 37.5 nM, 112.5 nM), and then stimulated them with LPS for the times indicated in the figure. Then, the concentrations of inflammatory cytokines in the supernatant were detected using the Luminex method.

[0117] As a result, it was found that the concentrations of TNF and IL-6 in the macrophage culture supernatant decreased significantly with increasing human-derived sCD4 protein concentration, as shown in Figure 2. Therefore, human-derived sCD4 suppresses macrophage inflammatory responses in a dose-dependent manner.

[0118] Example 3 sCD4 protein effectively reduces mortality in LPS sepsis The inventors intraperitoneally injected mice with mouse-derived sCD4 protein (15 μg / kg), followed 12 hours later by intraperitoneal injection of LPS, and monitored the survival rate of the mice at different time points. At the same time, serum samples were extracted from the mice and the concentrations of inflammatory cytokines TNF and IL-6 in the serum were analyzed at different time points.

[0119] Analysis of survival rates revealed that addition of sCD4 protein could significantly increase the survival rate of animals, as shown in Figure 3A.

[0120] Analysis of serum concentrations of the inflammatory cytokines TNF and IL-6 showed that, as shown in Figure 3B, both TNF and IL-6 concentrations decreased significantly 12 and 24 hours after LPS injection, with the decrease at 24 hours being particularly significant.

[0121] Example 4 sCD4 protein effectively reduces mortality in CLP sepsis In this example, the effects of sCD4 protein were analyzed using a mouse model of sepsis, such as cecal ligation and puncture (CLP).

[0122] We established a sepsis animal model by intraperitoneally injecting human sCD4 protein into mice and then performing CLP surgery on the mice. Serum was collected at the indicated time points, and splenocytes were isolated. The inflammatory cytokine response was measured using the Luminex method, and the activation of splenic macrophages was analyzed using flow cytometry.

[0123] The results of the Luminex assay, as shown in Figure 4A, showed that sCD4 significantly suppressed responses to the inflammatory cytokines TNF and IL-6 in mice.

[0124] As a result of flow cytometry analysis, as shown in Figure 4B, no significant change was observed in the number of resident macrophages in the sCD4 group, but among them, MHC II type increased.

[0125] Example 5: sCD4 suppression of TLR4-mediated inflammatory responses depends on MHC II The inventors administered mouse-derived sCD4 protein (25 nM) to WT or MHC II knockout (MHC II - / - After co-culture with mouse BMDM, the cells were stimulated with LPS. As shown in Figure 5A, after MHC II knockout, sCD4 was significantly reduced by MHC II. - / - sCD4 was unable to inhibit the inflammatory response of BMDM cells induced by TLR4, indicating that the suppression of TLR4-induced inflammatory responses by sCD4 is dependent on MHC II.

[0126] The inventors administered sCD4 protein (15 μg / kg) to WT or MHC II - / - Mice were intraperitoneally injected with sCD4 and challenged with LPS. The results, as shown in Figure 5B, showed that after MHC II knockout, sCD4 protein was not associated with MHC II. - / - It failed to reduce mortality or the inflammatory cytokine storm in mice.

[0127] Thus, sCD4 suppression of TLR4 inflammatory responses is MHC II dependent.

[0128] Example 6: sCD4 suppression of TLR4-induced inflammatory responses depends on macrophage MHC II molecules The inventors used clodronate liposomes to deplete mouse macrophages, and then isolated MHC II + / + or MHC II - / - The macrophages were then re-injected; mice were intraperitoneally injected with mouse-derived sCD4 protein, followed by intraperitoneal injection with LPS, and the survival rate and serum inflammatory cytokine levels of the mice were then monitored.

[0129] The results, as shown in Figure 6A-B, show that MHC II + / + After reinfusion of macrophages, sCD4 could significantly improve mouse survival and reduce inflammatory cytokine levels, whereas the changes were not significant when reinfused with MHC II- / - macrophages.

[0130] Thus, sCD4 suppression of TLR4-mediated inflammatory responses depends on macrophage MHC II molecules.

[0131] Example 7: sCD4 suppression of TLR4-induced inflammatory responses is independent of the CD40 signaling pathway We used mouse-derived soluble CD40 ligand protein (sCD40L, obtained from Sino Biological) as a control for either WT or MHC II. - / - After co-incubation with mouse bone marrow-derived BMDMs and stimulation with LPS, the levels of inflammatory cytokines in the supernatant were detected by Luminex analysis. As shown in Figure 7A, co-incubation with sCD40L significantly reduced the levels of inflammatory cytokines.

[0132] The inventors compared sCD4 with either WT or CD40 knockout (CD4O - / - ) BMDMs. After stimulation with LPS, the levels of inflammatory cytokines in the supernatant were detected by Luminex. The results, as shown in Figure 7B, showed that CD40 knockout (CD4O - / - ) showed a similar trend in inflammatory cytokine changes as WT.

[0133] Thus, sCD4 suppression of TLR4 inflammatory responses is independent of the CD40 signaling pathway.

[0134] Example 8 sCD4 suppresses TRAF6-mediated regulation of TLR4 inflammatory responses by activating SHP2 We co-incubated wild-type BMDMs with mouse-derived sCD4 protein (25 nM) and then stimulated them with LPS for different time periods. The phosphorylation level of SHP2 in the cell lysates was detected by protein blotting. The results, as shown in Figure 8A, demonstrated that co-incubation with sCD4 protein promoted SHP2 phosphorylation, which in turn activated SHP2.

[0135] Furthermore, we performed co-immunoprecipitation (co-IP) analysis of SHP2 and TRAF6 to measure their interaction, and the results, as shown in Figure 8B, showed that sCD4 protein significantly increased the interaction between SHP2 and TRAF6.

[0136] Furthermore, wild-type (SHP2 fl / fl ) or macrophage-specific knockout SHP2 gene (SHP2 - / - Mice were intraperitoneally injected with sCD4 protein and then with LPS, and their survival rate and inflammatory response ability were measured. The results, shown in Figure 8C, showed that the ability of sCD4 to suppress TLR4-mediated inflammatory responses was abolished by the deletion of the SHP2 gene in macrophages.

[0137] Furthermore, we also measured the phosphorylation levels of IRAK1 and IκBα. As shown in Figure 8D, the ability of sCD4 to suppress the phosphorylation of IRAK1 and IκBα was also lost when macrophages were deficient in the SHP2 gene.

[0138] Thus, by activating SHP2, sCD4 suppresses TRAF6 activation and exerts control over TLR4 / IRAK1 / NF-κB-mediated inflammatory responses.

[0139] Example 9: sCD4 suppression of TLR4-mediated inflammatory responses also depends on STING The inventors compared mouse-derived sCD4 protein with WT (STING + / + ) or STING gene knockout (STING - / - ) Mice were intraperitoneally injected with IgG, and then LPS was intraperitoneally injected, and the survival rate and inflammatory response ability of the mice were measured.

[0140] The results are shown in Figure 9. + / + ) administration of sCD4 significantly improved the survival rate of animals, reaching 100% survival. - / - There was no significant difference in the administration of sCD4 in the group.

[0141] Example 10: sCD4 disrupts the lipid raft region of the MHCII / TLR4 complex and suppresses pro-inflammatory TLR4 cell membrane distribution. BMDMs were pre-incubated with sCD4 and then LPS was added. Using the Duolink method, antibodies to MHC II, TLR, SHP2, phosphorylated SHP2, and STING were labeled with antibodies. Laser confocal imaging was then performed to quantitatively analyze the interactions between the protein molecules shown in Figure 10A (red dot structures). Alternatively, as shown in Figure 10B, colocalization analysis (yellow) was performed on the STING and SHP2 heterodimers stained with the Duolink method (green) and immunofluorescently stained MHC II molecules (red) in BMDMs.

[0142] These results demonstrate that sCD4 regulates the MHCII / STING / SHP2 complex. sCD4 significantly suppressed the MHCII / TLR4 inflammatory complex, reducing the binding of SHP2 and STING dimers to MHCII, but increasing the binding of activated SHP2 (pY580) to MHCII. This suggests that sCD4-MHCII binding disrupts the lipid raft domain of the MHCII / TLR4 complex and suppresses the pro-inflammatory TLR4 plasma membrane distribution.

[0143] Example 11 sCD4 specifically interferes with TLR4 activation but does not affect TNF inflammatory signaling The inventors co-incubated sCD4 protein with BMDMs and then stimulated them with TNF for different periods of time. Protein blotting was used to demonstrate changes in the inflammatory response in cell lysates, including the phosphorylation levels of JnK, p38, and IkBa.

[0144] The results, as shown in Figure 11, show that sCD4 specifically interferes with the activation of TLR4 but does not affect TNF inflammatory signals.

[0145] The above-described examples show some embodiments of the present invention, and although the explanations are more specific and detailed, this should not be construed as limiting the patent scope of the present invention.

[0146] Those skilled in the art can make some modifications and improvements without departing from the concept of the present invention, which fall within the protection scope of the present invention. Therefore, the patent protection scope of the present invention shall be subject to the scope of the appended claims. In addition, all references mentioned in this application are incorporated herein by reference as if they were incorporated individually by reference.

Claims

1. Use of a soluble CD4 recombinant protein or a construct expressing a soluble CD4 recombinant protein in the preparation of a pharmaceutical composition for treating or preventing an inflammatory response, wherein said inflammatory response is a septic hyperinflammatory response or a macrophage inflammatory response.

2. The use according to claim 1, characterized in that the macrophage inflammatory response includes inflammation caused by excessive expression of inflammatory cytokines mediated by macrophage TLR4 at the very early stage of sepsis onset; preferably, the inflammation caused by excessive expression of inflammatory cytokines mediated by macrophage TLR4 includes inflammation caused by excessive expression of TNF and / or IL-6.

3. The application of claim 1, characterized in that the soluble CD4 recombinant protein comprises the extracellular domain of a CD4 molecule; preferably, the extracellular domain of a CD4 molecule comprises D1, D2, D3 and D4 domains; preferably, the soluble CD4 recombinant protein is obtained by encoding it using a codon-optimized coding sequence of human or mouse origin.

4. The soluble CD4 recombinant protein comprises a protein having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a conservative variant thereof; preferably, the conservative variant is (1) a derived protein formed by substituting, deleting or inserting one or more amino acid residues from the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and retaining the protein function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; (2) a derived protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and that retains the protein function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; or (3) A protein formed by adding a tag sequence or a signal peptide sequence to a protein having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

3.

4. The application according to claim 1 or 3, characterized in that it comprises one selected from the group consisting of:

5. The application of claim 4, characterized in that the conservative variant is selected from variants containing an oligopeptide tag that contributes to the detection of the protein shown in SEQ ID NO: 1 or SEQ ID NO: 3, and / or Fc-fused recombinant variants that increase the stability of the protein shown in SEQ ID NO: 1 or SEQ ID NO:

3.

6. The application of claim 1, characterized in that the construct expressing the soluble CD4 comprises an expression vector; preferably, the expression vector comprises a viral expression vector or a non-viral expression vector; more preferably, the viral expression vector comprises an adeno-associated virus, a lentiviral vector, or an adenoviral vector.

7. The use of claim 1, characterized in that the soluble CD4 cross-suppresses the inflammatory response by binding to macrophage MHC II; preferably, the inflammatory response includes a sepsis hyperinflammatory response or a macrophage inflammatory response; more preferably, the macrophage inflammatory response is a macrophage TLR4 inflammatory response.

8. The use of claim 7, wherein the signaling pathway utilized in the TLR4 inflammatory response is the macrophage MHC II-TLR4 inflammatory response complex; preferably, the soluble CD4 recombinant protein or the construct expressing soluble CD4 upregulates MHC II, recruits and activates SHP-2, inhibits TRAF6, and cross-inhibits TLR4 / NF-κB-mediated inflammatory responses.

9. The application of a signal transduction pathway established artificially or present in a cell, or a system containing the signal transduction pathway; the application is to screen for substances (including compounds, compositions, drugs, etc.) that suppress macrophage inflammatory responses; the signal transduction pathway macrophage MHC II-SHP2 and / or STING-TRAF6-TLR4; macrophage MHC II-TLR4; macrophage SHP2-TRAF6-TLR4; Macrophage STING-TLR4 Selected from.

10. (1) A candidate substance is contacted with a system containing a signal transduction pathway selected from the following group: macrophage MHC II-SHP2 and / or STING-TRAF6-TLR4; macrophage MHC II-TLR4; macrophage SHP2-TRAF6-TLR4; macrophage STING-TLR4; (2) observing the system of (1) and screening for a substance that regulates the signal transduction pathway of (1), the substance being a substance (including a potential substance) useful for suppressing macrophage inflammatory responses; the regulation includes upregulating (e.g., activating or increasing expression) MHC II, upregulating SHP2, upregulating STING, and downregulating (e.g., suppressing or decreasing expression) TRAF6; A method for screening substances that suppress macrophage inflammatory responses.

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