Use of angptl8 neutralizing antibodies in the preparation of a medicament for treating cytokine storm syndrome

Abstract

The application provides an application of an Angptl8 neutralizing antibody in preparation of a drug for treating mouse cytokine storm syndrome. The present study proves that angiopoietin-like protein 8 (Angptl8) can promote macrophages to release inflammatory factors such as TNF-alpha, IL-6 and IL-1beta by using molecular biology means. In terms of mechanism, Angptl8 can cause the enhancement of the inflammatory factor storm of the body by promoting the polarization of M1 type macrophages and inhibiting the polarization of M2 type macrophages, and the knockout of Angptl8 can improve this pathological phenomenon. Therefore, we synthesize the Angptl8 neutralizing antibody and inject it into the CSS mouse to combine with the Angptl8 in the blood circulation of the mouse, successfully inhibit the large release of the inflammatory factors of the body, and improve the survival rate of the mouse within 72h to 60%. The application proves that the Angptl8 neutralizing antibody can effectively treat the mouse cytokine storm syndrome, and clarifies the molecular mechanism of the target Angptl8 in regulating the disease process, and provides an effective strategy for treating the CSS patient in the clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological technology, and particularly relates to the application of Angptl8 neutralizing antibody as an effective drug for treating cytokine storm by regulating macrophage polarization. BACKGROUND

[0002] Cytokine storm syndrome (CSS) is a systemic inflammatory response associated with infectious and non-infectious diseases, which can be induced by infection, drugs and other factors. In this process, a large number of cytokines participate in the process of immune regulation and inflammatory response of the body, thereby inducing cytokine storm, and then inducing the occurrence of CSS, leading to systemic inflammatory response and multiple organ failure, and even death. CSS is roughly divided into two categories, including those caused by infectious diseases and non-infectious diseases. Those induced by infectious diseases include those derived from bacteria such as Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumoniae and Yersinia pestis; and those derived from viruses. Those caused by non-infectious diseases include monogenic and autoimmune diseases. So far, the specific molecular mechanism of CSS has not been fully elucidated, and the clinical treatment means is single, mostly by using recombinant proteins, or monoclonal antibodies to neutralize cytokines to inhibit pro-inflammatory signals. For example, tocilizumab inhibits IL-6. Therefore, we urgently need to find an effective target for treating CSS and elucidate its mechanism.

[0003] Cytokine storm is different from ordinary inflammatory response, and some literatures have proposed three criteria for distinguishing cytokine storm from ordinary inflammatory response: 1. elevated circulating cytokine levels; 2. acute systemic inflammatory response; 3. occurrence of secondary organ dysfunction (usually kidney, liver or lung) caused by inflammation, and the degree of inflammatory response exceeds the normal immune response to pathogens. Cytokine storm includes several immune disorders characterized by systemic inflammation and multiple organ dysfunction, in which the body can rapidly develop into disseminated intravascular coagulation, accompanied by vascular occlusion or massive hemorrhage, dyspnea, hypoxemia, hypotension, hemostatic imbalance, vasodilatory shock, etc., and in the late stage of the disease or during recovery, it worsens into acute respiratory distress syndrome (ARDS) and multiple organ failure, which can cause the patient to die in a short time.

[0004] The development of cytokine storm is closely related to the immune system. In the early stage of inflammatory response, the body suppresses invasive pathogens by activating innate and adaptive immune responses. On the one hand, the immune system needs to produce enough cytokines to eliminate pathogens, on the other hand, the body needs to avoid producing a large amount of cytokines, which will lead to excessive inflammatory response, and the balance between the two should be maintained. Usually, cytokines play a key role in the regulation of signals in anti-pathogen and immune response amplification, but in excessive inflammatory response, a large amount of cytokines will cause significant collateral damage to the body. Innate immune system cells are the first line of defense against pathogens, which recognize pathogens through pattern recognition receptors, produce cytokines to activate adaptive immune system cells to recognize and respond to various microbial invasions. The most common innate cells involved in the pathogenesis of cytokine storm include neutrophils, macrophages and NK cells. Neutrophils can produce neutrophil extracellular traps, which are a kind of fiber network that helps thrombosis and amplifies cytokine production during cytokine storm. Macrophages are tissue-resident cells, usually derived from circulating monocytes, which do not divide, and they have multiple functions, playing a key role in the process of aging cell clearance, tissue repair, immune regulation and antigen presentation. In cytokine storm, macrophages are activated and secrete excessive cytokines, which eventually lead to severe tissue damage and cause organ failure. Hemophagocytosis is often observed in bone marrow biopsy specimens of cytokine storm patients. Interferon-gamma can induce macrophages to produce erythrophagocytosis, which may be the main cause of common cytopenia in cytokine storm patients. Therefore, macrophages can be an effective target for treating cytokine storm.

[0005] The liver is not only an organ for regulating emotions, but also plays an important role in innate immunity. The innate immune system of the liver contains physical and chemical barriers, humoral factors, phagocytes and lymphocytes (NK and NKT cells), which quickly respond to pathogenic attacks and are the first key line of defense against infection.

[0006] A large number of studies have shown that Angptl8, a liver-derived factor, is an important molecule for regulating glucose and lipid metabolism, and is closely related to the occurrence of various diseases, such as atherosclerosis, hyperlipidemia, NAFLD and type 2 diabetes, but the relationship between Angptl8 and the immune response process of the body is still unknown. SUMMARY

[0007] To solve the above technical problems in the prior art, the application provides the use of an Angptl8 neutralizing antibody in the preparation of a drug for treating cytokine storm syndrome, and provides the molecular function and regulation mechanism of Angptl8 in the CSS disease process.

[0008] A first object of the present application is to provide an Angptl8 neutralizing antibody comprising a heavy chain 1, a light chain 1, a heavy chain 2 and a light chain 2;

[0009] The amino acid sequences of the variable region of the heavy chain 1 are respectively:

[0010] VHCDR1: GFDFSRYW (SEQ ID NO. 5),

[0011] VHCDR2: IIPDSSTI (SEQ ID NO. 6),

[0012] VHCDR3: ASISTVVGRDWYFDV (SEQ ID NO. 7);

[0013] The amino acid sequences of the variable region of the light chain 1 are respectively:

[0014] VLCDR1: TGAVTTSN (SEQ ID NO. 8),

[0015] VLCDR2: GTS,

[0016] VLCDR3: ALWYSTHYV (SEQ ID NO. 9);

[0017] The amino acid sequences of the variable region of the heavy chain 2 are respectively:

[0018] VHCDR4: GFDFSRYW (SEQ ID NO. 10),

[0019] VHCDR5: IIPDSSTI (SEQ ID NO. 11),

[0020] VHCDR6: ASISTVVGRDWYFD (SEQ ID NO. 12);

[0021] The amino acid sequences of the variable region of the light chain 2 are respectively:

[0022] VLCDR4: QNVDTY (SEQ ID NO. 13),

[0023] VLCDR5: SAS,

[0024] VLCDR6: QQYNSFPL (SEQ ID NO. 14).

[0025] Further, the amino acid sequence of the heavy chain 1 is shown as SEQ ID NO. 1:

[0026] MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGK

[0027] GLEWIGEIIPDSSTINYTPSLKDKFIISRDNARNTLYLQMTKVRSEDTALYYCASISTVVGRDW

[0028] YFDVWGAGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLS

[0029] SGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKC

[0030] PAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHR

[0031] EDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEE

[0032] EMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0033] The amino acid sequence of the light chain 1 is shown in SEQ ID NO. 2:

[0034] MAWTSLILSLLALCSGASSQAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYATWVQEKPDH

[0035] LFTGLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHYVFGGGTKV

[0036] TVLGQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEGNKFMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL.

[0037] The amino acid sequence of heavy chain 2 is shown as SEQ ID NO. 3:

[0038] MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGK

[0039] GLEWIGEIIPDSSTINYTPSLKDKFIISRDNARNTLYLQMTKVRSEDTALYYCASISTVVGRDW

[0040] YFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSL

[0041] SSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICT

[0042] VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFN

[0043] STFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAK

[0044] DKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0045] The amino acid sequence of light chain 2 is shown as SEQ ID NO. 4:

[0046] MESQTQVFLSLLLWVSGTCGDIVITQTPKFMSTSVGDRVSVTCKASQNVDTYVAWYQQKPG

[0047] QSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSFPLTFGSGTKLQ

[0048] IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS

[0049] KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0050] A second object of the present application is to provide an application of the substance inhibiting Angptl8 expression in treating cytokine storm syndrome.

[0051] Further, the substance inhibiting Angptl8 expression is the aforementioned Angptl8 neutralizing antibody.

[0052] Further, the substance treats cytokine storm syndrome by inhibiting M1 polarization of macrophages in the body and promoting M2 polarization of macrophages.

[0053] Further, the Angptl8 neutralizing antibody is administered at a dose of 10 μg-50 μg / 25 g.

[0054] The present application expands the molecular function of Angptl8 and clarifies the relationship between Angptl8 and the occurrence and development of CSS. The research results show that Angptl8 plays an important role in the process of cytokine storm. On the one hand, Angptl8 promotes M1 polarization of macrophages in the body to release TNF-α, IL-6, IL-1β, etc., and on the other hand, it reduces the anti-inflammatory function of the body by inhibiting M2 polarization of macrophages.

[0055] In summary, Angptl8 promotes the process of CSS by regulating the phenotype conversion of macrophages. On this basis, in order to treat CSS through the potential target Angptl8, an Angptl8 neutralizing antibody is synthesized, and the treatment effect of the Angptl8 neutralizing antibody on CSS is verified.

[0056] In summary, the present application proves that Angptl8 is an important molecule for regulating CSS in the body, and the Angptl8 neutralizing antibody can effectively treat cytokine storm syndrome in mice.

[0057] The present application has the following beneficial effects:

[0058] The present application verifies the close correlation between Angptl8 and cytokine storm through molecular biology means, expands the molecular function of Angptl8, and clarifies the regulatory role of Angptl8 in the occurrence and development of CSS.

[0059] The present application also provides and verifies that the Angptl8 neutralizing antibody can significantly improve LPS-induced cytokine storm syndrome, thereby providing an effective treatment strategy for treating severe patients with CSS in clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1Serum Angptl8 levels in mice injected with LPS in Example 1;

[0061] Figure 2 mRNA expression of Angptl8 in liver of mice injected with LPS in Example 1;

[0062] Figure 3 mRNA expression of Angptl8 in bone marrow of mice injected with LPS in Example 1;

[0063] Figure 4 Protein expression of Angptl8 in liver of mice injected with LPS in Example 1;

[0064] Figure 5 Angptl8 expression in primary hepatocytes stimulated with different concentrations of LPS in Example 1;

[0065] Figure 6 Angptl8 expression in primary hepatocytes stimulated with LPS for different lengths of time in Example 1;

[0066] Figure 7 mRNA expression of Angptl8 in primary macrophages stimulated with LPS for different lengths of time in Example 1;

[0067] Figure 8 Protein expression of Angptl8 in primary macrophages stimulated with LPS for different lengths of time in Example 1;

[0068] Figure 9 WT and Angptl8 - / - Number of leukocytes in blood of mice in Example 2;

[0069] Figure 10 WT and Angptl8 - / - Number and proportion of neutrophils in blood of mice in Example 2;

[0070] Figure 11 WT and Angptl8 - / - Number and proportion of monocytes in blood of mice in Example 2;

[0071] Figure 12 WT and Angptl8 - / - TNF-α, IL-6 and IFN-γ levels in serum of mice in Example 2;

[0072] Figure 13 WT and Angptl8 - / - H&E staining of liver and spleen tissue sections of mice in Example 2;

[0073] Figure 14WT and Angptl8 in Example 2 - / - TUNEL staining of mouse liver tissue sections;

[0074] Figure 15 WT and Angptl8 in Example 2 - / - mRNA expression levels of Inos and proinflammatory factors Tnf-a, IL-6, IL-1b in mouse bone marrow tissues;

[0075] Figure 16 WT and Angptl8 in Example 2 - / - mRNA expression levels of Inos and proinflammatory factors Tnf-a, IL-6, IL-1b in mouse spleen tissues;

[0076] Figure 17 WT and Angptl8 in Example 2 - / - mRNA expression levels of Inos and proinflammatory factors Tnf-a, IL-6, IL-1b in mouse liver tissues;

[0077] Figure 18 WT and Angptl8 in Example 3 - / - M1 polarization ratio of mouse primary macrophages after LPS stimulation;

[0078] Figure 19 WT and Angptl8 in Example 3 - / - mRNA expression levels of Inos and proinflammatory factors Tnf-a, IL-6, IL-1b in mouse primary macrophages;

[0079] Figure 20 WT and Angptl8 in Example 3 - / - mRNA expression levels of proinflammatory factors Tnf-a, IL-6, IL-1b in mouse primary spleen cells;

[0080] Figure 21 WT and Angptl8 in Example 3 - / - mRNA expression levels of Inos and proinflammatory factors Tnf-a, IL-6, IL-1b in mouse primary hepatocytes;

[0081] Figure 22 M1 polarization ratio of mouse primary macrophages after stimulation with different concentrations of Angptl8 purified protein in Example 4;

[0082] Figure 23 mRNA expression levels of Inos and proinflammatory factors Tnf-a, IL-6, and IL-1b in mouse primary macrophages in Example 4;

[0083] Figure 24Protein expression of proinflammatory factors Tnf-a, IL-6 and IL-1 b in mouse primary macrophages in Example 4;

[0084] Figure 25 mRNA expression of proinflammatory factors Tnf-a, IL-6 and IL-1 b in mouse primary splenocytes in Example 4;

[0085] Figure 26 Protein expression of proinflammatory factors Tnf-a, IL-6 and IL-1 b in mouse primary splenocytes in Example 4;

[0086] Figure 27 mRNA expression of proinflammatory factors Tnf-a, IL-6 and IL-1 b in mouse primary hepatocytes in Example 4;

[0087] Figure 28 Protein expression of proinflammatory factors Tnf-a, IL-6 and IL-1 b in mouse primary hepatocytes in Example 4;

[0088] Figure 29 The polarization ratio of mouse primary macrophages after stimulation of IL-4 and different concentrations of Angptl8 purified protein in Example 5;

[0089] Figure 30 mRNA expression of Mrc1 in mouse primary macrophages after stimulation of IL-4 and different concentrations of Angptl8 purified protein in Example 5;

[0090] Figure 31 mRNA expression of Arg1 in mouse primary macrophages after stimulation of IL-4 and different concentrations of Angptl8 purified protein in Example 5;

[0091] Figure 32 mRNA expression of Ym1 in mouse primary macrophages after stimulation of IL-4 and different concentrations of Angptl8 purified protein in Example 5;

[0092] Figure 33 mRNA expression of Fizz1 in mouse primary macrophages after stimulation of IL-4 and different concentrations of Angptl8 purified protein in Example 5;

[0093] Figure 34 mRNA expression of Mrc1 and Arg1 in bone marrow tissues of WT and Angptl8 - / - mice in Example 5;

[0094] Figure 35 mRNA expression of Mrc1 and Arg1 in spleen tissues of WT and Angptl8 - / - mice in Example 5;

[0095] Figure 36 WT and Angptl8 in Example 5 - / - mRNA expression levels of Mrc1 and Arg1 in mouse liver tissues;

[0096] Figure 37 WT and Angptl8 in Example 5 - / - M2 polarization ratio of mouse primary macrophages after IL-4 stimulation;

[0097] Figure 38 WT and Angptl8 in Example 5 - / - mRNA expression levels of Mrc1 and Arg1 in mouse primary macrophages after IL-4 stimulation;

[0098] Figure 39 Survival rate of CSS mice injected with Angptl8 neutralizing antibody in Example 6;

[0099] Figure 40 TNF-a, IL-6 and IFN-g contents in serum of CSS mice injected with Angptl8 neutralizing antibody in Example 6;

[0100] Figure 41 H&E staining of liver and spleen of CSS mice injected with Angptl8 neutralizing antibody in Example 6;

[0101] Figure 42 Number of white blood cells, number and ratio of neutrophils and monocytes in blood of CSS mice injected with Angptl8 neutralizing antibody in Example 6;

[0102] Figure 43 Macrophage immunofluorescence (F4 / 80) of liver and adipose tissues of CSS mice injected with Angptl8 neutralizing antibody in Example 6;

[0103] Figure 44 mRNA expression levels of Inos, pro-inflammatory factors Tnf-a, IL-6 and IL-1b in bone marrow tissues of CSS mice injected with Angptl8 neutralizing antibody in Example 6;

[0104] Figure 45 mRNA expression levels of Mrc1, Arg1, Fizz1 and Ym1 in bone marrow tissues of CSS mice injected with Angptl8 neutralizing antibody in Example 6. DETAILED DESCRIPTION

[0105] The present application is further explained in conjunction with the following examples, which do not limit the application in any way.

[0106] Example 1. Liver Angptl8 is closely related to the CSS course

[0107] To further explore the relationship between Angptl8 and CSS, we used a lipopolysaccharide (LPS) stimulated CSS model in both in vitro and animal experiments.

[0108] In in vivo experiments, we collected serum and tissues from C57BL / 6J mice 6h after intraperitoneal injection of LPS (25mg / kg). The results showed that the content of Angptl8 in the serum of the model group mice increased significantly (as shown in Figure 1 , and the content of Angptl8 in liver and bone marrow tissues also increased significantly (as shown in Figure 2 , 3, 4).

[0109] As shown in Figure 5 and Figure 6 , in primary hepatocytes, Angptl8 mRNA and protein expression levels were found to be significantly elevated by LPS stimulation in a dose-dependent and time-dependent manner. The experimental results showed that LPS induced endogenous expression of Angptl8 in macrophages. The expression of Angptl8 mRNA in macrophages increased significantly after 6, 12h of LPS stimulation of BMDMs, and the expression of Angptl8 mRNA decreased sharply after 24h (as shown in Figure 7 , while the protein level of Angptl8 gradually increased over time, and the protein expression reached a maximum at 24h (as shown in Figure 8 ). Therefore, we concluded from the above results that Angptl8 is closely related to the occurrence and development of CSS.

[0110] Example 2. Angptl8 deficiency improves LPS-induced CSS in mice

[0111] To further explore the molecular function of Angptl8 in mouse CSS, our team has successfully constructed a model of Angptl8 systemic knockout mice (knockout model see Zhang Z, Yuan Y, Hu L, Tang J, Meng Z, Dai L, Gao Y, Ma S, Wang X, Yuan Y, Zhang Q, Cai W, Ruan X, Guo X. ANGPTL8 accelerates liver fibrosis mediated by HFD-induced inflammatory activity via LILRB2 / ERK signaling pathways. J Adv Res. 2023 May;47:41-56.). In the course of CSS, a large number of inflammatory cytokines are derived from various immune cells, and the white blood cells of patients with acute infection will significantly increase, and the number of neutrophils and mononuclear cells in white blood cell classification will also increase sharply. The results of blood routine test showed that compared with wild type (WT) mice, the number of white blood cells (WBC), especially the number and proportion of neutrophils (NEU) and mononuclear cells (MON) in the blood of Angptl8 knockout mice (Angptl8- / -) were significantly improved (as shown in Figure 9 , 10 and 11).

[0112] Further, we detected the content of various inflammation-related factors in mouse serum by ELISA method. The results showed that the pro-inflammatory cytokines TNF-α, IL-6 and macrophage activation factor IFN-γ in the serum of model mice were significantly increased, and the release of these pro-inflammatory factors was significantly inhibited in Angptl8 knockout (as shown in Figure 12 ). At the histopathological level, we compared the pathophysiological phenotypes of liver and spleen of mice in each group. H&E staining sections showed that the liver lobule structure of model mice was severely damaged, accompanied by bridging necrosis and a large number of inflammatory cell infiltration, while the damage to the liver tissue structure of Angptl8- / -mice was significantly improved (as shown in Figure 13 ). At the same time, in the spleen tissue, the model mice showed blurred boundary between white pulp and red pulp, lymphocyte necrosis and other pathological phenomena, which were relieved to some extent in Angptl8- / - -mice. In addition, the results of liver TUNEL staining showed that Angptl8- / -mice could significantly reduce the hepatocyte apoptosis induced by LPS (as shown in Figure 14mRNA expression of proinflammatory cytokines and Ml -type macrophage marker iNOS in bone marrow, spleen and liver tissues of mice. The results showed that the above-mentioned indicators of model mice were significantly increased, while Angptl8 - / - mice showed significant improvement (e.g. Figure 15 , 16 and 17). In summary, our research results confirmed that Angptl8 deletion has a significant protective effect on CSS mice.

[0113] Example 3. Angptl8 deletion improves LPS-induced CSS at in vitro level

[0114] In in vitro experiments, we isolated primary hepatocytes and primary macrophages from WT mice and Angptl8 - / - mice. Angptl8 - / - Mice macrophages significantly improved LPS-induced Ml -type polarization (e.g. Figure 18 shown). At the same time, Angptl8 - / - mice primary hepatocytes, splenocytes and macrophages were significantly resistant to LPS-induced proinflammatory cytokine expression (e.g. Figure 19 , 20 and 21).

[0115] Example 4. Angptl8 induces inflammatory cytokine expression at in vitro level

[0116] We isolated primary macrophages, primary splenocytes and primary hepatocytes and added different concentrations of Angptl8 purified protein for stimulation. The results showed that Angptl8 purified protein can stimulate a significant increase in the proportion of Ml -type macrophages in a concentration-dependent manner (e.g. Figure 22 shown). Angptl8 also induced a significant increase in the mRNA expression of iNOS and proinflammatory cytokines Tnf-a, IL-6, IL-1 β in primary macrophages in a concentration-dependent manner (e.g. Figure 23 shown), and the same trend was observed in protein expression (e.g. Figure 24 shown). Similarly, adding Angptl8 purified protein to primary splenocytes and primary hepatocytes can induce the release of inflammatory cytokines Tnf-a, IL-6, IL-1 β, etc. (e.g. Figure 25 , 26, 27 and 28). In summary, Angptl8 can significantly promote Ml -type macrophage polarization and induce cell inflammation to worsen.

[0117] Example 5. Angptl8 inhibits IL-4-induced M2-type macrophage polarization

[0118] The above experiments have verified that Angptl8 can induce macrophages to polarize to a proinflammatory phenotype, but whether Angptl8 can inhibit M2 macrophage polarization is unknown. Flow cytometry detection showed that different concentrations of Angptl8 purified protein can significantly inhibit the proportion of M2 macrophage polarization and show a dose-dependent effect (as shown in Figure 29 Meanwhile, Angptl8 inhibited the mRNA expression of M2 macrophage marker genes Mrc1, Arg1, Ym1 and Fizz1 (as shown in Figure 30 , 31, 32 and 33).

[0119] In addition, intraperitoneal injection of LPS into WT mice can reduce the expression of M2 macrophage-related genes in the liver, spleen and bone marrow, while the expression of Mrc1 and Arg1 in Angptl8- / mice is restored (as shown in Figure 34 , 35 and 36). Similarly, primary macrophages isolated from WT mice and Angptl8- / mice were stimulated with IL-4 for 24 h to induce M2 macrophage polarization, and the results showed that Angptl8 - / - promotes IL-4-mediated M2 macrophage polarization (as shown in Figure 37 and 38 . In summary, we have shown in both in vivo and in vitro experiments that Angptl8 can inhibit IL-4-induced M2 macrophage polarization, and that knocking out Angptl8 can restore the decrease in the proportion of LPS-mediated M2 macrophages.

[0120] Example 6. Angptl8 neutralizing antibody significantly improves mouse CSS symptoms

[0121] The above experimental results all demonstrate that Angptl8 plays an important molecular function in the cytokine storm and can be used as a potential target for treating CSS. Therefore, we injected the Angptl8 neutralizing antibody described in this patent into the tail vein of mice (10 μg / 25 g, 50 μg / 25 g), and 1 h later, intraperitoneally injected LPS. After 6 h, the serum and related tissues were collected for detection. The results showed that all mice in the model group died at 66 h, while the survival rates of mice injected with low and high doses of Angptl8 neutralizing antibody reached 30% and 60% within 72 h, respectively (as shown in Figure 39 . Injection of a high dose of Angptl8 neutralizing antibody can significantly inhibit the release of proinflammatory factors TNF-α, IL-6 and IFN-γ in the CSS course of mice, and the low dose group also has a certain therapeutic effect (as shown in Figure 40The results showed that the mice in the model group had obvious bridging necrosis in the liver, hepatocyte damage, and a large number of inflammatory cell infiltration; the spleen had a large number of red blood cell aggregation, severe congestion, and the boundary between the white pulp and the red pulp was blurred, and phenomena such as lymphocyte necrosis and nuclear fragmentation appeared. The above phenomena were alleviated to varying degrees in the low-dose group and the high-dose group (as shown in FIG. 3). Figure 41 The blood routine report of the mice showed that the number of white blood cells in the LPS group of mice increased significantly, and the number and proportion of neutrophils and monocytes increased significantly, and the injection of neutralizing antibodies improved the above-mentioned phenomena (as shown in FIG. 4). Figure 42 The results of tissue immunofluorescence showed that the LPS group of mice had a large number of macrophage infiltration in the liver and fat, and the injection of neutralizing antibodies alleviated the degree of macrophage infiltration (as shown in FIG. 5). Figure 43 Meanwhile, we also detected the expression of macrophage markers, and the results showed that the mRNA expression of the bone marrow macrophage markers Inos and the inflammatory factors Tnfα and Il1b of the LPS group of mice increased significantly, and the injection of neutralizing antibodies inhibited the expression of the above genes (as shown in FIG. 6). Figure 44 The mRNA expression of the M2 macrophage markers Mrc1, Arg1, Fizz1 and Ym1 of the bone marrow of the mice in the model group decreased significantly, and the injection of neutralizing antibodies increased the expression of the above genes (as shown in FIG. 7). Figure 45 In summary, the neutralizing antibody of Angptl8 described in the present patent can effectively treat cytokine storm syndrome.

[0122] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An Angptl8 neutralizing antibody, characterized by, The Angptl8 neutralizing antibody comprises heavy chain 1, light chain 1, heavy chain 2 and light chain 2; The amino acid sequences of the three CDRs contained in the variable region of the heavy chain 1 are respectively: VHCDR1: GFDFSRYW, VHCDR2: IIPDSSTI, VHCDR3: ASISTVVGRDWYFDV; The amino acid sequences of the three CDRs contained in the variable region of the light chain 1 are respectively: VLCDR1: TGAVTTSN, VLCDR2: GTS, VLCDR3: ALWYSTHYV; The amino acid sequences of the three CDRs contained in the variable region of the heavy chain 2 are respectively: VHCDR4: GFDFSRYW, VHCDR5: IIPDSSTI, VHCDR6: ASISTVVGRDWYFDV; The amino acid sequences of the three CDRs contained in the variable region of the light chain 2 are respectively: VLCDR4: QNVDTY, VLCDR5: SAS, VLCDR6: QQYNSFPL.

2. The Angptl8-neutralizing antibody according to claim 1, characterized by, The amino acid sequence of the heavy chain 1 is shown as SEQ ID NO. 1, the amino acid sequence of the light chain 1 is shown as SEQ ID NO. 2, the amino acid sequence of the heavy chain 2 is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain 2 is shown as SEQ ID NO.

4.

3. Use of a substance that inhibits the expression of Angptl8 in the manufacture of a medicament for treating cytokine storm syndrome, characterized in that, The substance for inhibiting the expression of Angptl8 is the Angptl8 neutralizing antibody of claim 1.

4. Use according to claim 3, characterized in that, The substance treats cytokine storm syndrome by inhibiting the polarization of M1 type of macrophages in the body and promoting the polarization of M2 type of macrophages.

5. Use according to claim 3, characterized in that, The administration dose of the Angptl8 neutralizing antibody is 10 μg~50 μg / 25 g.

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