Application of Angpt18 neutralizing antibody in preparation of medicine for treating cytokine storm syndrome

Abstract

The invention provides an application of an Angpt18 neutralizing antibody in preparation of a medicine for treating mouse cytokine storm syndrome. The research proves that the angiogenin-like protein 8 (Angpt18) can promote macrophages to release inflammatory factors such as TNF-alpha (Tumor Necrosis Factor-alpha), IL-6 (Interleukin-6), IL-1beta (Interleukin-1beta) and the like by using a molecular biology means. In the aspect of mechanism, the Angpt18 promotes polarization of M1 type macrophages and inhibits M2 type polarization of the M1 type macrophages to cause enhancement of body inflammatory factor storm, and the pathological phenomenon can be improved by knocking out the Angpt18. Therefore, the Angpt18 neutralizing antibody is synthesized and injected into a CSS mouse so as to be combined with Angpt18 in blood circulation of the CSS mouse, massive release of inflammatory factors of the organism is successfully inhibited, and the survival rate of the mouse within 72 hours is increased to 60%. The invention proves that the Angpt18 neutralizing antibody can effectively treat the mouse cytokine storm syndrome, and clarifies the molecular mechanism of the target Angpt18 of the Angpt18 neutralizing antibody for regulating the disease progress, thereby providing an effective strategy for clinically treating CSS patients.

Description

Technical Field

[0001] The present invention relates to the field of biological technology, and in particular to the use of Angptl8 neutralizing antibodies as an effective drug for treating cytokine storms by regulating macrophage polarization. Background Art

[0002] Cytokine Storm Syndrome (CSS) is a systemic inflammatory response related to infectious and non-infectious diseases, which can be induced by multiple factors such as infection and drugs. In this process, a large number of cytokines participate in the body's immune regulation and inflammatory response process, thereby triggering a cytokine storm, which in turn induces the occurrence of CSS, leading to systemic inflammatory response and multiple organ failure, and even death. CSS can be roughly divided into two categories, including those caused by infectious and non-infectious diseases. Infectious disease-induced infections include: from bacteria such as Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumoniae and Yersinia pestis; from viruses. Non-infectious diseases include monogenic and autoimmune diseases. So far, the specific molecular mechanism of CSS has not been fully elucidated, and clinical treatment methods are 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 the treatment of CSS and clarify its mechanism.

[0003] Cytokine storm is different from ordinary inflammatory response. Some literatures have proposed three criteria for distinguishing cytokine storm from ordinary inflammatory response: 1. Elevated levels of circulating cytokines; 2. Acute systemic inflammatory response; 3. The occurrence of secondary organ dysfunction caused by inflammation (usually kidney, liver or lung), 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 this process, the body can rapidly develop disseminated intravascular coagulation, accompanied by vascular occlusion or massive bleeding, dyspnea, hypoxemia, hypotension, hemostasis imbalance, vasodilatory shock, etc., and deteriorate to acute respiratory distress syndrome (ARDS) and multiple organ failure in the late stage of the disease or during recovery, which can cause death of patients in a short period of time.

[0004] The occurrence and development of the course of cytokine storm is closely related to the immune system. In the early stage of inflammatory response, the body inhibits invasive pathogens by activating innate and adaptive immune responses. On the one hand, the immune system needs to produce enough cytokines to eliminate pathogens, and on the other hand, the body also needs to avoid producing a large number of cytokines, which will lead to excessive inflammatory response. A proper balance should be maintained between the two. Normally, cytokines play a key role in regulating signals against pathogens and amplifying immune responses, but in excessive inflammatory responses, the production of a large number of cytokines will cause significant collateral damage to the body. Cells of the innate immune system are the first line of defense against pathogens. They recognize pathogens through pattern recognition receptors and produce cytokines that activate cells of the adaptive immune system to recognize and respond to various microbial invasions. The innate cells most commonly involved in the pathogenesis of cytokine storms include neutrophils, macrophages, and NK cells. Neutrophils can produce neutrophil extracellular traps, which are a fibrous network that contributes to thrombosis and amplifies the production of cytokines during cytokine storms. Macrophages are tissue-resident cells that usually originate from circulating monocytes and do not divide. They have multiple functions and play a key role in the clearance of senescent cells, tissue repair, immune regulation, and antigen presentation. In a cytokine storm, macrophages are activated and secrete excessive cytokines, which ultimately leads to severe tissue damage and organ failure. Hemophagocytic cells are often observed in bone marrow biopsy specimens of patients with cytokine storm. Interferon-γ can induce macrophages to produce erythrophagocytosis, which may be the main cause of the common cell reduction in patients with cytokine storm. Therefore, macrophages may become an effective target for the treatment of cytokine storm.

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

[0006] A large number of studies have shown that the liver-derived factor Angptl8 is an important molecule that regulates glucose and lipid metabolism and is closely related to the occurrence of various diseases, such as atherosclerosis, hyperlipidemia, NAFLD and type 2 diabetes, but its connection with the body's immune response process remains unknown. Summary of the invention

[0007] In order to solve the above-mentioned technical problems existing in the prior art, the present invention provides the use of Angptl8 neutralizing antibodies in the preparation of drugs for treating cytokine storm syndrome, and provides the molecular function and regulatory mechanism of Angptl8 in the course of CSS.

[0008] The first object of the present invention is to provide an Angptl8 neutralizing antibody, wherein the Angptl8 neutralizing antibody comprises a heavy chain 1, a light chain 1, a heavy chain 2 and a light chain 2;

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

[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 light chain 1 variable region are:

[0014] VLCDR1:TGAVTTSN(SEQ ID NO.8)、

[0015] VLCDR2: GTS,

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

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

[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 light chain 2 variable regions are:

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

[0023] VLCDR5:SAS,

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

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

[0026] MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGK

[0027] GLEWIGEIIPDSSTINYTPSLKDKFIISRDNARNTLYLQMTKVRSEDTALYYCASISTVVGRDW

[0028] YFDVWGAGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLS

[0029] SGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKC

[0030] PAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHR

[0031] EDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEE

[0032] EMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0033] The amino acid sequence of 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 in 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 in SEQ ID NO.4:

[0046] MESQTQVFLSLLLWVSGTCGDIVITQTPKFMSTSVGDRVSVTCKASQNVDTYVAWYQQKPG

[0047] QSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSFPLTFGSGTKLQ

[0048] IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS

[0049] KDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0050] The second object of the present invention is to provide use of a substance that inhibits the expression of Angptl8 in the treatment of cytokine storm syndrome.

[0051] Furthermore, the substance that inhibits the expression of Angptl8 is the aforementioned Angptl8 neutralizing antibody.

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

[0053] Furthermore, the dosage of the Angptl8 neutralizing antibody is 10 μg to 50 μg / 25g.

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

[0055] In summary, Angptl8 promotes CSS by regulating macrophage phenotypic transformation. On this basis, in order to treat CSS through Angptl8 as a potential target, Angptl8 neutralizing antibodies were synthesized and their therapeutic effect on CSS was confirmed.

[0056] In summary, the present invention demonstrates that Angptl8 is an important molecule that regulates CSS in the body, and that Angptl8 neutralizing antibodies can effectively treat cytokine storm syndrome in mice.

[0057] The beneficial effects of the present invention are:

[0058] The present invention confirms the close correlation between Angptl8 and cytokine storm by molecular biological means, expands the molecular function of Angptl8, and clarifies its regulatory role in the occurrence and development of CSS.

[0059] The present invention also provides and verifies that Angptl8 neutralizing antibodies can significantly improve LPS-induced cytokine storm syndrome, providing an effective treatment strategy for the clinical treatment of severe CSS patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1The Angptl8 level in the serum of mice after LPS injection in Example 1;

[0061] Figure 2 is the expression level of Angptl8 mRNA in the liver of mice after LPS injection in Example 1;

[0062] Figure 3 is the expression level of Angptl8 mRNA in the bone marrow of mice after LPS injection in Example 1;

[0063] Figure 4 is the expression level of Angptl8 protein in the liver of mice after LPS injection in Example 1;

[0064] Figure 5 is the expression level of Angptl8 in primary hepatocytes after stimulation with different concentrations of LPS in Example 1;

[0065] Figure 6 is the expression level of Angptl8 in primary hepatocytes after LPS stimulation for different time periods in Example 1;

[0066] Figure 7 is the Angptl8 mRNA expression level of primary macrophages after LPS stimulation for different time periods in Example 1;

[0067] Figure 8 is the expression level of Angptl8 protein in primary macrophages after LPS stimulation for different time periods in Example 1;

[0068] Fig. 9 WT and Angptl8 in Example 2 - / - The number of white blood cells in the mouse blood;

[0069] Fig.10 WT and Angptl8 in Example 2 - / - The number and proportion of neutrophils in mouse blood;

[0070] Fig.11 WT and Angptl8 in Example 2 - / - The number and proportion of monocytes in mouse blood;

[0071] Fig.12 WT and Angptl8 in Example 2 - / - TNF-α, IL-6, and IFN-γ levels in mouse serum;

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

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

[0074] Fig.15 WT and Angptl8 in Example 2 - / - mRNA expression of Inos and proinflammatory factors Tnf-α, IL-6, and IL-1β in mouse bone marrow tissue;

[0075] Fig.16 WT and Angptl8 in Example 2 - / - mRNA expression levels of Inos and proinflammatory factors Tnf-α, IL-6, and IL-1β in mouse spleen tissue;

[0076] Fig.17 WT and Angptl8 in Example 2 - / - mRNA expression levels of Inos and proinflammatory factors Tnf-α, IL-6, and IL-1β in mouse liver tissue;

[0077] Fig.18 WT and Angptl8 in Example 3 - / - The proportion of M1 polarization of mouse primary macrophages after LPS stimulation;

[0078] Fig.19 WT and Angptl8 in Example 3 - / - mRNA expression of Inos and proinflammatory factors Tnf-α, IL-6, and IL-1β in primary mouse macrophages;

[0079] Fig. 20 WT and Angptl8 in Example 3 - / - mRNA expression of proinflammatory factors Tnf-α, IL-6, and IL-1β in mouse primary splenocytes;

[0080] Fig.21 WT and Angptl8 in Example 3 - / - mRNA expression of Inos and proinflammatory factors Tnf-α, IL-6, and IL-1β in primary mouse hepatocytes;

[0081] Fig. 22 is the polarization ratio of primary mouse macrophages stimulated by different concentrations of purified Angptl8 protein in Example 4;

[0082] Fig.23 The mRNA expression levels of Inos and proinflammatory factors Tnf-α, IL-6 and IL-1β in primary mouse macrophages in Example 4;

[0083] Fig.24The protein expression levels of Inos and pro-inflammatory factors Tnf-α, IL-6 and IL-1β in primary mouse macrophages in Example 4;

[0084] Fig.25 The mRNA expression levels of proinflammatory factors Tnf-α, IL-6 and IL-1β in primary splenocytes of mice in Example 4;

[0085] Fig.26 The protein expression levels of proinflammatory factors Tnf-α, IL-6 and IL-1β in primary splenocytes of mice in Example 4;

[0086] Fig. 27 is the mRNA expression of pro-inflammatory factors Tnf-α, IL-6 and IL-1β in primary mouse hepatocytes in Example 4;

[0087] Fig.28 The protein expression levels of proinflammatory factors Tnf-α, IL-6 and IL-1β in primary mouse hepatocytes in Example 4;

[0088] Fig.29 is the polarization ratio of primary mouse macrophages after stimulation with IL-4 and different concentrations of purified Angptl8 protein in Example 5;

[0089] Fig.30 is the mRNA expression level of Mrc1 in primary mouse macrophages after stimulation with IL-4 and different concentrations of purified Angptl8 protein in Example 5;

[0090] Fig.31 is the mRNA expression level of Arg1 in primary mouse macrophages after stimulation with IL-4 and different concentrations of purified Angptl8 protein in Example 5;

[0091] Fig.32 is the mRNA expression level of mouse primary macrophage Ym1 after stimulation with IL-4 and different concentrations of purified Angptl8 protein in Example 5;

[0092] Fig.33 is the mRNA expression level of Fizz1 in primary mouse macrophages after stimulation with IL-4 and different concentrations of purified Angptl8 protein in Example 5;

[0093] Fig.34 WT and Angptl8 in Example 5 - / - Mrc1 and Arg1 mRNA expression in mouse bone marrow tissue;

[0094] Fig.35 WT and Angptl8 in Example 5 - / - Mrc1 and Arg1 mRNA expression in mouse spleen tissue;

[0095] Fig.36 WT and Angptl8 in Example 5 - / - Mrc1 and Arg1 mRNA expression in mouse liver tissue;

[0096] Fig.37 WT and Angptl8 in Example 5 - / - The proportion of M2 polarization of mouse primary macrophages after IL-4 stimulation;

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

[0098] Fig.39 is the survival rate of CSS mice injected with Angptl8 neutralizing antibody in Example 6;

[0099] Fig.40 The levels of TNF-α, IL-6 and IFN-γ in the serum of CSS mice injected with Angptl8 neutralizing antibodies in Example 6;

[0100] Fig.41 H&E staining of the liver and spleen of CSS mice injected with Angptl8 neutralizing antibodies in Example 6;

[0101] Fig.42 The number and ratio of leukocytes, neutrophils and monocytes in the blood of CSS mice injected with Angptl8 neutralizing antibodies in Example 6;

[0102] Fig.43 Immunofluorescence analysis of liver and adipose tissue macrophages of CSS mice injected with Angptl8 neutralizing antibodies in Example 6 (F4 / 80);

[0103] Fig.44 The mRNA expression levels of Inos and pro-inflammatory factors Tnf-α, IL-6, and IL-1β in the bone marrow tissue of CSS mice injected with Angptl8 neutralizing antibodies in Example 6;

[0104] Fig.45 The mRNA expression levels of Mrc1, Arg1, Fizz1 and Ym1 in the bone marrow tissue of CSS mice injected with Angptl8 neutralizing antibodies in Example 6. DETAILED DESCRIPTION

[0105] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

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

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

[0108] In the in vivo experiment, we injected LPS (25 mg / kg) into C57BL / 6J mice intraperitoneally and collected serum and tissues 6 hours later. The Angptl8 content in mouse serum was detected by ELISA. The results showed that the Angptl8 content in the serum of the model group mice increased significantly (e.g. Figure 1 ), while the content of Angptl8 in liver and bone marrow tissues increased significantly (as shown Figure 2 ,3,4).

[0109] like Figure 5 and Figure 6 As shown in Figure 2, in primary hepatocytes, the expression of Angptl8 mRNA and protein was significantly increased by LPS stimulation, and was dose-dependent and time-dependent. The experimental results showed that LPS induced the endogenous expression of Angptl8 in macrophages. After 6 and 12 hours of LPS stimulation of BMDMs, the expression of Angptl8 mRNA in macrophages increased significantly, and after 24 hours, the expression of Angptl8 mRNA decreased sharply (as shown in Figure 2). Figure 7 However, the Angptl8 protein level gradually increased over time, reaching the highest level at 24 h (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] In order to further explore the molecular function of Angptl8 in mouse CSS, our team has successfully constructed a systemic knockout mouse model of Angptl8 (for the 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 / ERKsignaling pathways. J Adv Res. 2023 May; 47: 41-56.). During the course of CSS, a large number of inflammatory cytokines are derived from a variety of immune cells. The blood white blood cells of patients with acute infection will increase significantly, and the number of neutrophils and monocytes in the white blood cell classification will also increase sharply. The results of routine blood tests showed that compared with wild-type (WT) mice, after injection of LPS (25 mg / kg), Angptl8 knockout mice (Angptl8- / -) showed significant improvement in the number of white blood cells (WBC), especially the number and ratio of neutrophils (NEU) and monocytes (MON). Fig. 9 , as shown in 10 and 11).

[0112] Furthermore, we used ELISA to detect the levels of various inflammatory factors in the serum of mice. The results showed that the pro-inflammatory cytokines TNF-α, IL-6 and macrophage activation factor IFN-γ in the serum of the model group mice were significantly increased, while Angptl8 knockout significantly inhibited the release of these pro-inflammatory factors (such as Fig.12 At the histopathological level, we compared the pathophysiological phenotypes of the liver and spleen of mice in each group. H&E staining sections showed that the liver lobule structure of mice in the model group was severely damaged, accompanied by bridging necrosis and a large number of inflammatory cell infiltration, while Angptl8- / -The liver tissue structural damage of mice was significantly improved (such as Fig.13 At the same time, in the spleen tissue, the model group mice showed pathological phenomena such as blurred boundaries between white pulp and red pulp and lymphocyte necrosis. These phenomena were not observed in Angptl8- / - In addition, liver TUNEL staining results showed that Angptl8- / - mice were able to significantly reduce LPS-induced hepatocyte apoptosis (eg Fig.14In order to gain a deeper understanding of the molecular mechanism of this phenomenon, we used RT-qPCR to detect the mRNA expression of pro-inflammatory factors and M1 macrophage marker nitric oxide synthase (iNOS) in the bone marrow, spleen and liver tissues of mice. The results showed that the above indicators of mice in the model group were significantly increased, while Angptl8 - / - mice showed significant improvement (e.g. Fig.15 , 16 and 17). Taken together, our results confirm that Angptl8 deficiency has a significant protective effect in CSS mice.

[0113] Example 3. Angptl8 deficiency improves LPS-induced CSS in vitro

[0114] In in vitro experiments, we isolated 1447 WT mice and Angptl8 - / - Primary hepatocytes and primary macrophages were isolated from mice. - / - Mouse macrophages significantly improved LPS-induced M1 polarization (e.g. Fig.18 At the same time, Angptl8 - / - Primary hepatocytes, splenocytes, and macrophages of mice were significantly resistant to the expression of pro-inflammatory cytokines induced by LPS (e.g. Fig.19 , as shown in 20 and 21).

[0115] Example 4. Angptl8 induces expression of inflammatory factors in vitro

[0116] We isolated primary macrophages, primary splenocytes and primary hepatocytes and added different concentrations of purified Angptl8 protein for stimulation. The results showed that purified Angptl8 protein could stimulate a significant increase in the proportion of M1 macrophages in a concentration-dependent manner (e.g. Fig. 22 Angptl8 can also induce a significant increase in the mRNA expression of nitric oxide synthase (iNOS) and proinflammatory cytokines Tnf-α, IL-6, and IL-1β in primary macrophages in a concentration-dependent manner (as shown in Fig.23 The protein expression also showed the same trend (as shown in Fig.24 Similarly, adding purified Angptl8 protein to primary spleen cells and primary hepatocytes can induce them to release inflammatory factors such as Tnf-α, IL-6, and IL-1β (as shown in Fig.25 ,26,27 and 28). In summary, Angptl8 can significantly promote the polarization of M1 macrophages and induce aggravated cellular inflammatory response.

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

[0118] The above experiments have verified that Angptl8 can induce macrophage polarization to a pro-inflammatory phenotype, but whether Angptl8 can inhibit M2 macrophage polarization remains unknown. Flow cytometry showed that different concentrations of Angptl8 purified protein can significantly inhibit the polarization ratio of M2 macrophages in a dose-dependent manner (e.g. Fig.29 At the same time, Angptl8 inhibited the mRNA expression of M2 macrophage marker genes Mrc1, Arg1, Ym1 and Fizz1 (as shown in Fig.30 , 31, 32 and 33).

[0119] In addition, intraperitoneal injection of LPS into WT mice reduced the expression of M2 macrophage-related genes in the liver, spleen, and bone marrow, whereas Angptl8- / -Mrc1 and Arg1 expression were restored in mice (e.g. Fig.34 , 35 and 36). Similarly, from WT mice and Angptl8- / - Primary macrophages were isolated from mice and stimulated with IL-4 for 24 h to induce macrophage polarization to M2 type. The results showed that Angptl8 - / - Promote IL-4-mediated M2 macrophage polarization (such as Fig.37 and 38 In summary, our in vivo and in vitro experiments demonstrated that Angptl8 could inhibit IL-4-induced M2 macrophage polarization, and knockout of Angptl8 could restore LPS-mediated decrease in the proportion of M2 macrophages.

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

[0121] The above experimental results all prove that Angptl8 plays an important molecular function in cytokine storm and can be used as a potential target for the treatment of CSS. Therefore, we injected the Angptl8 neutralizing antibody (10μg / 25g, 50μg / 25g) described in this patent into the tail vein of mice, injected LPS intraperitoneally 1 hour later, and collected serum and related tissues for testing after 6 hours. The results showed that all mice in the model group died within 66 hours, while the survival rates of mice injected with low-dose and high-dose Angptl8 neutralizing antibodies reached 30% and 60% respectively within 72 hours (as shown in Figure 2). Fig.39 Injection of high-dose Angptl8 neutralizing antibody can significantly inhibit the release of pro-inflammatory factors TNF-α, IL-6 and IFN-γ during the course of CSS in mice, while the low-dose group also has a certain therapeutic effect (as shown in Figure 2). Fig.40). Pathological sections of mouse tissues showed that the liver of the model group mice had obvious bridging necrosis, hepatocyte damage, and a large number of inflammatory cells infiltrated; and a large number of red blood cells aggregated in the spleen, with severe congestion, blurred boundaries between the white pulp and the red pulp, and lymphocyte necrosis and nuclear fragmentation. The above phenomena were alleviated to varying degrees in the low-dose group and the high-dose group (such as Fig.41 The blood routine test of mice showed that the number of white blood cells in the LPS group mice increased significantly, among which the number and proportion of neutrophils and monocytes increased significantly, and there was a significant improvement after the injection of neutralizing antibodies (as shown in Figure 2). Fig.42 Tissue immunofluorescence showed that there were a large number of macrophage infiltrations in the liver and fat of mice in the LPS group, and the injection of neutralizing antibodies reduced the degree of macrophage infiltration (as shown in Fig.43 At the same time, we also detected the expression of macrophage markers. The results showed that the mRNA expression of bone marrow macrophage marker Inos and inflammatory factors Tnfα and Il1b in the LPS group mice increased significantly, while the injection of neutralizing antibodies inhibited the expression of the above genes (as shown in Figure 2). Fig.44 The mRNA expression of bone marrow M2 macrophage markers Mrc1, Arg1, Fizz1 and Ym1 in the model group mice was significantly decreased, and the injection of neutralizing antibodies increased the expression of the above genes (as shown in Fig.45 In summary, the Angptl8 neutralizing antibody described in this patent can effectively treat cytokine storm syndrome.

[0122] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

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

4.

3. Use of substances that inhibit the expression of Angptl8 in the preparation of drugs for the treatment of cytokine storm syndrome.

4. The use according to claim 3, characterized in that: The substance that inhibits the expression of Angptl8 is the Angptl8 neutralizing antibody according to claim 1.

5. The use according to claim 3, characterized in that: The substance treats cytokine storm syndrome by inhibiting the polarization of M1 macrophages and promoting the polarization of M2 macrophages.

6. The use according to claim 4, characterized in that: The dosage of the Angptl8 neutralizing antibody is 10 μg to 50 μg / 25g.

Images