Medication combinations for reducing unwanted humoral immune responses

Abstract

The present invention provides a polypeptide derived from wild-type TNFα that inhibits cell necrosis, the polypeptide comprising 10-200 amino acid residues and the sequence QLVVPSE. The present invention also provides a pharmaceutical composition comprising the polypeptide. The polypeptide of the present invention and the pharmaceutical composition thereof can treat inflammatory diseases by inhibiting cell necrosis.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine, specifically to a protein capable of neutralizing cellular necroptosis-promoting antibodies and its use in the treatment of inflammation. [Background technology]

[0002] Tumor necrosis factor alpha (TNF) is a multifunctional cytokine secreted by various cells, mainly macrophages and T cells. TNF exerts many biological functions through TNF receptors 1 and 2 (TNFR1 and TNFR2). Many TNF functions are mainly three intracellular events: 1) stimulating the transcription factor nuclear factor kappa B (NF-κB), leading to cell activation and cytokine production, 2) induction of the extrinsic pathway of cell apoptosis, and 3) induction of necrosis. The intracellular signaling pathways of these activities share some components but lead to different outcomes. Activation of NF-κB leads to secretion of proinflammatory factors and cell survival and activation. Apoptosis is a state of cell death characterized by activation of caspase-3, nuclear fragmentation, early and intact cell membranes, and little or no inflammatory response. However, necrosis is another mechanism of cell death in which activation of caspase-3 is absent and cell membrane integrity is compromised.

[0003] Necrosis leads to the release of intracellular substances that stimulate intense immune and inflammatory responses (4, 5). In apoptotic conditions, the inhibition of caspases results in a form of programmed cell death that, when it occurs, has characteristics of necrosis and is called "necroptosis" (6-11). Such necroptosis is an inflammatory process and is therefore clinically relevant in diseases such as rheumatoid arthritis, Crohn's disease, and psoriasis. However, to date, the factors that initiate necroptosis in these diseases have not yet been identified.

[0004] Neutralizing TNF by directly targeting the TNF molecule (monoclonal antibodies) or as a pseudoreceptor antagonist has revolutionized the treatment of inflammatory diseases (12), but complete blockade of TNF function leads to life-threatening side effects, such as infections and tumors (13). TNF plays an important pathological role in many inflammatory diseases (e.g., rheumatoid arthritis, inflammatory bowel disease, psoriasis, etc.). In clinical practice, the treatment of inflammatory diseases with anti-TNF drugs is highly effective. Anti-TNF biologics include anti-TNF monoclonal antibodies, which can neutralize TNF, free TNF receptors, etc. The market for anti-TNF drugs is worth $15 billion annually.

[0005] Despite these achievements, the mechanisms of cell and tissue destruction in inflammatory diseases are still unknown. Induction of NF-κB promotes survival and does not directly lead to cell death; in fact, NF-κB signaling inhibits apoptosis in inflammatory bowel disease (14). Apoptosis has been proposed to suppress inflammation (15). Necroptosis is a possible mechanism of inflammation and cell / tissue destruction, but evidence of clinical relevance in humans is lacking. For example, it is not known what factors outside target cells can cause necroptosis in non-viral inflammatory pathogenesis (14, 16). Of course, the identification of these factors would provide biomarkers for the diagnosis and treatment of many inflammatory diseases.

[0006] In sepsis patients, TNF is significantly increased and positively correlated with the severity of the disease. However, neither traditional TNF blocking antibodies nor free receptors can effectively treat sepsis, suggesting that other factors or factors play an important role in cooperation with TNF in the pathogenesis process. Thus, there is a great need in the art for material means to modulate and therefore reverse cell necrosis so that inflammatory diseases can be treated. Summary of the Invention

[0007] It is an object of the present invention to provide a material means for the regulation and therefore reversal of cell necrosis so that inflammatory diseases can be treated.

[0008] In a first aspect, the present invention provides a polypeptide that inhibits cell necrosis, the polypeptide being derived from wild-type TNFα and comprising 10 to 200 amino acid residues and the sequence QLVVPSE, wherein said polypeptide does not bind to TNFR1 but competes with TNF to bind to a cell necrosis-promoting antibody. In a specific embodiment, the wild-type TNFα is human-derived TNFα whose amino acid sequence is set forth in SEQ ID NO:1. In a specific embodiment, the cell necrosis-promoting antibody has a light chain set forth in SEQ ID NO:9 and / or a heavy chain set forth in SEQ ID NO:10. In a specific embodiment, the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NOs:2-8. In a preferred embodiment, the polypeptide is a soluble polypeptide. In specific embodiments, the amino acid sequence of the polypeptide is set forth in SEQ ID NO:2, 3 or 4.

[0009] In a second aspect, the present invention provides the use of a polypeptide according to the first aspect of the invention in the manufacture of a medicament for inhibiting cell necrosis or for treating a disease associated with cell necrosis. In a specific embodiment, said cell necrosis associated disease is an inflammatory disease. In specific embodiments, said inflammatory diseases include, but are not limited to, rheumatoid arthritis, Crohn's disease, psoriasis, and sepsis.

[0010] In a third aspect, the present invention provides a pharmaceutical composition for inhibiting cell necrosis comprising a therapeutically effective amount of a polypeptide according to the first aspect of the invention and a pharma- ceutical acceptable carrier. In a preferred embodiment, the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NOs:2-8. In a preferred embodiment, the polypeptide is a soluble polypeptide. In a preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO:2, 3 or 4. In a specific embodiment, the pharmaceutical composition is an injection for intravenous administration.

[0011] In a fourth aspect, the present invention provides a dialysis machine for treating the blood of a patient, the dialysis machine comprising a polypeptide according to the first aspect of the invention. In a preferred embodiment, the dialysis device is used to remove or neutralize pro-necrosis antibodies in the patient's blood. In a preferred embodiment, the cell necrosis-promoting antibody has a light chain as set forth in SEQ ID NO:9 and / or a heavy chain as set forth in SEQ ID NO:10. In a preferred embodiment, the dialysis device is a dialysis column.

[0012] In a fifth aspect, the present invention provides a method of removing or neutralising pro-necrosis antibodies in the blood of a patient, the method comprising the step of suppressing or neutralising pro-necrosis antibodies in the blood of said patient with a polypeptide according to the first aspect of the invention. In a preferred embodiment, the cell necrosis-promoting antibody has a light chain as set forth in SEQ ID NO:9 and / or a heavy chain as set forth in SEQ ID NO:10.

[0013] In a preferred embodiment, the method comprises the step of suppressing or neutralising pro-necrosis antibodies in the blood of a patient in need thereof by administering to the patient, for example by injection, a polypeptide according to the first aspect of the invention. In a preferred embodiment, the method comprises the step of suppressing or neutralising pro-necrosis antibodies in the patient's blood by dialysis with a polypeptide according to the first aspect of the invention.

[0014] In a preferred embodiment, the method comprises the step of suppressing or neutralising pro-necrosis antibodies in the patient's blood with a dialysis device comprising a polypeptide according to the first aspect of the invention. In a preferred embodiment, the dialysis device is a dialysis column. In a preferred embodiment, the method is used to treat a disease associated with cell necrosis. In a preferred embodiment, the cell necrosis associated disease to be treated is an inflammatory disease. In a preferred embodiment, the inflammatory disease includes, but is not limited to, rheumatoid arthritis, Crohn's disease, psoriasis, and sepsis.

[0015] Of course, it is understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (for example, in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be described here one by one due to space limitations. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 shows the selection of monoclonal antibodies capable of binding to TNF from 33 monoclonal antibodies, in which ELISA test demonstrated that 244-12 had the strongest binding to TNF. [Diagram 2] Figure 2 shows that the binding of antibody 244-12 to TNF does not affect the binding of TNF to its cell surface receptors. Here, "Merge" refers to the merge, and "alone" refers to antibody 244-12 alone. [Diagram 3] Figure 3 shows that antibody 244-12 blocks TNF-induced cell apoptosis (i.e., expression of active caspase-3) in both cell lines, the top panel being L929 cells (mouse fibroblast tumor cells) and the bottom panel being C28I2 cells (chondrocytes).

[0017] [Figure 4] Figure 4 shows necrosis of L929 cells caused by antibody 244-12 and TNF. In the figure, the ordinate represents cell apoptosis (active caspase-3) and the abscissa represents necroptosis (destruction of cell membrane integrity). Here, TNF caused cell apoptosis (top right). When the control antibody M26 was added (blocking TNF binding to the receptor), L929 cells survived (middle right). When 244-12 and TNF were added, L929 cells became necrotic (bottom right). [Diagram 5]Figure 5 shows that necrosis caused by the antibody 244-12 + TNF of the present invention is suppressed by Nec-1. Here, after 244-12 + TNF is administered with Nec-1 (top right), cell necrosis is also converted to cell apoptosis. It was demonstrated that cell necrosis is caused by signal transduction, i.e., necroptosis.

[0018] [Figure 6] FIG. 6 shows autoantibody-mediated necroptosis in synovial fluid of rheumatoid arthritis patients. [Figure 7] Figure 7 shows the average levels of antibodies competing with the antibody 244-12 of the present invention for the antigen-binding site in cured (n = 5) and deceased (n = 19) patients with sepsis. The difference between the two is highly significant. P<0.01. [Figure 8] FIG. 8 is a schematic diagram of the pNIC28Bsa4 vector showing the important components involved in cloning and expression.

[0019] [Figure 9] Figure 9 shows that the polypeptide P11-17 of the present invention can bind to purified necrosis-promoting antibody (244-12) and also to autoantibodies in the synovial fluid of arthritis patients with necrosis-promoting antibodies (SF024, SF045). However, the reaction of the synovial fluid of a control patient without necrosis-promoting antibodies was negative (SF010). Microplates were coated with 2 μg / ml P11-17, blocked with BSA, and incubated with 244-12, SF024, SF045, and SF010, respectively, at room temperature for 1 hour, and then recognized by horseradish peroxidase-labeled anti-murine or anti-human IgG secondary antibodies.

[0020] [Figure 10] 10 shows that both the polypeptide P11-17 of the present invention and the polypeptide shown in SEQ ID NO: 2 can effectively inhibit cell necrosis. Cell necrosis may be induced by purified cell necrosis-promoting antibody (244-12) or by cell necrosis-promoting antibody in the patient's synovial fluid (SF002, SF045). [Figure 11] FIG. 11 shows that the polypeptides P12-10, P13-1, P13-2 and P13-3 of the present invention can competitively inhibit the binding of TNF to monoclonal antibody 244-12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Through extensive and in-depth research, the inventor unexpectedly found that antibody 244-12 can convert TNF-induced apoptosis to necroptosis in the presence of TNF, and found that some inflammatory patients, such as arthritis and sepsis, have autoantibodies that induce similar necroptosis, and the pathology of these patients is significantly more severe. Furthermore, the inventor has produced a polypeptide that can block the patient's autoantibody, and thus can treat inflammation without causing severe side effects. Based on this, the present invention has been completed.

[0022] Apoptosis or cell apoptosis as described herein refers to programmed cell death, i.e., cell death by signal transduction. It is characterized by cell shrinkage, cell nucleus fragmentation, but cell membrane intact. Cell apoptosis does not cause inflammation, because apoptotic cells with intact cell membranes are quickly engulfed by macrophages. Necrosis or cell necrosis as described herein refers to the destruction and death of cells caused by external forces, characterized by cell fragmentation and cell membrane destruction and incompleteness. During necrosis, cells release many inflammatory substances, such as nucleic acids, uric acid, HMGB1, etc., which causes an inflammatory response.

[0023] Necroptosis or cell necroptosis as described herein refers to cell signaling-induced death. It is characterized by cell fragmentation and cell membrane destruction and incompleteness. As with the above-mentioned cell necrosis, in the process of necroptosis, necrotic cells release many inflammatory substances, such as nucleic acids, uric acid, HMGB1, etc., which causes inflammatory responses.

[0024] TNF and its functions TNF is a multifunctional cytokine secreted primarily by macrophages and T cells. TNF exerts many biological functions through TNF receptors 1 and 2 (TNFR1 and TNFR2), including stimulation of the transcription factor nuclear factor kappa B (NF-κB), induction of the extrinsic pathway of cell apoptosis, and induction of necrosis.

[0025] TNF is a multifunctional cytokine secreted primarily by macrophages and T cells. TNF exerts many biological functions through TNF receptors 1 and 2 (TNFR1 and TNFR2), including stimulation of the transcription factor nuclear factor kappa B (NF-κB), induction of the extrinsic pathway of cell apoptosis, and induction of necrosis.

[0026] All current studies emphasize the downstream consequences after TNF-TNFR1 binding. However, the subtle molecular basis at the level of TNF-TNFR1 interaction, where different cellular functions can be observed, has not been studied. The conventional concept is that TNF-TNFR1 binding is generally sufficient to initiate all TNF functions, including stimulation of NF-κB and induction of cell death. In addition, existing techniques have investigated short peptides derived from human TNFα, some of which significantly induce cell apoptosis, while others significantly induce cell necrosis. And the solubility of each short peptide is significantly different, which affects the drug-likeness of the short peptide.

[0027] antibody As used herein, the terms "antibody", "monoclonal antibody", "244-12" or "autoantibody" have the same meaning and refer to an antibody capable of binding to TNF, in particular specifically to the sequence QLVVPSE, i.e., the binding epitope is QLVVPSE. It has been found that said antibody is capable of inducing necroptosis of cells. As used herein, the antibody includes not only a complete monoclonal antibody, but also an antibody fragment having immunological activity, such as a Fab or (Fab')2 fragment, an antibody heavy chain, and an antibody light chain. In a specific embodiment, the antibody has a light chain shown in SEQ ID NO:9 and / or a heavy chain shown in SEQ ID NO:10.

[0028] Polypeptides of the Invention In order to provide a material means for regulating and therefore reversing cell necrosis, the present invention provides polypeptides that bind to the above-mentioned autoantibodies capable of inducing cell necroptosis, but do not bind to TNFR1, thereby inhibiting cell necrosis and treating or alleviating inflammation. In a specific embodiment, the present invention provides a polypeptide that inhibits cell necrosis, which is derived from wild-type TNFα and comprises 10 to 200 amino acid residues (preferably 10 to 180, more preferably 10 to 160 amino acid residues) and the sequence QLVVPSE. In a specific embodiment, the wild-type TNFα is human-derived TNFα, the amino acid sequence of which is set forth in SEQ ID NO:1.

[0029] In a preferred embodiment, the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NOs: 2 to 8. In a further preferred embodiment, the polypeptide of the present invention is a soluble polypeptide, which can provide excellent drug-like properties. In specific embodiments, the amino acid sequence of the polypeptide is set forth in SEQ ID NO:2, 3 or 4.

[0030] Drug Compositions Based on the polypeptide of the present invention, the present invention further provides a pharmaceutical composition for inhibiting cell necrosis, comprising a therapeutically effective amount of the polypeptide of the present invention and a pharma- ceutically acceptable carrier. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount that has a function or activity in humans and / or animals and that can be tolerated by humans and / or animals for use.

[0031] As used herein, the term "pharmaceutically acceptable" refers to a substance that, when administered to humans and / or mammals, does not cause undue adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for delivery of a therapeutic agent, and includes various excipients and diluents.

[0032] The pharmaceutical composition of the present invention comprises a safe and effective amount of the polypeptide of the present invention as an active ingredient and a pharma- ceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof. In general, pharmaceutical preparations are based on the mode of administration, and the pharmaceutical composition of the present invention may be in the form of a solid or a solution, preferably in the form of a solution, such as an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, or a sustained release agent. For example, it can be prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other auxiliary agents. The pharmaceutical composition is preferably prepared under aseptic conditions.

[0033] The effective amount of the active ingredient of the present invention can be varied depending on the mode of administration and the severity of the disease to be treated. The selection of a suitable effective amount can be determined by those skilled in the art based on various factors (e.g., clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc., the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc. Depending on the requirements of the treatment situation, it can be administered in several divided doses or in proportionally decreasing doses every day.

[0034] The pharma- ceutically acceptable carriers, effective amounts of active ingredients and modes of administration described in the present invention are all familiar to those skilled in the art.

[0035] In view of its function, those skilled in the art will understand that the polypeptide and pharmaceutical composition of the present invention can inhibit cell necrosis, thereby treating diseases associated with cell necrosis, including but not limited to inflammatory diseases. In a specific embodiment, the inflammatory diseases include but are not limited to rheumatoid arthritis, Crohn's disease, psoriasis, and sepsis.

[0036] Based on the polypeptide and pharmaceutical composition of the present invention, the present invention further provides a method for inhibiting cell necrosis or treating a cell necrosis-associated disease, such as an inflammatory disease, comprising administering to a subject in need thereof a polypeptide of the present invention or a pharmaceutical composition comprising a polypeptide of the present invention.

[0037] The main advantages of the present invention are: 1. In the present invention, it was found for the first time that autoantibodies that cause cellular necroptosis can be neutralized. 2. The results of this invention have significant clinical significance. 3. The polypeptides of the present invention are capable of treating, alleviating or reducing an inflammatory response. 4. The polypeptide of the present invention does not bind to TNF receptors, so it does not affect the normal function of TNF. It does not cause side effects such as tuberculosis infection or tumor development by suppressing TNF function, unlike anti-TNF monoclonal antibodies or anti-inflammatory therapies using TNF-releasing receptors currently used in clinical practice.

[0038] The present invention will be described in more detail below with reference to specific examples. It is understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. Experimental methods for which detailed conditions are not shown in the following examples are usually performed according to standard conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts are calculated by weight. All experimental materials used in the examples of the present invention are commercially available unless otherwise specified.

[0039] Materials and Methods patient Patients were recruited from rheumatology clinics or intensive care units, and all samples were stored at -80°C until analysis. Synovial fluid was aspirated from knee joints of patients with inflammatory disease as part of therapeutic arthrocentesis. Rheumatoid arthritis was defined according to the 1987 American College of Rheumatology or the 2010 ACR / EULAR classification criteria. Other inflammatory arthritis was diagnosed by clinical and radiographic criteria. All rheumatoid arthritis patients were seropositive for rheumatoid factor and had moderate disease activity. Patients with sepsis were diagnosed according to the criteria established by the American Thoracic Society in 1992. Samples and / or data obtained were collected with the donors' informed consent and in full compliance with national and institutional ethical requirements.

[0040] Cell Lines, TNF and Polypeptides Mouse fibrosarcoma L929 cells and human lymphocyte Jurkat A3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Human C28I2 chondrocytes were provided by Dr. Mary B. Goldring. Human SaOs-2 osteoblastic cells were purchased from Sigma. TNF was purchased from Immunotools (Germany).

[0041] Apoptosis and necrosis assays Cells were incubated overnight (or for the times indicated) in the presence of TNF (Immunotools, Friesoythe, Germany) or TNF peptides with or without 20 μM z-VAD-FMK (R&D Systems, Minneapolis, MN) or 20 μM Necrostatin-1 (PeproTech, Rocky Hill, NJ). In some experiments, TNF was co-incubated with cells and the indicated concentrations of monoclonal antibodies or synovial fluid. Cells were stained with a live / dead cell staining kit (Invitrogen, Paisley, UK) and fixed with a cytofix / cytoperm fixation / permeabilization solution kit (BD Pharmingen, Oxford, UK) following the instructions in the manufacturer's brochure. Intracellular staining was then performed with a FITC-coupled anti-caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA). Cells were acquired using a CyAn flow cytometer (Beckman Coulter, Fullerton, Calif.) and data were analyzed using Flowjo (Tree Star Inc. Ashland, Oregon).

[0042] ELISA Monoclonal antibody selection / suppression testing ELISA plates were coated with 2 μg / ml TNF overnight at 4°C or for 2 h at 37°C, followed by incubation with monoclonal antibodies for 1 h at 37°C. A secondary antibody coupled to HRP was used to detect the reaction. For inhibition studies, TNFR1 or synovial fluid was plated together with the monoclonal antibodies.

[0043] Binding of TNF to TNFR1 Binding of TNFR1 to TNF was measured by ELISA as follows: ELISA plates were coated with 1.5 μg / ml TNF or mTNF-HA at 4°C overnight or 2 h at 37°C. After 2 h incubation with TNFR1 (1 μg / ml) at 37°C, they were incubated with anti-TNFR1 or anti-HA antibodies for another 2 h and then detected with a secondary antibody-mouse IgG antibody coupled to HRP for 30 min and its substrate. Color was developed and detected at OD 450 nm by a Wallace Victor2 1420 multilabel counter (PerkinElmer, Massachusetts, USA).

[0044] Immunofluorescence microscopy of L929 cells TNF at 20 ng / ml was incubated with 2 μg / ml mAb M26 or 244-12 for 1 h at room temperature. The TNF / M26 or TNF / 244-12 mixture was incubated with L929 cells grown on coverslips for 15 min on ice. Cells were fixed with 4% paraldehyde (prepared in PBS) for 10 min, blocked with phosphate buffer containing 0.5% BSA and 0.1% cold water fish gelatin for 15 min, and further incubated with rabbit anti-TNFR1 antibody (Abcam) for 1 h. Cells were washed with PBS and then incubated with the relevant secondary antibody coupled to Alexa Fluor 488 or Alexa Fluor 568 (Invitrogen). PBS-washed samples were then mounted in Gelvatol / DABCO (Sigma-Aldrich). DNA was counterstained with DAPI (Sigma-Aldrich). All samples were analyzed by fluorescence microscopy using a Nikon Eclipse 80i at 60x. Images were acquired using a Hamamatsu camera with NIS-Elements AR3.0 software.

[0045] Polypeptide and protein synthesis and detection methods: The coding nucleotide sequences corresponding to the amino acid sequences shown in SEQ ID NOs: 2 to 4 were synthesized by a conventional method, then expressed in E. coli and purified. The purified products were confirmed by SDS electrophoresis and mass spectrometry (Bruker Daltonics Ultraflex TOF / TOF mass spectrometer). The amino acid sequences shown in SEQ ID NOs: 5 to 8 and their modified sequences, for example those biotinylated at the N-terminus or C-terminus (if necessary), were synthesized using an APEX396 automated polypeptide synthesizer according to the Fmoc protocol. The synthesized polypeptides were confirmed using a mass spectrometer (Bruker Daltonics Ultraflex TOF / TOF mass spectrometer). EXAMPLES

[0046] Example 1. Production of monoclonal antibody 244-12 and identification of its binding epitope The inventors prepared 33 strains of monoclonal antibodies by conventional methods and detected the binding state between the prepared monoclonal antibodies and TNF. Among them, the ELISA test proved that the binding between monoclonal antibody 244-12 and TNF was the strongest. The inventors then identified the binding epitope of monoclonal antibody 244-12 to be QLVVPSE.

[0047] Example 2. Conversion of TNF-associated apoptosis to necroptosis by monoclonal antibody 244-12 The inventors have studied the effect of monoclonal antibody (mAb) 244-12 on the function of TNF. First, the confocal microscope photograph in Figure 2 shows that the binding of monoclonal antibody 244-12 to TNF does not affect the binding of TNF to the TNF receptor. In the confocal microscope, the TNF receptor was stained red, and 244-12 was stained green. Here, the upper figure shows that when TNF is present, the green and red overlap (yellow), explaining that 244-12 overlaps with TNF and the TNF receptor. The lower figure shows that when TNF is not present, only red is present, explaining that 244-12 does not directly bind to the cell surface.

[0048] The inventor then observed that TNF stimulated L929 cells to cause cell apoptosis (expression of active caspase-3), but when monoclonal antibody 244-12 was added, cell apoptosis was suppressed (active caspase-3 was suppressed). Figure 3 shows that monoclonal antibody 244-12 blocked cell apoptosis (i.e., expression of active caspase-3) induced by TNF in both cell lines, L929 cells (mouse fibroblast tumor cells, upper figure) and C28I2 cells (chondrocytes, lower figure).

[0049] However, unexpectedly, when monoclonal antibody 244-12 was administered to L929 cells with TNF, apoptosis was inhibited, but the cells underwent necrosis. Figure 4 shows necrosis of L929 cells induced by antibody 244-12 and TNF. Here, TNF caused apoptosis of the cells (top right). When control antibody M26 was administered (blocking TNF binding to the receptor), L929 cells survived (middle right). When 244-12 and TNF were administered, L929 cells underwent necrosis (bottom right).

[0050] The inventors further studied and proved that necrosis caused by 244-12 + TNF was caused by signal transduction. Figure 5 shows that necrosis caused by the antibody 244-12 + TNF of the present invention was inhibited by Nec-1. Here, after 244-12 + TNF induces Nec-1, necroptosis was also converted to cell apoptosis (right of the figure above). It was explained that cell necrosis was caused by signal transduction, i.e., necroptosis.

[0051] Example 3. Autoantibody-induced necroptosis in synovial fluid of rheumatoid arthritis patients The inventors unexpectedly found that large amounts of antibodies are present in the synovial fluid of some untreated patients with rheumatoid arthritis or osteoarticular disease (Figure 6A). These antibodies can compete with 244-12 for the antigen-binding site (Figure 6B). Synovial fluid containing these antibodies can induce necroptosis of cells (Figure 6C).

[0052] Here, Figure 6A shows that anti-TNF autoantibodies were present in the synovial fluid of some arthritis patients. The synovial fluid of patient SF8 contained a large amount of autoantibodies. 6B shows that the autoantibodies competed with 244-12 for the TNF binding site. In a binding test (ELISA) between 244-12 and TNF, the addition of SF8 synovial fluid inhibited the binding of 244-12 to TNF. 6C shows that SF8 synovial fluid induced necroptosis in the presence of TNF. TNF induced apoptosis of L929 cells (top right), but the addition of SF8 synovial fluid converted the cells to necroptosis (middle right). On the other hand, the control synovial fluid SF4 did not induce necrosis (bottom right). The inventor further observed 24 sepsis patients and found that 14 patients had antibodies that compete with 244-12 for the antigen-binding site, while the other 10 patients did not contain similar antibodies. The 14 patients who contained antibodies that compete with 244-12 for the antigen-binding site were given the best possible care and treatment, but all of them died. Meanwhile, of the other 10 patients, 5 died, and the remaining 5 were fully recovered and discharged from the hospital after 2-3 weeks. The antibody levels of the deceased patients were significantly higher than those of the fully recovered group (p<0.01) (Figure 7).

[0053] Example 4. The inventors have further investigated antibodies that specifically bind to other TNF molecules and fragments and have not found that these antibodies have any effect on reversing TNF-associated apoptosis to necroptosis. Furthermore, the present inventors screened full-length TNF and found that an antibody that specifically binds to QLVVPSE has the effect of reversing TNF-associated apoptosis to necroptosis.

[0054] Example 5. Production of the polypeptide of the present invention As described in "Synthesis and detection methods of polypeptides and proteins" in "Materials and Methods," the polypeptides of the present invention shown below were produced, detected, and identified.

[0055] SEQ ID NO:2: VRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELR DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VFHQTKVNLL SAIKSPCQRE TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL, P11-17: QLQWLNRRAN ALLANGVELR DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE (SEQ ID NO: 3), P11-17 (mutated, SY mutation): QLQWLNRRAN ALLANGVELR DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VFHQTKVNLL SAIKSPCQRE (SEQ ID NO: 4); P12-10: RDNQLVVPSE (SEQ ID NO: 5), P13-1: DNQLVVPSEG (SEQ ID NO: 6), P13-2: NQLVVPSEGL (SEQ ID NO: 7), P13-3: QLVVPSEGLY (sequence number 8).

[0056] Example 6. Neutralization of the activity of cell necrosis-promoting antibodies by polypeptides of the present invention The present inventors further investigated the binding of the polypeptide of the present invention to a cell necrosis-promoting antibody, and the ability to competitively inhibit the binding of TNF to a cell necrosis-promoting antibody, and the ability of the polypeptide of the present invention to inhibit cell necrosis. As shown in Figure 9, the polypeptide P11-17 (SEQ ID NO: 3) of the present invention was able to bind to purified pro-necrosis antibody (244-12) and to pro-necrosis antibodies (SF024, SF045) in the synovial fluid of arthritis patients. However, the reaction of the synovial fluid of a control patient without autoantibodies was negative (SF010). Microplates were coated with 2 μg / ml P11-17, blocked with BSA, and incubated with 244-12, SF024, SF045, and SF010 at room temperature for 1 hour, respectively, and then recognized by horseradish peroxidase-labeled anti-murine or human IgG secondary antibodies.

[0057] 10 shows that both the polypeptide P11-17 of the present invention and SEQ ID NO: 2 were able to effectively inhibit cell necrosis, which may be due to purified cell necrosis-promoting antibody (244-12) or due to cell necrosis-promoting antibody in the patient's synovial fluid (SF002, SF045). Figure 11 shows that the polypeptides P12-10, P13-1, P13-2 and P13-3 of the present invention can competitively inhibit the binding of TNF and monoclonal antibody 244-12. P12-10, P13-1, P13-2 and P13-3 significantly inhibited the binding of TNF and monoclonal antibody 244-12. Method: TNF was coated on a microplate, and the indicated polypeptides were added to each well, followed by monoclonal antibody 244-12. When the polypeptides bind to 244-12, the binding of 244-12 to TNF was inhibited.

[0058] The binding ability of the polypeptide of the present invention to the cell necrosis-promoting antibody and the ability of the polypeptide of the present invention to inhibit cell necrosis are summarized in the table below.

[0059] [Table 1]

[0060] Consider Based on the findings of the present invention, the inventors have proposed a new treatment for inflammation. The mechanism of necroptosis involves autoantibodies and TNF. Necroptosis is one of the causes of inflammation. Currently, one of the strategies for treating inflammatory diseases is to block TNF by inhibiting TNF. This strategy is effective in suppressing inflammation, but leads to life-threatening side effects, such as TB or lymphoma. The inventors have put forward an alternative method of blocking autoantibodies by the polypeptide of the present invention (which does not bind to TNFR1). The result of this strategy is to reduce the inflammatory load while simultaneously inhibiting necroptosis, but keeping TNF intact, which may prevent tumors and serious infections, such as TB.

[0061] All documents related to the present invention are incorporated herein by reference as if each document were incorporated individually. After reading the above content of the present invention, it will be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalent forms thereof are within the scope of the claims of the present invention.

[0062] References 1. Chan, K.F., Siegel, M.R., and Lenardo, J.M. 2000. Signaling by the TNF receptor superfamily and T cell homeostasis. Immunity 13:419-422. 2. Thoma, B., Grell, M., Pfizenmaier, K., and Scheurich, P. 1990. Identification of a 60-kD tumor necrosis factor (TNF) receptor as the major signal transducing component in TNF responses. J Exp Med 172:1019-1023. 3. Idriss, H.T., and Naismith, J.H. 2000. TNF alpha and the TNF receptor superfamily: structure-function relationship(s). Microsc Res Tech 50:184-195. 4. Van Herreweghe, F., Festjens, N., Declercq, W., and Vandenabeele, P. 2010. Tumor necrosis factor-mediated cell death: to break or to burst, that's the question. Cell Mol Life Sci 67:1567-1579. 5. Kono, H., and Rock, K.L. 2008. How dying cells alert the immune system to danger. Nat Rev Immunol 8:279-289. 6. Declercq, W., Vanden Berghe, T., and Vandenabeele, P. 2009. RIP kinases at the crossroads of cell death and survival. Cell 138:229-232. 7. Hitomi, J., Christofferson, D.E., Ng, A., Yao, J., Degterev, A., Xavier, R.J., and Yuan, J. 2008. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135:1311-1323. 8. Galluzzi, L., and Kroemer, G. 2008. Necroptosis: a specialized pathway of programmed necrosis. Cell 135:1161-1163. 9. Vandenabeele, P., Galluzzi, L., Vanden Berghe, T., and Kroemer, G. 2010. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nature Reviews Molecular Cell Biology 11:700-714. 10. Berghe, T.V., Vanlangenakker, N., Parthoens, E., Deckers, W., Devos, M., Festjens, N., Guerin, C.J., Brunk, U.T., Declercq, W., and Vandenabeele, P. 2010. Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ 17:922-930. 11. Chan, F.K., Shisler, J., Bixby, J.G., Felices, M., Zheng, L., Appel, M., Orenstein, J., Moss, B., and Lenardo, M.J. 2003. A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J Biol Chem 278:51613-51621. 12. Feldmann, M. 2009. Translating molecular insights in autoimmunity into effective therapy. Annu Rev Immunol 27:1-27. 13. Scheinfeld, N. 2004. A comprehensive review and evaluation of the side effects of the tumor necrosis factor alpha blockers etanercept, infliximab and adalimumab. J Dermatolog Treat 15:280-294. 14. Gunther, C., Neumann, H., Neurath, M.F., and Becker, C. 2013. Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium. Gut 62:1062-1071. 15. Rock, K.L., and Kono, H. 2008. The inflammatory response to cell death. Annu Rev Pathol 3:99-126. 16. Linkermann, A., and Green, D.R. 2014. Necroptosis. N Engl J Med 370:455-465.

Claims

1. Use of a polypeptide in the manufacture of a medicament for inhibiting cell necrosis in the presence of a cell necrosis-promoting antibody, or in the manufacture of a medicament for inhibiting necroptosis by an autoantibody to treat an inflammatory disease, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 2, and the cell necrosis-promoting antibody has a light chain as set forth in SEQ ID NO: 9 and a heavy chain as set forth in SEQ ID NO:

10.

2. The use according to claim 1, wherein the inflammatory disease is selected from rheumatoid arthritis, Crohn's disease, psoriasis and sepsis.

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