WO2017088821A1 - 一种中和促细胞程序性坏死抗体的蛋白及其应用 - Google Patents

一种中和促细胞程序性坏死抗体的蛋白及其应用 Download PDF

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WO2017088821A1
WO2017088821A1 PCT/CN2016/107291 CN2016107291W WO2017088821A1 WO 2017088821 A1 WO2017088821 A1 WO 2017088821A1 CN 2016107291 W CN2016107291 W CN 2016107291W WO 2017088821 A1 WO2017088821 A1 WO 2017088821A1
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polypeptide
tnf
necrosis
cell
antibody
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PCT/CN2016/107291
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French (fr)
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姜石松
逯文姝
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姜石松
逯文姝
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Priority to JP2018546736A priority Critical patent/JP2019500412A/ja
Priority to EP16868044.5A priority patent/EP3381934B1/en
Priority to US15/779,282 priority patent/US11434270B2/en
Publication of WO2017088821A1 publication Critical patent/WO2017088821A1/zh
Priority to HK18116228.0A priority patent/HK1257223A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
    • A61M1/16—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3687—Chemical treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biomedicine; in particular, the present invention relates to a protein capable of neutralizing a pro-apoptotic antibody and its use in the treatment of inflammation.
  • Tumor necrosis factor alpha is a multifunctional cytokine secreted by various cells, mainly macrophages and T cells. TNF exerts a variety of biological functions through TNF receptors 1 and 2 (TNFR1 and TNFR2) (1-3).
  • TNF functions are mainly involved in three intracellular events: 1) stimulation of the transcription factor nuclear factor kappa B (NF- ⁇ B), which leads to cell activation and cytokine production; 2) external pathways that induce apoptosis; and 3) induced necrosis .
  • NF- ⁇ B activation leads to pro-inflammatory cytokine secretion and cell survival and activation
  • apoptosis is a state of cell death characterized by caspase-3 activation Nuclear rupture, with intact cell membranes at an early stage, with little or no inflammatory response; however, necrosis is another mechanism of cell death in which there is no activation of caspase-3 and impaired cell membrane integrity.
  • necrosis results in the release of intracellular substances that stimulate strong immune and inflammatory responses (4, 5). Under apoptotic conditions, inhibition of caspase results in a form of programmed cell death that is characterized by necrosis when it occurs, termed "programmed necrosis" (6-11). Because such programmed necrosis is an inflammatory process, it may be clinically associated with diseases such as rheumatoid arthritis, Crohn's disease, and psoriasis. However, to date, factors that trigger programmed necrosis in these diseases have not been identified.
  • TNF inflammatory diseases
  • Anti-TNF biologics include: anti-TNF monoclonal antibodies capable of neutralizing TNF, free TNF receptors and the like. The market for anti-TNF agents is $15 billion per year.
  • NF- ⁇ B signaling was found to prevent senescence in inflammatory bowel disease Dead (14). It is believed that apoptosis inhibits inflammation (15). Programmed necrosis may be a possible mechanism of inflammation and cell/tissue destruction, but lacks evidence of human clinical relevance. For example, in a non-viral inflammatory pathogenesis, it is not known which factors outside the target cell trigger programmed necrosis (14, 16). Clearly, the identification of these factors provides a biomarker for the diagnosis and treatment of many inflammatory diseases.
  • TNF is significantly elevated and positively correlated with the severity of the disease.
  • traditional TNF-blocking antibodies and free receptors are not effective in treating sepsis, suggesting that other factors or factors play a key role in TNF synergy during pathogenesis.
  • the present invention provides a polypeptide which inhibits cell necrosis, which polypeptide is derived from wild-type TNF ⁇ and comprises 10-200 amino acid residues and the sequence QLVVPSE, wherein the polypeptide does not bind to TNFR1 but competes with TNF Binding to promote cell necrosis antibodies.
  • the wild-type TNF ⁇ is human TNF ⁇ having the amino acid sequence set forth in SEQ ID NO: 1.
  • the pro-cellular necrosis antibody has: the light chain of SEQ ID NO: 9; and/or the heavy chain of SEQ ID NO: 10.
  • amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NOs: 2-8.
  • the polypeptide is a soluble polypeptide.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 2, 3 or 4.
  • the present invention provides the use of the polypeptide of the first aspect of the invention for the preparation of a medicament for inhibiting cell necrosis or treating a cell necrosis-related disease.
  • the cell necrosis related disease is an inflammatory disease.
  • the inflammatory disease includes, but is not limited to, rheumatoid arthritis, Crohn's disease, psoriasis, sepsis.
  • the present invention provides a pharmaceutical composition for inhibiting cell necrosis, comprising a therapeutically effective amount of the polypeptide of the first aspect of the invention and a pharmaceutically acceptable carrier.
  • amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NOs: 2-8.
  • the polypeptide is a soluble polypeptide.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 2, 3 or 4.
  • the pharmaceutical composition is an intravenously administered injection.
  • the invention provides a dialysis device for treating blood of a patient, the dialysis device comprising the polypeptide of the first aspect of the invention.
  • the dialysis device is used to remove or neutralize pro-cellular necrotic antibodies in the blood of the patient.
  • the pro-cellular necrosis antibody has: the light chain of SEQ ID NO: 9; and/or the heavy chain of SEQ ID NO: 10.
  • the dialysis device is a dialysis column.
  • the present invention provides a method of inhibiting or neutralizing a cell-promoting antibody in a blood of a patient, comprising inhibiting or neutralizing a cell-promoting antibody in the blood of the patient using the polypeptide of the first aspect of the invention .
  • the pro-cellular necrosis antibody has: the light chain of SEQ ID NO: 9; and/or the heavy chain of SEQ ID NO: 10.
  • the method comprises administering a polypeptide of the first aspect of the invention, for example by injection, to a patient in need thereof to inhibit or neutralize a pro-cellular necrotic antibody in the blood of the patient.
  • the method comprises dialysis using the polypeptide of the first aspect of the invention to inhibit or neutralize pro-cellular necrotic antibodies in the blood of the patient.
  • the method comprises using a dialysis device comprising the polypeptide of the first aspect of the invention to inhibit or neutralize pro-cellular necrotic antibodies in the blood of the patient.
  • the dialysis device is a dialysis column.
  • the method is for treating a cell necrosis related disorder.
  • the therapeutic cell necrosis related disease is an inflammatory disease.
  • the inflammatory disease includes, but is not limited to, rheumatoid arthritis, Crohn's disease, psoriasis, sepsis.
  • Figure 1 shows screening of monoclonal antibodies capable of binding to TNF from 33 monoclonal antibodies, wherein ELISA assays demonstrated that 244-12 binds TNF most strongly;
  • FIG. 2 shows that binding of antibody 244-12 to TNF does not affect TNF binding to cell surface receptors, where "Merge” means conjugation and “alone” means only antibody 244-12;
  • Figure 3 shows that antibody 244-12 blocks TNF-induced apoptosis (i.e., active caspase-3 expression) in both cell lines.
  • TNF-induced apoptosis i.e., active caspase-3 expression
  • L929 cells mouse fibroblastoma cells
  • C28I2 cells chondrocytes
  • FIG. 4 shows that antibody 244-12 and TNF cause necrosis of L929 cells.
  • the ordinate indicates apoptosis (active caspase-3); the abscissa indicates cell necrosis (destruction of cell membrane integrity).
  • TNF caused apoptosis (above right); addition of control antibody M26 (blocking TNF-binding receptor), L929 cells survived (middle); 244-12 and TNF, L929 cell necrosis (bottom right) ;
  • Figure 5 shows that necrosis by antibody 244-12 + TNF can be inhibited by Nec-1.
  • 244-12+TNF was added to Nec-1 (right panel)
  • cell necrosis was transformed into apoptosis. It indicates that cell necrosis is through signal transduction, ie programmed necrosis.
  • Figure 6 shows autoantibodies in synovial fluid of patients with rheumatoid arthritis triggering programmed necrosis
  • Figure 7 shows the average level of antibodies that compete with the antibody 244-12 of the present invention for antigen binding sites in patients with sepsis (5 patients) and death patients (19 patients). The difference between the two is very significant. P ⁇ 0.01;
  • Figure 8 is a schematic representation of the pNIC28Bsa4 vector showing important components associated with clonal expression.
  • Figure 9 shows that the polypeptide P11-17 of the present invention is capable of binding to both the purified cell-producing antibody (244-12) and the autoantibody (SF024, SF045) in the joint fluid of patients with osteoblastic antibody-producing arthritis.
  • the reaction with the joint fluid of the control patient without the cell-producing necrotic antibody was negative (SF010).
  • the plate was coated with 2 ⁇ g/ml of P11-17. After blocking with BSA, it was incubated with 244-12, SF024, SF045 and SF010 for 1 hour at room temperature, then labeled with horseradish superoxide dismutase. The secondary antibody of IgG was identified.
  • Figure 10 shows that both the polypeptides of the present invention P11-17 and SEQ ID NO: 2 are effective in inhibiting cell necrosis. Whether cell necrosis is caused by purified pro-cellular necrosis antibody (244-12) or by cell necrosis antibodies in patients' joint fluid (SF002, SF045).
  • Figure 11 shows that the polypeptides P12-10, P13-1, P13-2 and P13-3 of the present invention are capable of competitively inhibiting the binding of TNF to mAb 244-12.
  • autoantibody 244-12 can convert TNF-induced apoptosis into programmed necrosis in the presence of TNF, and then found some patients with inflammation such as arthritis and sepsis. There are similar autoantibodies that cause programmed necrosis in the body, and this part of the patient's condition is significantly worse. Further, the inventors prepared a polypeptide capable of blocking the autoantibody of a patient, which in turn can treat inflammation without causing serious side effects. The present invention has been completed on this basis.
  • Apoptosis or apoptosis as described herein refers to programmed cell death, i.e., death of cells through signaling. It is characterized by cell atrophy, nuclear rupture, but cell membrane integrity. Since the cell membrane is intact and the apoptotic cells are quickly phagocytosed by macrophages, apoptosis does not cause inflammation.
  • necrosis or necrosis as described herein refers to the death of cells by external force and is characterized by cell disruption and destruction of the membrane. During necrosis, the cells release many inflammatory substances such as nucleic acids, uric acid, HMGB1, etc., which cause an inflammatory reaction.
  • programmed necrosis or programmed cell necrosis refers to the death of a cell through signaling. It is characterized by cell disruption and destruction of the cell membrane. Like the above-mentioned cell necrosis, during the process of programmed necrosis, necrotic cells release many inflammatory substances such as nucleic acid, uric acid, HMGB1, etc., and thus cause an inflammatory reaction.
  • TNF is a multifunctional cytokine secreted mainly by macrophages and T cells. It exerts a variety of biological functions through TNF receptors 1 and 2 (TNFR1 and TNFR2), including stimulation of the transcription factor nuclear factor kappa B. (NF- ⁇ B); an external pathway that induces apoptosis; and induces necrosis.
  • TNFR1 and TNFR2 TNF receptors 1 and 2
  • NF- ⁇ B transcription factor nuclear factor kappa B.
  • TNF-stimulated cells primarily activate NF- ⁇ B for cell survival. Apoptosis and necrosis are only triggered when the NF- ⁇ B pathway is inhibited (24). It has been suggested that TNF stimulates membrane-bound complex I (25), which initiates NF- ⁇ B activation but does not initiate apoptosis/necrosis. However, if NF- ⁇ B activation is blocked, TNF stimulates its target cells to form a second complex (complex II) in the cytoplasm, which directs the signaling pathway to cell death.
  • complex II complex II
  • TNF-TNFR1 binding is sufficient to initiate all TNF functions, including stimulation of NF- ⁇ B and induction of cell death.
  • the prior art also studied short peptides derived from human TNF ⁇ , some of which significantly caused apoptosis, and some caused cell necrosis. And the solubility of various short peptides may be significantly different, which may affect the drug-forming properties of short peptides.
  • antibody As used herein, “antibody”, “monoclonal antibody”, “244-12” or “autoantibody” have the same meaning and refers to the ability to bind to TNF, in particular to specifically bind to the sequence QLVVPSE, ie, the binding epitope is QLVVPSE antibody.
  • the antibody can cause programmed necrosis of the cells.
  • the antibody includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • the antibody has: the light chain of SEQ ID NO: 9; and/or the heavy chain of SEQ ID NO: 10.
  • the present invention provides a polypeptide which is capable of binding to the above-mentioned autoantibody which causes apoptosis of the cells without binding to TNFR1, thereby inhibiting cell necrosis. In turn, it can treat or reduce inflammation.
  • the present invention provides a polypeptide for inhibiting cell necrosis derived from wild-type TNF ⁇ , the polypeptide comprising 10-200 amino acid residues (preferably 10-180, more preferably 10-160 amino acid residues) Base) and sequence QLVVPSE.
  • the wild-type TNF ⁇ is human TNF ⁇ having the amino acid sequence set forth in SEQ ID NO: 1.
  • the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NOs: 2-8.
  • the polypeptide of the invention is a soluble polypeptide, thereby providing excellent drug-forming properties.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 2, 3 or 4.
  • the invention further provides a pharmaceutical composition for inhibiting cell necrosis comprising a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier.
  • ⁇ ективное amount or “therapeutically effective amount” as used herein refers to a function or activity that can be exerted on a human and/or animal. And the amount acceptable to humans and / or animals.
  • pharmaceutically acceptable is a substance that is suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation, and allergies), i.e., materials having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise a safe and effective amount of a polypeptide of the present invention as an active ingredient together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched with the administration mode, and the pharmaceutical composition of the present invention may be in a solid form or a solution form, preferably in the form of a solution, such as an injection, an oral preparation (tablet, capsule, oral solution), a transdermal agent, Sustained release agent.
  • it is prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc. Depending on the condition of the treatment, several separate doses may be administered per day, or the dose may be proportionally reduced.
  • the polypeptides and pharmaceutical compositions of the present invention are capable of inhibiting cell necrosis, thereby treating diseases associated with cell necrosis, including but not limited to inflammatory diseases.
  • the inflammatory disease includes, but is not limited to, rheumatoid arthritis, Crohn's disease, psoriasis, sepsis.
  • the present invention also provides a method for inhibiting cell necrosis or treating a cell necrosis-related disease, such as an inflammatory disease, comprising administering a polypeptide of the present invention or a medicament comprising the polypeptide of the present invention to a subject in need thereof, based on the polypeptide of the present invention and a pharmaceutical composition. combination.
  • the present invention finds for the first time a polypeptide capable of neutralizing autoantibodies that cause programmed necrosis of cells;
  • the polypeptide of the invention is capable of treating, ameliorating or ameliorating an inflammatory response.
  • the polypeptide of the present invention does not bind to the TNF receptor, so it does not affect the normal function of TNF; unlike the current anti-TNF monoclonal antibody or TNF free receptor anti-inflammatory therapy, it causes tuberculosis infection and tumor due to inhibition of TNF function. Other side effects occur.
  • 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 osteoblasts were purchased from Sigma. TNF was purchased from Immunotools (Germany).
  • TNF (Immunotools, Friesoythe, Germany) or TNF peptide with or without 20 ⁇ M z-VAD-FMK (R&D Systems, Minneapolis, Minnesota) or 20 ⁇ M Necrotatin-1 (PeproTech, Rocky Hill, NJ) Incubate the cells overnight (or incubate the time shown in the figure) in the presence.
  • TNF was co-cultured with cells and monoclonal antibodies or synovial fluid at the indicated concentrations. Cells were stained with live/dead cell staining kit (Invitrogen, Paisley, UK) according to the manufacturer's manual and fixed using the cytofix/cytoperm immobilization/permeabilization solution kit (BD Pharmingen, Oxford, UK).
  • FITC-conjugated anti-caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA) was then used for intracellular staining. Cells were harvested using a CyAn flow cytometer (Beckman Coulter, Fullerton, Calif.) and analyzed using Flowjo (Tree Star Inc. Ashland, Oregon).
  • the ELISA plate was coated with 2 ⁇ g/ml TNF overnight at 4 ° C, or at 37 ° C for 2 hours, and then incubated with monoclonal antibody for 1 hour at 37 ° C. A second antibody conjugated to HRP is used to detect the reaction. For the inhibition assay, TNFR1 or synovial fluid is added along with the monoclonal antibody.
  • TNF binds to TNFR1
  • TNFR1 binding to TNF was tested by ELISA as follows: ELISA plates were coated with 1.5 ⁇ g/ml TNF or mTNF-HA overnight at 4 °C, or coated at 37 °C for 2 hours. After incubation with TNFR1 (1 ⁇ g/ml) for 2 hours at 37 ° C, it was incubated with anti-TNFR1 or anti-HA antibody for an additional two hours, and a second antibody coupled to HRP was added. Mouse IgG antibody was incubated for 30 minutes and then added to its substrate for detection. Color development was performed and detected at OD 450 nm using a Wallace Victor 2 1420 multi-label counter (PerkinElmer, Massachusetts, MA, USA).
  • TNF 20 ng/ml of TNF was incubated with 2 ⁇ g/ml of mAb M26 or 244-12 for 1 hour at room temperature.
  • Cells were fixed in 4% paraformaldehyde (PBS) for 10 minutes, blocked with phosphate buffer containing 0.5% BSA and 0.1% cold water fish gelatin for 15 minutes, and then incubated with rabbit anti-TNFR1 antibody (Abeam). 1 hour.
  • PBS paraformaldehyde
  • Abeam rabbit anti-TNFR1 antibody
  • the corresponding nucleotide sequence of the amino acid sequence shown by SEQ ID NO: 2-4 was synthesized by a conventional method, and then expressed and purified using Escherichia coli. The purified product was confirmed by SDS electrophoresis and mass spectrometry (Bruker Daltonics Ultraflex TOF/TOF mass spectrometer).
  • amino acid sequences set forth in SEQ ID NOS: 5-8 and modified sequences thereof, such as N-terminal or C-terminal biotinylation (if desired) were synthesized on an APEX 396 automated polypeptide synthesizer according to the Fmoc strategy.
  • the synthesized polypeptide was confirmed by a mass spectrometer (Bruker Daltonics Ultraflex TOF/TOF mass spectrometer).
  • the inventors prepared 33 monoclonal antibodies by a conventional method, and examined the binding of the prepared monoclonal antibodies to TNF, wherein the ELISA test demonstrated that the monoclonal antibody 244-12 binds to TNF most strongly.
  • the confocal micrograph in Figure 2 shows that binding of monoclonal antibody 244-12 to TNF does not affect TNF binding to the TNF receptor.
  • the TNF receptor was stained red and 244-12 stained green.
  • the above figure shows that when TNF is present, green and red overlap (yellow), indicating that 244-12 overlaps with TNF and TNF receptors; the figure below shows that when there is no TNF, only red, indicating that 244-12 cannot directly bind to cells surface.
  • FIG. 3 shows that monoclonal antibody 244-12 blocks TNF-induced apoptosis in both cell lines, L929 cells (mouse fibroblastoma cells, top panel) and C28I2 cells (chondrocytes, lower panel) (ie, , active caspase-3 expression).
  • necrosis caused by 244-12+TNF is signal transduced.
  • Figure 5 shows that the necrosis caused by the antibody 244-12 + TNF of the present invention can be inhibited by Nec-1.
  • 244-12+TNF was added to Nec-1, cell necrosis was transformed into apoptosis (above). It indicates that cell necrosis is through signal transduction, ie programmed necrosis.
  • FIG. 6A shows the presence of anti-TNF autoantibodies in the joint fluid of some arthritic patients.
  • the joint fluid of patient SF8 contains high autoantibodies.
  • 6B shows that autoantibodies compete with 244-12 for TNF binding sites.
  • ELISA TNF binding assay
  • binding of 244-12 to TNF was inhibited.
  • 6C showed that SF8 joint fluid caused programmed necrosis in the presence of TNF.
  • TNF caused apoptosis in L929 cells (right panel).
  • SF8 joint fluid was added, the cells turned into programmed necrosis (middle in the middle).
  • the control joint fluid SF4 did not cause necrosis (right in the figure below).
  • the inventors also observed 24 patients with sepsis, 14 patients detected antibodies that competed with 244-12 for antigen binding sites, and 10 patients did not contain similar antibodies. Fourteen patients with antibodies that competed with 244-12 for antigen binding sites were all killed despite careful care and treatment. Five of the other 10 patients died, and the other five were cured after 2-3 weeks. Antibody levels were significantly higher in the dead patients than in the disease group (p ⁇ 0.01) ( Figure 7).
  • the present inventors further studied antibodies which specifically bind to other TNF molecules and fragments, and found that these antibodies have an effect of converting TNF-related apoptosis into programmed necrosis;
  • the present inventors screened with full-length TNF and found that an antibody that specifically binds to QLVVPSE has an effect of converting TNF-related apoptosis into programmed necrosis.
  • polypeptide of the present invention shown below is prepared as described in “Polypeptide and Protein Synthesis and Detection Method” in “Materials and Methods” and is detected and confirmed:
  • SEQ ID NO: 2 VRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRANALLANGVELR DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIAVFHQTKVNLL SAIKSPCQRE TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL;
  • P11-17 mutant, SY mutation: QLQWLNRRAN ALLANGVELR DNQLVVPSEGLYLIYSQVLF KGQGCPSTHV LLTHTISRIA VFHQTKVNLL SAIKSPCQRE (SEQ ID NO: 4);
  • P12-10 RDNQLVVPSE (SEQ ID NO: 5);
  • P13-1 DNQLVVPSEG (SEQ ID NO: 6);
  • NQLVVPSEGL SEQ ID NO: 7
  • P13-3 QLVVPSEGLY (SEQ ID NO: 8).
  • Example 6 Neutralizing the activity of a cell-producing antibody against a polypeptide of the present invention
  • the present inventors further studied the binding of the polypeptide of the present invention to a cell-promoting antibody, thereby competing for the ability to inhibit the binding of TNF to a cell-producing antibody and the ability of the polypeptide of the present invention to inhibit cell necrosis.
  • the polypeptide of the present invention P11-17 (SEQ ID NO: 3) is capable of binding to purified pro-cellular necrosis antibody (244-12) and can bind to pro-cellular necrosis antibodies present in the joint fluid of arthritic patients. (Sample SF024, sample SF045). However, the joint fluid reaction was negative in the control patients without autoantibodies (SF010).
  • the plate was coated with 2 ⁇ g/ml P11-17, and after blocking with BSA, it was incubated with 244-12, SF024, SF045, SF010 for 1 hour at room temperature, and then labeled with horseradish superoxide dismutase-labeled anti-mouse or human IgG. The secondary antibody is identified.
  • Figure 10 shows that both the polypeptides P11-17 and SEQ ID NO: 2 of the present invention are effective in inhibiting cell necrosis. Whether cell necrosis is caused by purified pro-cellular necrosis antibody (244-12) or by cell-producing antibodies in the joint fluid of patients (SF002, SF045).
  • Figure 11 shows that the polypeptides P12-10, P13-1, P13-2 and P13-3 of the present invention are capable of competing to inhibit the binding of TNF to mAb 244-12.
  • P12-10, P13-1, P13-2 and P13-3 significantly inhibited the binding of TNF to monoclonal antibody 244-12.
  • METHODS TNF was coated with enzyme-linked plates, and the indicated polypeptides were added to each well, followed by the addition of monoclonal antibody 244-12. If the polypeptide binds to 244-12, inhibition of 244-12 binding to TNF.
  • the inventors have proposed a new therapeutic strategy for inflammation.
  • the mechanism of programmed necrosis involves autoantibodies and TNF.
  • Programmed necrosis is one of the causes of inflammation.
  • One of the current treatment strategies for inflammatory diseases is to block TNF by inhibiting TNF. This strategy is effective in inhibiting inflammation, but can lead to life-threatening side effects such as TB or lymphoma.
  • the inventors propose an alternative method of blocking autoantibodies (without binding to TNFRl) by the polypeptides of the invention. The result of this strategy may be to suppress programmed necrosis while reducing the inflammatory load, but to maintain TNF in place, thereby preventing tumors and serious infections such as TB.
  • TNF tumor necrosis factor
  • TNF alpha and the TNF receptor superfamily structure-function relationship(s). Microsc Res Tech 50: 184-195.

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Abstract

本发明提供了一种衍生自野生型TNFα的抑制细胞坏死的多肽，所述多肽包含10-200个氨基酸残基和序列QLVVPSE。本发明还提供包含所述多肽的药物组合物。本发明的多肽及其药物组合物能够抑制细胞坏死，进而治疗炎性疾病。

Description

一种中和促细胞程序性坏死抗体的蛋白及其应用 技术领域
本发明涉及生物医药领域；具体地说，本发明涉及一种能够中和促细胞程序性坏死抗体的蛋白及其在炎症治疗中的应用。
背景技术
肿瘤坏死因子α(TNF)是由各种细胞，主要是巨噬细胞和T细胞分泌的多功能细胞因子。TNF通过TNF受体1和2(TNFR1和TNFR2)行使多种生物学功能(1-3)。
许多TNF功能主要涉及三种胞内事件：1)刺激转录因子核因子kappa B(NF-κB)，从而导致细胞激活和细胞因子产生；2)诱导细胞凋亡的外部途径；和3)诱导坏死。这些活性的胞内信号传导途径共有一些组分但导致不同的结果：NF-κB激活导致促炎细胞因子分泌和细胞存活及激活；凋亡是细胞死亡的状态，特征在于胱冬酶-3激活、核断裂、在早期含有完整的细胞膜、炎性反应很少或没有；然而，坏死是细胞死亡的另一机制，其中没有胱冬酶-3的激活，细胞膜的完整性受损。坏死导致刺激强烈免疫和炎性反应的胞内物质释放(4,5)。在凋亡条件下，抑制胱冬酶可导致一种程序性细胞死亡形式，其发生时具备坏死的特征，称为“程序性坏死”(6-11)。由于此类程序性坏死是炎性过程，因此在临床上可能与诸如类风湿性关节炎、克罗恩病和银屑病等疾病相关。然而，迄今为止，尚未鉴定到触发这些疾病中程序性坏死的因子。
通过直接靶向TNF分子(单克隆抗体)或用作假受体的拮抗剂来中和TNF使得炎性疾病的治疗得到根本性变革(12)，但完全阻断TNF功能可能导致威胁生命的副作用，例如感染和肿瘤(13)。TNF在很多炎症性疾病(例如风湿性关节炎、炎性肠炎、牛皮癣等等)中都起很重要的病理作用。临床上用抗TNF治疗炎症性疾病非常有效。抗TNF的生物制剂包括：能够中和TNF的抗TNF的单克隆抗体，游离的TNF受体等等。抗TNF制剂的市场是每年150亿美元。
虽然有以上成果，但炎性疾病中细胞和组织破坏的机制仍未知：诱导NF-κB是促生存的，不直接导致细胞死亡-实际上，发现NF-κB信号在炎性肠病中阻止凋亡(14)。据信，凋亡是抑制炎症的(15)。程序性坏死可能是炎症和细胞/组织破坏的可能机制，但缺乏人临床相关性的证据。例如，在非病毒炎性致病机理中，不知道靶细胞外的何种因子触发程序性坏死(14,16)。显然，鉴定这些因子为诊断和治疗许多炎性疾病提供了生物标志物。
在脓毒症病人中，TNF显著升高并且与病情严重程度呈正相关。然而传统的TNF阻断型抗体和游离的受体均不能有效治疗脓毒症，暗示有其他因素或因子在致病过程中协同TNF起关键作用。
因此，本领域急需调控、甚至逆转细胞坏死的物质手段，从而能治疗炎性疾病。
发明内容
本发明的目的在于提供调控、甚至逆转细胞坏死的物质手段，从而能治疗炎性疾病。
在第一方面，本发明提供一种抑制细胞坏死的多肽，所述多肽衍生自野生型TNFα，并且包含10-200个氨基酸残基和序列QLVVPSE，其中，所述多肽不结合TNFR1但与TNF竞争结合促细胞坏死抗体。
在具体的实施方式中，所述野生型TNFα是氨基酸序列如SEQ ID NO:1所示的人源TNFα。
在具体的实施方式中，所述促细胞坏死抗体具有：SEQ ID NO:9所示的轻链；和/或SEQ ID NO:10所示的重链。
在具体的实施方式中，所述多肽的氨基酸序列选自下组：SEQ ID NO:2-8。
在优选的实施方式中，所述多肽是可溶性多肽。
在具体的实施方式中，所述多肽的氨基酸序列如SEQ ID NO:2、3或4所示。
在第二方面，本发明提供本发明第一方面所述的多肽的用途，所述多肽用于制备抑制细胞坏死或治疗细胞坏死相关疾病的药物。
在具体的实施方式中，所述细胞坏死相关疾病是炎性疾病。
在具体的实施方式中，所述炎性疾病包括但不限于类风湿性关节炎、克罗恩病、银屑病、脓毒症。
在第三方面，本发明提供一种抑制细胞坏死的药物组合物，所述药物组合物包含治疗有效量的本发明第一方面所述的多肽和药学上可接受的载体。
在优选的实施方式中，所述多肽的氨基酸序列选自下组：SEQ ID NO:2-8。
在优选的实施方式中，所述多肽是可溶性多肽。
在优选的实施方式中，所述多肽的氨基酸序列如SEQ ID NO:2、3或4所示。
在具体的实施方式中，所述药物组合物是静脉内给药的注射剂。
在第四方面，本发明提供一种处理患者血液的透析装置，所述透析装置包含本发明第一方面所述的多肽。
在优选的实施方式中，所述透析装置用于去除或中和所述患者血液中的促细胞坏死抗体。
在优选的实施方式中，所述促细胞坏死抗体具有：SEQ ID NO:9所示的轻链；和/或SEQ ID NO:10所示的重链。
在优选的实施方式中，所述透析装置是透析柱。
在第五方面，本发明提供一种抑制或中和患者血液中的促细胞坏死抗体的方法，包括利用本发明第一方面所述的多肽抑制或中和所述患者血液中的促细胞坏死抗体。
在优选的实施方式中，所述促细胞坏死抗体具有：SEQ ID NO:9所示的轻链；和/或SEQ ID NO:10所示的重链。
在优选的实施方式中，所述方法包括将本发明第一方面所述的多肽给予，例如通过注射给予有此需要的患者以抑制或中和患者血液中的促细胞坏死抗体。
在优选的实施方式中，所述方法包括利用本发明第一方面所述的多肽进行透析来抑制或中和所述患者血液中的促细胞坏死抗体。
在优选的实施方式中，所述方法包括利用包含本发明第一方面所述的多肽的透析装置来抑制或中和所述患者血液中的促细胞坏死抗体。
在优选的实施方式中，所述透析装置是透析柱。
在优选的实施方式中，所述方法用于治疗细胞坏死相关疾病。
在优选的实施方式中，所述治疗细胞坏死相关疾病是炎性疾病。
在优选的实施方式中，所述炎性疾病包括但不限于类风湿性关节炎、克罗恩病、银屑病、脓毒症。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示从33株单克隆抗体中筛选能够结合TNF的单抗，其中ELISA试验证明244-12结合TNF最强；
图2显示抗体244-12与TNF结合不影响TNF与细胞表面受体结合，其中“Merge”表示合并，“alone”表示仅有抗体244-12；
图3显示抗体244-12在两个细胞株都阻断TNF引起的细胞凋亡(即活性胱冬酶-3表达)。其中，上图是L929细胞(小鼠成纤维细胞瘤细胞)；下图是C28I2细胞(软骨细胞)；
图4显示抗体244-12与TNF引起L929细胞坏死。图中纵坐标表示细胞凋亡(活性胱冬酶-3)；横坐标表示细胞坏死(细胞膜完整性破坏)。其中，TNF引起细胞凋亡(上图右)；加入对照抗体M26(阻断TNF结合受体)，L929细胞存活(中图右)；加入244-12与TNF，L929细胞坏死(下图右)；
图5显示抗体244-12+TNF引起的坏死可以被Nec-1抑制。其中，244-12+TNF加入Nec-1后(上图右)，细胞坏死又转变成细胞凋亡。说明细胞坏死是通过信号传导，即程序性坏死。
图6显示类风湿性关节炎患者滑液中的自身抗体触发程序性坏死；
图7显示脓毒症病愈患者(5人)与死亡患者(19人)中与本发明抗体244-12竞争抗原结合位点的抗体平均水平。二者相差非常显著。P<0.01；
图8是pNIC28Bsa4载体的示意图，其中显示了与克隆可表达相关的重要组成部分。
图9显示本发明的多肽P11-17既能够结合纯化的促细胞坏死抗体(244-12)，又能够结合有促细胞坏死抗体关节炎病人关节液中的自身抗体(SF024，SF045)。但与不含促细胞坏死抗体的对照病人关节液反应为阴性(SF010)。酶标板用2μg/ml的P11-17包被，BSA封闭后分别与244-12、SF024、SF045、SF010在室温下孵育1小时，然后用辣根过氧化物歧化酶标记的抗鼠或人IgG的二抗进行识别。
图10显示本发明的多肽P11-17及SEQ ID NO:2所示多肽都可以有效抑制细胞坏死。不论细胞坏死是由纯化的促细胞坏死抗体(244-12)引起的，还是由病人关节液中(SF002，SF045)促细胞坏死抗体引起的。
图11显示本发明的多肽P12-10、P13-1、P13-2及P13-3能够竞争抑制TNF与单抗244-12的结合。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现自身抗体244-12在TNF存在下可以将TNF-诱导的凋亡转为程序性坏死，继而发现部分炎症病人如关节炎、脓毒症病人体内存在类似的引起程序性坏死的自身抗体，且这部分病人病情明显加剧。进一步地，发明人制备了能够阻断患者的自身抗体的多肽，进而可以治疗炎症但不产生严重的副作用。在此基础上完成了本发明。
本文所述的凋亡或细胞凋亡是指细胞程序性死亡，即，细胞通过信号传导的死亡。其特征在于细胞萎缩、细胞核断裂、但细胞膜完整。由于细胞膜完整而且凋亡的细胞很快被巨噬细胞吞噬，因此细胞凋亡不引起炎症。
本文所述的坏死或细胞坏死是指细胞受外力破坏而死亡，其特征在于细胞破碎，胞膜破坏而不完整。坏死时细胞释放出很多炎性物质比如核酸、尿酸、HMGB1等等，因此会引起炎症反应。
本文所述的程序性坏死或细胞程序性坏死是指细胞通过信号传导的死亡。其特征在于细胞破碎，细胞膜破坏而不完整。同上述细胞坏死一样，在程序性坏死过程中，坏死细胞释放出很多炎性物质，例如核酸、尿酸、HMGB1等等，因此会引起炎症反应。
TNF及其功能
TNF是主要由巨噬细胞和T细胞分泌的多功能细胞因子。其通过TNF受体1和2(TNFR1和TNFR2)行使多种生物学功能，包括刺激转录因子核因子kappa B (NF-κB)；诱导细胞凋亡的外部途径；和诱导坏死。
在大多数情况中，TNF刺激细胞主要激活NF-κB以供细胞存活。凋亡和坏死仅在NF-κB途径受抑制时触发(24)。有人提出TNF刺激膜结合复合物I(25)，其启动NF-κB激活但不启动凋亡/坏死。然而，如果NF-κB激活受阻止，TNF刺激其靶细胞在胞质中形成第二复合物(复合物II)，其将信号途径导向细胞死亡。
目前的所有研究强调TNF-TNFR1结合后的下游结果。然而，没有在可能导致观察到不同细胞功能的TNF-TNFR1相互作用的水平上研究过微妙的分子基础。常规的概念一般是，TNF-TNFR1结合足以启动所有的TNF功能，包括刺激NF-κB和诱导细胞死亡。现有技术还研究了衍生自人TNFα的短肽，有的明显引起细胞凋亡，有的明显引起细胞坏死。并且各种短肽的溶解性可能有显著不同，从而会影响短肽的成药性能。
抗体
在本文中，“抗体”、“单克隆抗体”、“244-12”或“自身抗体”具备相同的含义，是指能够结合TNF，特别是能够特异性结合序列QLVVPSE，即，结合表位为QLVVPSE的抗体。所述抗体能引起细胞的程序性坏死。
在本文中，所述抗体不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体片段，如Fab或(Fab’)2片段；抗体重链；抗体轻链。在具体的实施方式中，所述抗体具有：SEQ ID NO:9所示的轻链；和/或SEQ ID NO:10所示的重链。
本发明的多肽
为提供一种调控、甚至逆转细胞坏死的物质手段，本发明提供了这样一种多肽，所述多肽能够结合能引起细胞程序性坏死的上述自身抗体而不结合TNFR1，从而能抑制细胞的坏死，进而能治疗或减轻炎症。
在具体的实施方式中，本发明提供一种衍生自野生型TNFα的抑制细胞坏死的多肽，所述多肽包含10-200个氨基酸残基(优选10-180个、更优选10-160个氨基酸残基)和序列QLVVPSE。在具体的实施方式中，所述野生型TNFα是氨基酸序列如SEQ ID NO:1所示的人源TNFα。
在优选的实施方式中，所述多肽的氨基酸序列选自下组：SEQ ID NO:2-8。在进一步优选的实施方式中，本发明的多肽是可溶性多肽，从而能够提供优异的成药性能。
在具体的实施方式中，所述多肽的氨基酸序列如SEQ ID NO:2、3或4所示。
药物组合物
在本发明的多肽的基础上，本发明进一步提供一种抑制细胞坏死的药物组合物，所述药物组合物包含治疗有效量的本发明多肽和药学上可接受的载体。
本文所用的术语“有效量”或“治疗有效量”是指可对人和/或动物产生功能或活性 的且可被人和/或动物所接受的用量。
本文所用的术语“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物组合物含有安全有效量的本发明多肽作为活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型可以为固体形式或溶液形式，优选溶液形式，例如注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。根据治疗状况的要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明所述的药学上可接受的载体、活性成分的有效量以及给药方式都是本领域的普通技术人员所熟知的。
鉴于其功能，本领域技术人员知晓本发明的多肽以及药物组合物能够抑制细胞坏死，进而治疗细胞坏死相关的疾病，包括但不限于炎性疾病。在具体的实施方式中，所述炎性疾病包括但不限于类风湿性关节炎、克罗恩病、银屑病、脓毒症。
基于本发明的多肽以及药物组合物，本发明还提供了抑制细胞坏死或治疗细胞坏死相关疾病，例如炎性疾病的方法，包括给予有此需要的对象本发明的多肽或包含本发明多肽的药物组合物。
本发明的主要优点在于：
1.本发明首次发现能够中和引起细胞的程序性坏死的自身抗体的多肽；
2.本发明的成果具备显著的临床意义；
3.本发明的多肽能够治疗、缓解或减轻炎症反应。
4.本发明的多肽不与TNF受体结合，所以不影响TNF的正常功能；不像目前临床用的抗TNF单抗或TNF游离受体的抗炎症疗法因抑制TNF功能而引起结核感染及肿瘤发生等副作用。
下面结合具体实施例，进一步详述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按 照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。本发明实施例中所用的实验材料如无特殊说明均可从市售渠道获得。
材料与方法
患者
患者由风湿病诊所或ICU病房募集，所有样品保存在-80℃直至分析。作为治疗性关节穿刺术的一部分，滑液从炎性疾病患者的膝关节吸取。根据1987美国风湿病学院或2010ACR/EULAR分类标准定义类风湿性关节炎；其它炎性关节病基于临床和X光照相标准诊断。所有风湿病患者呈风湿病因子血清反应阳性，具有中等疾病活性。脓毒症患者按照1992年美国胸科医师协会确立标准诊断。在捐献者知情同意下，完全按照国家和制度伦理要求收集获得的样品和/或数据。
细胞系、TNF和多肽
小鼠纤维肉瘤L929细胞和人淋巴细胞Jurkat A3细胞购自美国模式培养物保藏所(ATCC,Manassas,VA)。人C28I2软骨细胞由Mary B.Goldring博士提供。.人SaOs-2成骨细胞购自Sigma。TNF购自Immunotools(德国)。
凋亡和坏死试验
在含或不含20μM z-VAD-FMK(R&D Systems,明尼阿波利斯,明尼苏达州)或20μM Necrotatin-1(PeproTech,Rocky Hill,新泽西州)的TNF(Immunotools,Friesoythe,德国)或TNF肽存在下，温育细胞过夜(或温育图中所示的时间)。在一些实验中，TNF与细胞和所示浓度的单克隆抗体或滑液共培养。根据生产商手册的指导，利用活/死细胞染色试剂盒(Invitrogen,Paisley,英国)染色细胞，利用cytofix/cytoperm固定/透化溶液试剂盒(BD Pharmingen,牛津,英国)固定。然后利用FITC-偶联的抗-胱冬酶-3抗体(Cell Signaling Technology,Danvers,马萨诸塞州,美国)作胞内染色。用CyAn流式细胞仪(Beckman Coulter,Fullerton,加利福尼亚州)获取细胞，利用Flowjo(Tree Star Inc.Ashland,俄勒冈州)分析数据。
ELISA
筛选单克隆抗体/抑制试验
4℃，用2μg/ml TNF包被ELISA平板过夜，或在37℃包被2小时，然后在37℃与单克隆抗体温育1小时。与HRP偶联的第二抗体用于检测反应。对于抑制试验，将TNFR1或滑液与单克隆抗体一起加入。
TNF结合TNFR1
如下所示通过ELISA测试TNFR1与TNF的结合：4℃，用1.5μg/ml TNF或mTNF-HA包被ELISA平板过夜，或在37℃包被2小时。37℃与TNFR1(1μg/ml)温育2小时后，与抗-TNFR1或抗-HA抗体再温育两小时，加入与HRP偶联的第二抗- 小鼠IgG抗体，30分钟，然后加入其底物以供检测。显色，并利用Wallace Victor2 1420多标记计数器(PerkinElmer,Massachusetts,马萨诸塞州,美国)在OD 450nm下检测。
L929细胞免疫荧光显微术
室温下，将20ng/ml的TNF与2μg/ml的mAb M26或244-12温育1小时。TNF/M26或TNF/244-12的混合物在冰上与生长在盖玻片上的L929细胞再温育15分钟。用4％低聚甲醛(PBS配制)固定细胞10分钟，用含0.5％BSA和0.1％冷水鱼明胶的磷酸盐缓冲液阻断15分钟，然后再与家兔抗-TNFR1抗体(Abcam)温育1小时。用PBS洗涤细胞，然后与偶联于Alexa Fluor 488或Alexa Fluor 568(Invitrogen)的相关第二抗体温育。然后用Gelvatol/DABCO(Sigma-Aldrich)安装PBS-洗涤的样本。用DAPI(Sigma-Aldrich)复染DNA。利用Nikon Eclipse 80i，以60x通过荧光显微术分析所有样品。利用Hamamatsu照相机，以NIS-Elements AR3.0软件获取图像。
多肽及蛋白合成及检测方法：
采用常规方法，合成SEQ ID NO:2-4所示氨基酸序列相应的编码核苷酸序列，然后利用大肠杆菌表达并纯化。纯化的产物经SDS电泳及质谱仪(Bruker Daltonics Ultraflex TOF/TOF mass spectrometer)确认。
SEQ ID NO:5-8所示氨基酸序列及其修饰序列，例如N端或C端生物素化(如果需要的话)是根据Fmoc策略在APEX396自动多肽合成仪上合成。用质谱仪(Bruker Daltonics Ultraflex TOF/TOF mass spectrometer)确认合成的多肽。
实施例
实施例1.单克隆抗体244-12的制备及其结合表位的鉴定
发明人通过常规方法制备了33株单克隆抗体，并检验所制得的单克隆抗体与TNF的结合情况，其中，ELISA试验证明单克隆抗体244-12结合TNF最强。
随后，发明人鉴定了单克隆抗体244-12的结合表位，为QLVVPSE。
实施例2.单克隆抗体244-12将TNF相关凋亡转为程序性坏死
发明人研究了单克隆抗体(mAb)244-12对TNF功能的影响。
首先，图2中的共聚焦显微镜照片显示单克隆抗体244-12结合TNF并不影响TNF结合TNF受体。在共聚焦显微镜下，TNF受体染红色，244-12染绿色。其中，上图显示当有TNF存在时，绿色与红色重叠(黄色)，说明244-12与TNF及TNF受体重叠；下图显示没有TNF时，只有红色，说明244-12不能直接结合到细胞表面。
随后，发明人观察到TNF刺激L929细胞引起细胞凋亡(活性胱冬酶-3表达)，而当加入单抗244-12时，细胞凋亡受到抑制(活性胱冬酶-3抑制)。图3显示单克隆抗体244-12在两个细胞株，L929细胞(小鼠成纤维细胞瘤细胞，上图)和C28I2细胞(软骨细胞，下图)都阻断TNF引起的细胞凋亡(即，活性胱冬酶-3表达)。
然而，出乎意料的是，当单克隆抗体244-12加入TNF与L929细胞中时，虽然 凋亡被抑制，但是细胞却发生了坏死。图4显示抗体244-12与TNF引起L929细胞坏死。其中，TNF引起细胞凋亡(上图右)；加入对照抗体M26(阻断TNF结合受体)，L929细胞存活(中图右)；加入244-12与TNF，L929细胞坏死(下图右)。
发明人经过进一步研究证明，244-12+TNF引起的坏死是通过信号传导的。图5显示本发明的抗体244-12+TNF引起的坏死可以被Nec-1抑制。其中，244-12+TNF加入Nec-1后，细胞坏死又转变成细胞凋亡(上图右)。说明细胞坏死是通过信号传导，即程序性坏死。
实施例3.类风湿性关节炎患者滑液中的自身抗体触发程序性坏死
我们意外的发现，在一些未经治疗的风湿性关节炎或骨关节病的患者关节液中存在着很高的抗体(图6A)。这些抗体可以与244-12竞争抗原结合位点(图6B)。这些含有抗体的关节液能够引起细胞程序性坏死(图6C)。
其中，图6A显示在一些关节炎病人的关节液存在抗TNF的自身抗体。患者SF8的关节液含有很高的自身抗体。6B显示自身抗体与244-12竞争TNF结合点。在244-12与TNF结合试验(ELISA)中加入SF8关节液，则244-12与TNF结合被抑制。6C显示SF8关节液在TNF存在下引起程序性坏死。TNF引起L929细胞凋亡(上图右)，当加入SF8关节液，细胞转变成程序性坏死(中图右)。而对照关节液SF4则不引起坏死(下图右)。
发明人又观察了24位脓毒症患者，14位患者检测出与244-12竞争抗原结合位点的抗体，另外10位患者不含类似的抗体。14位含有与244-12竞争抗原结合位点的抗体的患者虽经精心护理和治疗，仍然全部死亡。而其它10位患者有5位死亡，另外5位在2-3周后痊愈出院。死亡患者中抗体水平非常显著地高于病愈组(p<0.01)(图7)。
实施例4.
本发明人进一步研究了特异性结合其它TNF分子及片段的抗体，并没有发现这些抗体具有将TNF相关凋亡转为程序性坏死的效果；
此外，本发明人以全长TNF进行筛选，发现仅特异性结合QLVVPSE的抗体具有将TNF相关凋亡转为程序性坏死的效果。
实施例5.本发明多肽的制备
如“材料与方法”中“多肽及蛋白合成及检测方法”所述制备如下所示的本发明多肽并检测、确认：
SEQ ID NO:2：VRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRANALLANGVELR DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIAVFHQTKVNLL SAIKSPCQRE TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL；
P11-17：QLQWLNRRAN ALLANGVELR DNQLVVPSEG LYLIYSQVLFKGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE(SEQ ID NO:3)；
P11-17(突变型，SY突变)：QLQWLNRRAN ALLANGVELR DNQLVVPSEGLYLIYSQVLF 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(SEQ ID NO:8)。
实施例6.本发明多肽中和促细胞坏死抗体的活性
本发明人进一步研究了本发明多肽与促细胞坏死抗体的结合，进而竞争抑制TNF与促细胞坏死抗体结合的能力以及本发明多肽抑制细胞坏死的能力。
如图9所示，本发明多肽P11-17(SEQ ID NO:3)既能够结合纯化的促细胞坏死抗体(244-12)，又能够结合存在于关节炎病人关节液中的促细胞坏死抗体(样品SF024、样品SF045)。但与不含自身抗体的对照病人关节液反应为阴性(SF010)。酶标板用2μg/ml P11-17包被，BSA封闭后，分别与244-12、SF024、SF045、SF010室温孵育1小时，然后用辣根过氧化物歧化酶标记的抗鼠或人IgG的二抗进行识别。
图10显示，本发明的多肽P11-17和SEQ ID NO:2都可以有效抑制细胞坏死。不论细胞坏死是由纯化的促细胞坏死抗体(244-12)引起的，还是由病人关节液中(SF002、SF045)的促细胞坏死抗体引起的。
图11显示，本发明的多肽P12-10、P13-1、P13-2及P13-3能够竞争抑制TNF与单抗244-12的结合。P12-10、P13-1、P13-2及P13-3显著抑制TNF与单抗244-12的结合。方法：TNF包被酶联板，每孔分别加入所示多肽，然后加入单抗244-12。如果多肽与244-12结合，则抑制244-12与TNF结合。
本发明的多肽与促细胞坏死抗体的结合能力以及本发明多肽抑制细胞坏死的能力总结于下表。
Figure PCTCN2016107291-appb-000001
Figure PCTCN2016107291-appb-000002
讨论
基于本发明的发现，本发明人提出炎症的新治疗策略。程序性坏死的机制涉及自身抗体和TNF。程序性坏死是炎症的原因之一。目前炎性疾病的治疗策略之一是通过抑制TNF来阻断TNF。该策略对于抑制炎症有效，但可能导致危及生命的副作用，例如TB或淋巴瘤。本发明人提出通过本发明多肽阻断自身抗体(不结合TNFR1)的替代方法。该策略的结果可能是在降低炎性负荷的同时抑制程序性坏死，但将TNF维持在原位，从而阻止肿瘤和严重的感染，例如TB。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
参考文献
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8.Galluzzi,L.,and Kroemer,G.2008.Necroptosis:a specialized pathway of programmed necrosis.Cell 135:1161-1163.
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Claims (12)

  1. 一种抑制细胞坏死的多肽，所述多肽衍生自野生型TNFα，并且包含10-200个氨基酸残基和序列QLVVPSE，其中，所述多肽不结合TNFR1但与TNF竞争结合促细胞坏死抗体。
  2. 如权利要求1所述的多肽，其特征在于，所述野生型TNFα是氨基酸序列如SEQ ID NO:1所示的人源TNFα。
  3. 如权利要求1所述的多肽，其特征在于，所述促细胞坏死抗体具有：SEQ ID NO:9所示的轻链；和/或SEQ ID NO:10所示的重链。
  4. 如权利要求1-3中任一项所述的多肽，其特征在于，所述多肽的氨基酸序列选自下组：SEQ ID NO:2-8。
  5. 如权利要求4所述的多肽，其特征在于，所述多肽的氨基酸序列如SEQ ID NO:2、3或4所示。
  6. 如权利要求1-5中任一项所述的多肽的用途，所述多肽用于制备抑制细胞坏死或治疗细胞坏死相关疾病的药物。
  7. 如权利要求6所述的用途，其特征在于，所述细胞坏死相关疾病是炎性疾病。
  8. 如权利要求7所述的用途，其特征在于，所述炎性疾病包括但不限于类风湿性关节炎、克罗恩病、银屑病、脓毒症。
  9. 一种抑制细胞坏死的药物组合物，所述药物组合物包含治疗有效量的权利要求1-5中任一项所述的多肽和药学上可接受的载体。
  10. 如权利要求9所述的药物组合物，其特征在于，所述药物组合物是静脉内给药的注射剂。
  11. 一种处理患者血液的透析装置，所述透析装置包含权利要求1-5中任一项所述的多肽。
  12. 一种抑制或中和患者血液中的促细胞坏死抗体的方法，包括利用权利要求1-5中任一项所述的多肽抑制或中和所述患者血液中的促细胞坏死抗体。
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