WO2013125687A1 - 炎症疾患治療剤 - Google Patents
炎症疾患治療剤 Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Definitions
- the present invention relates to a novel inflammatory disease therapeutic agent comprising a monoclonal antibody or an antigen-binding fragment thereof as an active ingredient.
- DAMPs damage-associated molecular pattern molecules
- sepsis is caused by diseases such as infections, cirrhosis, renal failure, diabetes, abnormal labor, and injuries and diseases such as indwelling catheters, infusion devices, dialysis, and tracheostomy.
- infections such as infections, cirrhosis, renal failure, diabetes, abnormal labor, and injuries and diseases such as indwelling catheters, infusion devices, dialysis, and tracheostomy.
- indwelling catheters such as indwelling catheters, infusion devices, dialysis, and tracheostomy.
- a severe systemic infection in which bacteria constantly or intermittently enter the blood.
- Sepsis is not broadly limited to host invasion by microorganisms, but is a clinical symptom of infection, ie (1) body temperature> 38 ° C to ⁇ 36 ° C; (2) heart rate> 90 beats / minute; 3) Respiratory rate> 20 breaths / min to PaCO2 ⁇ 32 mmHg; (4) White blood cell count> 12000 / ⁇ l, ⁇ 4000 / ⁇ l, or rod-shaped neutrophil> 10% Yes. Recently, a pathological condition showing such symptoms is called systemic inflammatory response syndrome (SIRS) (Non-patent Document 1: Crit. Care Med., 20: 864-874, 1992).
- SIRS systemic inflammatory response syndrome
- sepsis includes organ dysfunction, hypoperfusion, or hypotension, severe sepsis, lactic acidosis, oliguria, septic shock complicated by disturbance of consciousness, etc.
- Non-Patent Document 2 Chest, 101: 1644-). 1655, 1992).
- the pathological condition leads to disseminated intravascular coagulation syndrome (DIC), adult respiratory distress syndrome (ARDS), and multiple organ dysfunction (MODS) from severe sepsis and septic shock.
- DIC intravascular coagulation syndrome
- ARDS adult respiratory distress syndrome
- MODS multiple organ dysfunction
- sepsis caused by these bacteria has decreased due to the development of various powerful antibiotics, but sepsis due to new pathogens that have acquired resistance genes such as MRSA is increasing.
- the incidence of sepsis tends to increase in large hospitals, reflecting the use of indwelling catheters and infusion devices, or the general use of invasive procedures and operations such as artificial dialysis and tracheostomy.
- the incidence of neonates, the elderly, and hematopoietic tumor patients with low resistance to infection, and the incidence of sepsis in patients who have been treated with corticosteroids or anticancer drugs and have weakened immunity has increased. .
- sepsis continues to increase against medical advances.
- the causative bacteria are detected and their antibiotic susceptibility is measured, and then the optimal antibiotic is administered to the causative bacteria, and fluid replacement, electrolyte correction, low protein
- methods for improving the defense power of the host such as improving blood glucose, supplementing nutrition, and administering ⁇ -globulin.
- treatments such as removal of lesions by surgery, improvement of circulatory disturbance, administration of opsonin activator, administration of corticosteroids, administration of synthetic protease inhibitors have been performed.
- the symptoms of the underlying disease and the symptoms of sepsis overlap, it is often difficult to make a clear diagnosis and it is difficult to prevent or treat sepsis.
- prevention and treatment of septic shock is difficult, and sepsis is still a disease that causes a high mortality rate.
- Septic mortality varies from 10% to 20% to 50% depending on reports. Septic shock is associated with 40% of septic cases, and the prognosis of shock cases is poor, with 77-90% mortality reported. Therefore, prevention of septic shock is the primary goal of treatment. In particular, if the change that occurs in the initial stage of shock can be grasped and early diagnosis can be made, early treatment is possible and prognosis can be expected. However, until now, the clinical effects of many anti-shock drugs and treatments that have been considered effective have been examined, but few of them were judged effective.
- TNF tumor necrosis factor
- interleukin produced excessively from monocytes, macrophages, vascular endothelial cells, etc. by infection stimuli (bacteria themselves, endotoxins, cell wall components of peptidoglycan / tycoic acid complex, and exotoxins). It has been thought to develop due to inflammatory cytokines such as 1 (IL-1), interleukin 6 (IL-6) and interleukin 8 (IL-8). Excessive inflammatory cytokines also release eicosanoids and lipid activators of platelet activating factor, and their interaction activates the cytokine network and amplifies the inflammatory response.
- IL-1 inflammatory cytokines
- IL-6 interleukin 6
- IL-8 interleukin 8
- the complement system, coagulation system, kinin system, and adrenocorticotropic hormone / endorphin system are also activated, and a systemic inflammatory reaction based on vascular endothelial damage is induced.
- a systemic inflammatory reaction based on vascular endothelial damage is induced.
- the involvement of granulocyte-derived elastase and active oxygen has been shown in the expression of circulatory and tissue disorders.
- Non-patent Document 3 Lancet, 351: 929-933, 1998, JAMA, 271: 1836-1843, 1994.
- ischemia-reperfusion injury has been reported as another inflammatory disease in which the damage-related molecular pattern molecule group is involved. It has also been reported that acute renal failure often occurs due to renal ischemia reperfusion injury. However, there are few effective treatments for such pathologies.
- the inventors of the present invention now have a monoclonal antibody or antigen-binding fragment thereof that can specifically recognize a peptide consisting of the amino acid sequence represented by SSVLYGGPPSAA (SEQ ID NO: 1), which is an excellent treatment for inflammatory diseases involving histones. I found out that there was an effect.
- the present invention is based on such knowledge. Therefore, an object of the present invention is to provide a novel therapeutic agent for inflammatory diseases.
- a monoclonal antibody or an antigen-binding fragment that binds to a peptide consisting of the amino acid sequence represented by SSVLYGGPPSAA (SEQ ID NO: 1) or a conjugate of the peptide and a pharmaceutically acceptable carrier.
- SSVLYGGPPSAA SEQ ID NO: 1
- An inflammatory disease therapeutic agent is provided.
- the monoclonal antibody or antigen-binding fragment of the present invention can effectively treat inflammatory diseases.
- the result of the isotype identification test of the monoclonal antibody of the present invention (hereinafter also referred to as “SSVmAb”) is shown.
- the result of the test which compared the binding affinity with respect to the histone H1, histone H2A, H2B, H3, or H4 regarding the monoclonal antibody (SSVmAb) of this invention is shown.
- the result of the mixed lymphocyte reaction (MLR) test using the monoclonal antibody (SSV mAb) and 16G9 mAb of the present invention is shown.
- the result of having compared the reactivity with respect to the T cell of the monoclonal antibody (SSVmAb) of this invention and 16G9mAb by flow cytometry is shown.
- FIG. 9A is a graph of histone H1 concentration in a blood sample.
- FIG. 9B is a graph of histone H1 concentration in the lung.
- FIG. 10A is a graph of histone H3 concentration in a blood sample.
- FIG. 10B is a graph of histone H3 concentration in the lung.
- FIG. 11A is a graph of histone H4 concentration in a blood sample.
- FIG. 11B is a graph of histone H4 concentration in the lung.
- FIG. 3 is a micrograph of lung tissue sections obtained and stained from healthy rats and rats of SSV mAb administration group and control group after completion of the test of Test Example 8.
- FIG. 12A is a photograph of a healthy rat.
- FIG. 12B is a photograph of the SSV mAb administration group.
- FIG. 12C is a photograph of the control group.
- FIG. 14A shows the TNF- ⁇ concentration in the blood sample.
- FIG. 14B shows the IL-1 ⁇ concentration in the blood sample.
- FIG. 14C shows IL-6 concentration in the blood sample.
- FIG. 14D shows the IL-10 concentration in the blood sample.
- the hybridoma of the present invention Mouse-Mouse hybridoma SSV-C93-3, has an original deposit date of August 17, 2010, and is based on the Patent Microorganism Depositary Center for Product Evaluation Technology (address: Kazusa, Kisarazu, Chiba, Japan). 2-5-8 Kamashita Biotechnology Headquarters) is deposited under the accession number NITE BP-972.
- the monoclonal antibody of the present invention or an antigen-binding fragment thereof binds to a peptide comprising the amino acid sequence represented by SSVLYGGPPSAA (SEQ ID NO: 1) or a conjugate of the peptide and a pharmaceutically acceptable carrier. It has one feature. It is a surprising fact that such a monoclonal antibody or an antigen-binding fragment thereof has an excellent therapeutic effect on inflammatory diseases.
- the inflammatory disease of the present invention is preferably an inflammatory disease involving histone, more preferably an acute inflammatory disease, more preferably sepsis, renal ischemia-reperfusion injury or renal failure, more preferably sepsis, renal ischemia. Reperfusion injury or acute renal failure, even more preferably sepsis.
- the antibody or antigen-binding fragment thereof is against a peptide consisting of the amino acid sequence represented by SSVLYGGPPSAA (SEQ ID NO: 1) or a pharmaceutically acceptable carrier.
- the antibody or antigen-binding fragment thereof specifically binds to histone H1, histone H3, and histone H4.
- the antibody or antigen-binding fragment thereof having such binding ability can be particularly advantageously used in the treatment of inflammatory diseases, as indicated by 8-2 in Examples described later.
- the antibody or antigen-binding fragment thereof has a higher binding affinity for the core histone than the binding affinity for the linker histone (histone H1).
- the core histone is histone H2A, H2B, H3 or H4, more preferably H2A, H3 or H4.
- the antibody of the present invention or the antibody-binding fragment thereof can contain a heavy chain and / or a light chain.
- Each light and heavy chain can have a variable region at its N-terminus, where each variable region alternates between 4 framework regions (FR) and 3 complementarity determining regions (CDRs). May be included.
- the residues in the variable region are conventionally numbered according to the system envisaged by Kabat et al. This system is described in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Departement of Health and Human Services, NIH, USA. This numbering system is used herein unless otherwise indicated. Such numbering based on the method of Kabat et al. Can be easily performed using, for example, the website http://www.bioinf.org.uk/abysis/tools/analyze.cgi .
- the Kabat residue nomenclature does not necessarily match directly with the linear numbering of amino acid residues.
- the actual linear amino acid sequence has fewer or additional amino acids than in strict Kabat numbering, corresponding to its truncation or insertion, whether it is a structural element, framework or CDR of the basic variable region structure Sometimes.
- the exact Kabat numbering of the residues will be determined for a given antibody by aligning the “standard” Kabat numbered sequence with homologous residues in the antibody sequence.
- the light chain variable region of the antibody of the present invention or the antigen-binding fragment thereof comprises a CDR1 consisting of an amino acid sequence represented by RASSSVSYMH (SEQ ID NO: 2), an amino acid sequence represented by ATSNLAS (SEQ ID NO: 3).
- CDR2 and CDR3 consisting of the amino acid sequence represented by QQWSSNPWT (SEQ ID NO: 4).
- the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 6 from No. 23 to No. 128.
- the heavy chain variable region of the antibody of the present invention or an antigen-binding fragment thereof is CDR1, NINPYYGSTSYNQKFKG (SEQ ID NO: 8) consisting of the amino acid sequence represented by GYNMN (SEQ ID NO: 7). It comprises CDR2 comprising the sequence and CDR3 comprising the amino acid sequence represented by SPYYSNYWRYFDY (SEQ ID NO: 9).
- the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 11 from No. 20 to No. 141.
- the antibody of the present invention or an antigen-binding fragment thereof is CDR1 consisting of an amino acid sequence represented by RASSSVSYMH (SEQ ID NO: 2), an amino acid sequence represented by ATSNLAS (SEQ ID NO: 3). And a light chain variable region comprising CDR3 comprising the amino acid sequence represented by QQWSSNPWT (SEQ ID NO: 4), and CDR1, NINPYYGSTSYNQKFGKG (SEQ ID NO: 8) comprising the amino acid sequence represented by GYNMN (SEQ ID NO: 7). And a heavy chain variable region comprising a heavy chain variable region comprising CDR3 comprising an amino acid sequence represented by SPYYSNYWRYFDY (SEQ ID NO: 9).
- the antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 6 from 23rd to 128th amino acid sequence, And a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 11 from No. 20 to No. 141.
- the monoclonal antibody or antigen-binding fragment thereof can down-regulate ATP synthase activity.
- the ATP synthase is a mitochondrial ATP synthase.
- the monoclonal antibody of the present invention is preferably a chimeric antibody, a humanized antibody or a fully human antibody.
- These antibodies include, for example, Morrison, SL, Oi, VT, “immunoglobulin genes” Academic Press (London), 260-274 (1989), Roguska, ML et. Al., Humanization of murine monoclonal antibodies through variable domain resurfacing, Proc . Natl. Acad. Sci. USA, 91, 969-973 (1994), Tomizuka, K. et.al. Functional expression and germline transmission of a human chromosome fragment in chimaeric mice, Nature Genet., 16, 133-143 ( 1997), Winter, G.
- the antigen-binding fragment is preferably Fab, Fab ′, (Fab ′) 2 , Fv or scFv.
- the monoclonal antibody or the antigen-binding fragment thereof is a hybridoma produced by Mouse-Mouse® hybridoma® SSV-C93-3.
- the monoclonal antibody or antigen-binding fragment thereof and hybridoma of the present invention can be produced, for example, as follows. That is, first, the hybridoma of the present invention uses a peptide consisting of the amino acid sequence represented by SSVLYGGPPSAA (SEQ ID NO: 1) or a conjugate of the peptide and a pharmaceutically acceptable carrier as a sensitizing antigen.
- the mammalian plasma cells (immune cells) immunized with the above can be fused with mammalian myeloma cells, and the resulting hybridomas are cloned and selected from the hybridomas.
- the monoclonal antibody of this invention can be obtained by culture
- a general administration method in the art can be used as a method for immunizing a mammal. Specifically, intraperitoneal injection, intrasplenic injection, intramuscular injection, subcutaneous injection, intradermal injection, oral administration, Although transmucosal administration and transdermal administration can be mentioned, intraperitoneal injection and intrasplenic injection are preferred.
- the interval between administrations of the sensitizing antigen is appropriately determined according to the dose of the sensitizing antigen, the type of mammal, and the like. For example, the interval can be several times during one month.
- the mammal to be immunized is not particularly limited, but is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion, and examples thereof include mice, rats, hamsters, etc. Is a mouse.
- spleen cells are preferably used as immune cells.
- myeloma cells used in the present invention include P3 (P3X63Ag8.653) (J. Immunol., 123, 1548, 1978), p3-U1 (Current Topics in Micro-biology and Immunology, 81, 1-7,1978). ), NS-1 (Eur. J. Immunol., 6, 511-519, 1976), MPC-11 (Cell, 8, 405-415, 1976), Sp2 / 0-Ag14 (Nature, 276, 269-270) , 1978), FO (J. Immunol. Meth., 35, 1-21, 1980), S194 (J. Exp. Med., 148, 313-323, 1978), and R210 (Nature, 277, 131-133). , 1979), and the like, preferably P3 or p3-U1, more preferably P3.
- Cell fusion between immune cells and myeloma cells can be performed, for example, according to the method of Milstein et al. (Methods Enzymol., 73, 3-46, 1981). Specifically, cell fusion can be performed, for example, by mixing immune cells and myeloma cells in a medium in the presence of a fusion promoter. In cell fusion, hybridomas can be generated by repeating the operation of adding a medium and centrifuging appropriately.
- Examples of the medium used for cell fusion include media usually used in cell fusion, such as RPMI-1640 medium and MEM medium. Moreover, serum supplements such as fetal bovine serum (FBS) can be used in combination as appropriate.
- the cell fusion temperature is preferably 25 to 37 ° C, more preferably 30 to 37 ° C.
- the mixing ratio of myeloma cells and immune cells is preferably about 1: 1 to 1:10.
- the fusion promoter examples include polyethylene glycol (PEG) and Sendai virus (HVJ), and PEG is preferred.
- PEG polyethylene glycol
- HVJ Sendai virus
- the molecular weight of PEG can be appropriately selected.
- the average molecular weight can be about 1,000 to 6,000.
- the concentration of PEG in the medium is preferably about 30 to 60% (W / V).
- auxiliary agents such as a dimethyl sulfoxide, can be suitably added to a culture medium if desired.
- the hybridoma obtained by cell fusion is cultured in a normal selective medium such as HAT medium, and is expressed by, for example, SSVLYGGPPSAA (SEQ ID NO: 1) using a normal limiting dilution method. It can be carried out by screening as an indicator such as an antibody titer against a peptide comprising an amino acid sequence or a conjugate of the peptide and a pharmaceutically acceptable carrier.
- the culture period in the HAT medium is a time sufficient for cells other than the target hybridoma (unfused cells) to die, and can usually be several days to several weeks.
- the hybridoma of the present invention thus obtained can be subcultured in a normal medium and can be stored for a long time in liquid nitrogen.
- the method for recovering the monoclonal antibody or antibody-binding fragment thereof of the present invention is, for example, a method of culturing a hybridoma according to a conventional method to obtain a monoclonal antibody or the like from the culture supernatant, or a hybridoma compatible with this.
- Examples thereof include a method for obtaining a monoclonal antibody from ascites by administering to a mammal and proliferating it.
- the former method is preferable for obtaining high-purity antibodies, while the latter method is preferable for producing antibodies in large quantities.
- the monoclonal antibody or the antibody-binding fragment thereof of the present invention can be purified with high purity by a method such as salting out, gel filtration, affinity chromatography.
- the monoclonal antibody or antigen-binding fragment thereof of the present invention has an excellent therapeutic effect on inflammatory diseases involving histones. Therefore, according to another aspect of the present invention, there is provided use of the monoclonal antibody of the present invention in the manufacture of a therapeutic agent for inflammatory diseases.
- the inflammatory disease is preferably an inflammatory disease involving histone, more preferably an acute inflammatory disease, more preferably sepsis, renal ischemia-reperfusion injury or renal failure, more preferably Sepsis, renal ischemia reperfusion injury or acute renal failure, and even more preferably sepsis.
- the monoclonal antibody or antigen-binding fragment thereof of the present invention may be used as it is, or may be used as a pharmaceutical composition together with pharmaceutically acceptable additives. Therefore, according to one aspect of the present invention, there is provided a pharmaceutical preparation for treating inflammatory diseases comprising the monoclonal antibody of the present invention or an antigen-binding fragment thereof.
- the therapeutic agent for sepsis of the present invention can be prepared by, for example, dissolving the monoclonal antibody of the present invention in physiological saline for injection, distilled water for injection, buffer solution for injection or the like.
- the immunosuppressive composition of the present invention includes an appropriate solvent, solubilizer, preservative, stabilizer, emulsifier, suspending agent, soothing agent, isotonic agent, buffer, excipient, A sticking agent, a coloring agent, a known carrier (various liposomes, polyamino acid carrier, synthetic polymer, natural polymer, etc.) and the like can be contained.
- a method for treating inflammatory diseases comprising administering an effective amount of the monoclonal antibody of the present invention or an antigen-binding fragment thereof to a subject.
- treatment means improving an established pathological condition.
- a method for reducing the risk of developing an inflammatory disease in a subject comprising administering an effective amount of the monoclonal antibody of the present invention or an antigen-binding fragment thereof to the subject. A method is provided comprising.
- the inflammatory disease is preferably an inflammatory disease involving histone, more preferably an acute inflammatory disease, more preferably sepsis, renal ischemia-reperfusion injury or renal failure, more preferably Sepsis, renal ischemia reperfusion injury or acute renal failure, and even more preferably sepsis.
- the monoclonal antibody of the present invention or the antigen binding breakage thereof can exert a remarkable immunosuppressive action. It is a surprising fact that the monoclonal antibody that can be used for the treatment of inflammatory diseases or its antigen binding can also cause an immunosuppressive action. Therefore, according to one embodiment, the monoclonal antibody of the present invention or an antigen binding fragment thereof is used as an immunosuppressant.
- the subject is preferably a mammal, more preferably a human.
- the monoclonal antibody or antigen-binding fragment thereof of the present invention may be administered to a mammal simultaneously or sequentially in combination with other drugs used for inflammatory diseases.
- the monoclonal antibody or antigen-binding fragment thereof of the present invention can be administered systemically or locally.
- Specific administration methods include infusion, intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection. , Oral administration, transmucosal administration, transdermal administration and the like.
- the effective amount of the monoclonal antibody or antigen-binding fragment of the present invention is not particularly limited, and is appropriately determined by those skilled in the art according to the type, nature, sex, age, symptoms, etc. of the subject.
- the effective amount includes 0.05 to 40 mg / kg body weight / day, preferably 2 to 10 mg / kg body weight / day once or several times.
- Example 1 Production of monoclonal antibody (SSVmAb) Production of antigenic substance
- SSVmAb monoclonal antibody
- antigenic substance a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and a conjugate of KLH were used.
- the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 was synthesized by Fmoc peptide solid phase synthesis method (manufacturing apparatus; manufactured by ABI430 type Applied Biosystems). Furthermore, the conjugate of the above peptide and KLH (manufactured by SIGMA) consists of about 5 mg of the above peptide, about 20 mg of KLH, and 30 ⁇ g of glutaraldehyde (Katayama Chemical Co., Ltd.) in a phosphate buffer (pH 8.0) at room temperature for about 6 hours. Synthesized with stirring.
- Hybridoma production 0.8 mL (antigen substance concentration: 0.5 mg / mL) of a solution in which an antigen substance is dissolved in immunized PBS and 0.8 mL of Freud complete adjuvant (manufactured by Wako Pure Chemical Industries, Ltd.) are mixed and a suspension ( Antigen concentration: 0.25 mg / mL).
- 0.2 mL of this suspension was intraperitoneally administered to BALB / c mice. Furthermore, this suspension was administered to mice in the same amount every 2 weeks.
- 0.2 mL of a solution in which the antigen was dissolved in PBS (antigen concentration: 600 to 1000 mg / mL) was finally administered intraperitoneally to the mouse.
- blood was collected from the fundus vein and the antibody titer was measured by ELISA.
- whole blood was collected, and the obtained blood was centrifuged (2000 rpm, 20 minutes) to obtain antiserum, which was used as a control antiserum for the following experiments. Further, after whole blood collection, the spleen was removed from the rat, and the obtained spleen cells were used for the following cell fusion.
- polyethylene glycol 4000 50% PBS solution
- IMDM IMDM
- GEBCO IMDM medium containing 15% FCS (manufactured by JRH BIOSCIENCES) at 37 ° C.
- the obtained suspension was dispensed into a 96-well culture plate by 100 mL and cultured in a 37 ° C./5% CO 2 incubator for 1 day. Further, 100 mL of HAT medium (HAT powder (HAT MEDIA SUPPLEMENT ( ⁇ 50), manufactured by SIGMA) dissolved in 10 mL of serum-free IMDM medium and diluted 50-fold with IMDM medium containing 10% FCS) was added, and 37 The cells were cultured in a ° C / 5% CO 2 incubator.
- HAT medium HAT medium
- HAT MEDIA SUPPLEMENT ⁇ 50
- the HAT medium is changed every 2 to 3 days. After 10 days, the HT medium (HT powder (HT MEDIA SUPPLEMENT, manufactured by SIGMA) is dissolved in 10 mL of serum-free IMDM medium and diluted 50-fold with IMDM medium containing 10% FCS. The culture was performed in a 37 ° C / 5% CO 2 incubator for 3 days. Thereafter, the medium (HT medium) was changed every 2-3 days. After confirming cell growth with a microscope, the culture supernatant (about 100 mL) was recovered. Using this culture supernatant, a hybridoma was screened by antibody titer measurement.
- HT powder HT MEDIA SUPPLEMENT, manufactured by SIGMA
- Hybridoma cell screening Antibody titer measurement Buffer solution (Bicarbonate buffer: 100 mM NaHCO 3 -NaOH, pH 9.2 to 9.5, peptide concentration: 1 ⁇ g / mL) containing the above antigenic substance (5 mg) is added to a 96-well flat bottom plate at 50 ⁇ L per well. Then, the coating was allowed to stand at room temperature for 2 hours. The plate was washed 3 times with a washing buffer (PBST), blocking buffer (3% skim milk 1% BSA, PBS) was added at 200 to 250 ⁇ L / well, reacted at 4 ° C. overnight, and then washed 3 times. .
- PBST washing buffer
- blocking buffer 3% skim milk 1% BSA, PBS
- the culture supernatant of the hybridoma was added at 100 ⁇ L / well and reacted at 37 ° C. for 4 hours or at 4 ° C. overnight.
- biotin-labeled anti-mouse IgG diluted 10,000 times with dilution buffer (10 mM Tris-HCl (pH 8.0), 0.9% (W / V) NaCl, 0.05% (W / V) Tween20) (Biotion-labeled anti-mouse IgG, SIGMA) was added at 50 ⁇ L / well and allowed to react at room temperature for 2 hours.
- alkaline phosphatase-labeled streptaridin (Streptaridin) diluted 1000-fold with a dilution buffer was added at 50 ⁇ L / well and allowed to react at room temperature for 1-2 hours. Thereafter, the plate was washed 6 times, and a fluorescent substrate buffer (Attophos substrate buffer, manufactured by Roche Diagnostics) was added at 50 ⁇ L / well to develop the color by shading the plate. The fluorescence intensity was measured with CytoFluorII (manufactured by Perceptive).
- IMDM medium containing 15% FCS 10% HCF (Hybridoma cloning factor, Origin) was added to obtain about 200 cells.
- HCF Hybridoma cloning factor
- the hybridoma Mouse-Mouse hybridoma SSV-C93-3 was cultured using RPMI medium containing 15% FCS (1 ⁇ 10 6 cells / mL). The hybridoma culture was then collected and filtered through a filter to remove dead cell debris. Next, ammonium sulfate was added to the culture supernatant to a final concentration of 40%, and the mixture was stirred at 40 ° C. for 1 hour. Next, centrifugation (3000 g, 30 minutes, 4 ° C.) was performed, and the supernatant was discarded to recover the precipitate. This precipitate was dissolved in 1/10 amount of PBS of the above culture supernatant and dialyzed overnight with PBS as an external solution.
- the precipitate was diluted 2-fold with 20 mM sodium phosphate buffer (pH 7.0) and added to a HiTrapNHS activation column together with 1 M Tris-HCl buffer. Further, the antibody was eluted with 0.1 M glycine-HCl solution (pH 2.7) and collected in a fraction tube.
- Test Example 1 Identification of SSVmAb Isotype
- SSVmAb monoclonal antibody
- An isotype identification test using Mouos Monoclonal Antibody Isotyping Reagents (Sigma) was performed. The result is as shown in FIG. 1, and IgG1 showed the highest value.
- Test Example 2 Confirmation of affinity of SSVmAb to core histone
- WO 2006/025580 discloses hybridoma 16G9 as an anti-H1 monoclonal antibody that can be used for immunosuppression and binds to a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
- a monoclonal antibody (16G9 mAb) produced by (Accession No. FERM BP-10413) has been reported.
- the antibody (16G9 mAb) described in WO2006 / 025580 was used as Reference Example 1, and the affinity for the antigen of the monoclonal antibody (SSVmAb) of Example 1 was compared.
- As the antigen histone H1, which is the antigen of Reference Example 1 (16G9mAb), and core histones H2A, H2B, H3, and H4, which are histone H1-like antigens, were selected.
- the affinity between histone H1 or core histone and SSV mAb was determined by ELISA.
- a 96 well microplate was coated with histone H1, H2A, H2B, H3 or H4. Each histone used was dissolved in 100 mM sodium carbonate buffer (pH 9.3). The plate was washed with PBS-tween20 (0.05%) and blocked with 3% skim milk and 1% BSA for 1 hour.
- SSVmAb 5 ⁇ g / mL was added to each well and incubated for 1 hour. Bound SSVmAb was detected using peroxidase (HRP) conjugated anti mouse IgG1 Ab (Sigma) and incubated for 1 hour.
- HRP peroxidase
- Bound SSVmAb was detected using ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-sulfonic acid)] substrate solution and absorbance at 405 nm using Multiskan Ascent (Thermo Fisher Scientific Inc., Waltham, MA) was measured.
- Test Example 3 MLR test Untreated DA rat-derived spleen lymphocytes (response cells) and spleen lymphocytes (stimulated cells) derived from LEW rats treated with mitomycin C (manufactured by Kyowa Hakko Kogyo Co., Ltd.) were used. Responder cells were adjusted to 5 ⁇ 10 5 cells / mL with 10% FCS-RPMI medium, and stimulator cells were adjusted to 8 ⁇ 10 6 cells / mL with 10% FCS-RPMI medium.
- the monoclonal antibody 16G9 mAb (0.1, 2) of Reference Example 1 was started at the start of mixed culture. 4 or 6 ⁇ g / mL / well) or the monoclonal antibody SSVmAb of Example 1 (4 ⁇ g / mL / well) was added and cultured at 37 ° C. under 5% CO 2 /95% air for 3.5 days or more. did.
- an immunosuppressant tacrolimus FK506: manufactured by Fujisawa Pharmaceutical Co., Ltd., 1 nM / well
- bromodeoxyuridine (BrdU) was added 15 hours before the end of the culture. Then, using BrdU Labeling & Detection Kit III (Roche Diagnostics), the proliferation ability of the cells treated with the immunosuppressive substance was measured using the amount of BrdU incorporated into the intracellular DNA as an index, It was used as an index of immunosuppression level.
- Example 1 SSVmAb
- the absorbance indicating BrdU incorporation was lower than that of Reference Example 1 (16G9 mAb) and tacrolimus (FK506).
- the average value of Example 1 was about 41% of that of Reference Example 1.
- Test Example 4 Confirmation of Reactivity to Monoclonal Antibody (SSVmAb) T Cells.
- SSVmAb Monoclonal Antibody
- the spleen was extracted from C57BL / 6 mice (5 weeks old, female, manufactured by Charles River Japan) by the following method to prepare whole splenocytes. . First, in a 5 ml culture dish (BD Bioscience FALCON 351007) containing 5 ml RPMI1640 medium (Sigma-Aldrich R-8758), loosen the spleen well with dissecting scissors and tweezers to suspend the spleen cells. After turbidity, it was transferred to a 15 ml centrifuge tube (BD Bioscience FALCON 352096).
- the 5 ml dish was washed several times with phosphate-buffered saline (PBS, Invitrogen 20012-027), and these were added to the previous cell suspension and allowed to stand. Collected in a centrifuge tube. Furthermore, 5 ml of RPMI1640 medium was again added to the residual insoluble spleen tissue, and after standing, only the supernatant was collected. The resulting suspension was combined with the above cell suspension, and centrifuged at 1,500 rpm for 5 min.
- PBS phosphate-buffered saline
- lysis buffer 150 mM NH 4 Cl / 15 mM NaHCO 3 /0.1 mM EDTA-Na 2 , pH 7.3
- PBS 1,500 rpm
- total T cells were purified from the splenocytes by magnetic sorting (MACS) using the Pan T Cell Isolation Kit, mouse (Miltenyi Biotec 130-090-861).
- MACS Magnetic Sorting
- spleen cells were suspended in MACS buffer (0.5% bovine serum albumin (BSA, Nacalai Tesque 08777-36) / PBS) at a rate of 5 x 10 7 cells / 200 ⁇ l, and 50 ⁇ l Biotin-antibody cocktail / 5 x 10 7 cells were added and incubated at 4 ° C. for 10 min.
- BSA bovine serum albumin
- each T cell sample (1 x 10 6 cells) was suspended in 89 ⁇ l of FACS buffer (0.5% FBS / PBS / 0.02% NaN 3 ) and then 1 ⁇ g of anti-mouse CD16 / 32-blocks Fc binding (eBioscience) 14-0161-85) was added and incubated at 4 ° C. for 20 min.
- FACS buffer 0.5% FBS / PBS / 0.02% NaN 3
- 1 ⁇ g of anti-mouse CD16 / 32-blocks Fc binding (eBioscience) 14-0161-85) was added and incubated at 4 ° C. for 20 min.
- 10 ⁇ l of 16G9 mAb or SSV mAb 100 ⁇ g / ml was added as a primary antibody and incubated at 4 ° C. for 60 min.
- the cells were again centrifuged and washed twice with FACS buffer, and then 100 ⁇ l of Streptavidin-PE-Cy7 (BD Biosciences 556463, 1 ⁇ g / ml) was added to the FITC-conjugated rat anti-mouse CD3 mAb (BD Biosciences 553062). ) was added to a final concentration of 1 ⁇ g / ml and incubated at 4 ° C. for 30 min.
- Test Example 5 Specific target of down-regulation by SSV mAb Seven candidate proteins to be down-regulated by Example 1 (SSV mAb) were identified by proteomic analysis. And it confirmed that ATP synthase was a target antigen of Example 1 (SSV mAb) among seven types of candidate proteins by the following method. First, T cells derived from Balb / c mice, in which mitochondrial ATP synthase was knocked down, were obtained using a Thermo Fisher siRNA kit. Next, according to the procedure of Test Example 2, the obtained T cells were used to conduct an MLR test using Example 1 (SSV mAb) as a test substance. Here, Isotype IgG1 (eBioscience) was used as a control reagent. As a control test, a similar test was performed using T cells derived from a mouse that did not knock down ATP synthase.
- Example 1 SSV mAb
- FIGS. 6A and B show that when ATP synthase was not knocked down, Example 1 (SSV mAb) significantly inhibited cell growth compared to Isotype IgG1.
- FIG. 6B when ATP synthase was knocked down, no significant difference was observed between Example 1 (SSV mAb) and Isotype IgG1 with respect to inhibition of cell proliferation.
- knockdown of ATP synthase reduces the immunosuppressive activity of SSV mAb, suggesting that SSV mAb down-regulates ATP synthase activity during immunosuppression. .
- Test Example 6 Identification of SSV mAb light and heavy chain variable region sequences Synthesis of hybridoma cDNA Total RNA was prepared from 1.6 ⁇ 10 7 cells of the hybridoma (Mouse-Mouse hybridoma SSV-C93-3) obtained in Test Example 1 using FastPure RNA Kit (TaKaRa). MRNA was prepared from 240 ⁇ g of total RNA using Poly (A) + Isolation Kit from Total RNA (manufactured by NIPPON GENE). Ethanol precipitation was performed using Etachinmate (manufactured by NIPPON GENE) to precipitate mRNA. After washing with 75% ethanol, the mRNA was dried.
- RNA solution was stored at ⁇ 80 ° C.
- SMARTer RACE cDNA Amplification Kit (Clontech)
- cDNA for 5′-RACE was synthesized from 1 ⁇ g of SSV hybridoma mRNA.
- the obtained cDNA solution was stored at -20 ° C.
- CDR complementarity determining regions
- the reaction solution was subjected to agarose electrophoresis, and an approximately 600 bp heavy chain 5′-RACE product and an approximately 550 bp light chain 5′-RACE product were separated from the gel using EZNA Gel Extraction Kit (manufactured by OMEGA bio-tek). Purified. This was ligated to pGEM-T Easy Vector (Promega) and transformed with Competent high E. coli DH5 ⁇ (TOYOBO). A plasmid was prepared from the obtained transformant using EZNA Plasmid Miniprep Kit I (manufactured by OMEGA bio-tek). Using the prepared plasmid as a template, a cycle reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems).
- the base sequences of the light chain and heavy chain variable regions were analyzed using a DNA sequencer (Applied Biosystems).
- the nucleotide sequence of the light chain variable region was represented by the 67th to 384th positions of SEQ ID NO: 5.
- the base sequence of the heavy chain variable region was represented by the 58th to 423rd positions of SEQ ID NO: 10.
- SEQ ID NO: 5 from 1 to 66 are the light chain signal peptide base sequence
- SEQ ID NO: 10 No. 1 to No. 57 were presumed to be base sequences of heavy chain signal peptides.
- amino acid sequences of the light chain and heavy chain variable regions were estimated from the obtained base sequences, and the CDR regions were identified according to the method of Kabat et al.
- the amino acid sequence of the variable region of the light chain was represented by the 23rd to 128th amino acids of SEQ ID NO: 6.
- No. 1 to No. 22 of SEQ ID NO: 6 are the amino acid sequences of the light chain signal peptide.
- CDR1 is represented by RASSSSVSYMH (SEQ ID NO: 2)
- CDR2 is represented by ATSNLAS (SEQ ID NO: 3)
- CDR3 is represented by QQWSSNPWT (SEQ ID NO: 4). It was confirmed that
- the amino acid sequence of the variable region of the heavy chain was represented by SEQ ID NO: 11 from No. 20 to No. 141.
- numbers 1 to 19 in SEQ ID NO: 11 are the amino acid sequences of heavy chain signal peptides.
- CDR1 is represented by GYNMN (SEQ ID NO: 7)
- CDR2 is represented by NINPYYGSTSYNQKFKG (SEQ ID NO: 8)
- CDR3 is represented by SPYYSNYWRYFDY (SEQ ID NO: 9). It was confirmed.
- LPS Lipopolysaccharide
- SSV mAb dissolved in physiological saline was intraperitoneally administered intraperitoneally 30 minutes before LPS administration, 6 hours after LPS administration, and 3 times after 12 hours, at an SSV mAb amount of 100 ⁇ g / dose. Subsequent survival of the animals was examined. As a control, IgG (manufactured by SIGMA) dissolved in physiological saline was similarly administered instead of SSV mAb. The results are shown in FIG. The survival rate at 70 hours after LPS administration (inducing sepsis) was 20% in the control group (IgG) (cumulative survival rate 0.2), compared to 70% in the SSV mAb administration group (cumulative survival rate 0.7). Met.
- the survival rate at 70 hours after LPS administration (inducing sepsis) was approximately 3.5 times higher in the SSV mAb administration group (SSV) than in the control group (IgG), and significantly higher in the SSV mAb administration group (p ⁇ 0.05).
- LPS popolysaccharide
- SSV mAb dissolved in physiological saline was intraperitoneally administered intraperitoneally 30 minutes before LPS administration, 6 hours after LPS administration, and 3 times after 12 hours, at an SSV mAb amount of 100 ⁇ g / dose. Subsequent survival of the animals was examined.
- IgG manufactured by SIGMA
- physiological saline was similarly administered instead of SSV mAb.
- the result is shown in FIG.
- the survival rate 24 hours after LPS administration (inducing sepsis) was 20% in the control group (IgG), but 70% in the SSV mAb administration group.
- the survival rate at 70 hours after LPS administration (inflammation induction) was about 3.5 times higher in the SSV mAb administration group (SSV) than in the control group (IgG), and significantly higher in the SSV mAb administration group (p ⁇ 0.05).
- FIG. 9A blood-derived serum
- FIG. 9B lung tissue
- the histone H1 concentration in the blood sample of the SSV mAb administration group remained almost constant without increasing during the test.
- the histone H1 concentration in the blood sample of the control group increased or decreased during the test period. Further, it was confirmed that the histone H1 concentration in the inflamed tissue (lung) was significantly decreased in the SSV mAb administration group than in the control group.
- FIG. 10A blood-derived serum
- FIG. 10B lung tissue
- the histone H3 concentration of the blood sample and the inflamed tissue (lung) tended to be lower in the SSV mAb administration group than in the control group.
- FIG. 11A blood-derived serum
- FIG. 11B lung tissue
- the histone H3 concentration of the blood sample and the inflamed tissue (lung) tended to be lower in the SSV mAb administration group than in the control group.
- lung tissue sections were obtained from the rats of the control group and the SSV mAb administration group, respectively, and Hasegawa et al. (Surg Res. 2012 May 1; 174 (1): 136-41.) was stained using a hematoxylin / eosin staining solution (Wako Pure Chemical Industries, Ltd.). Next, a micrograph of the obtained sample was taken. As a reference, photographs of lung tissue fragments were also taken for healthy rats.
- FIGS. 12A to 12C The result was as shown in FIGS. 12A to 12C.
- FIG. 12A healthy rat
- FIG. 12B SSV mAb administration group
- inflammation in the lung was not observed.
- FIG. 12C control group
- inflammation was confirmed in the lung.
- the SSV mAb administration group showed significantly lower values in all of congestion, edema, inflammation, and hemorrhage than in the control group.
- FIGS. 14A to 14C Regard the inflammatory cytokines TNF- ⁇ , IL-1 ⁇ and IL-6, the value of the SSV mAb-administered group was significantly decreased as compared with the control group.
- FIG. 14D regarding the inhibitory cytokine IL-10, the value of the SSV mAb administration group was significantly increased as compared with the control group. From the above results, it was confirmed that inflammation was suppressed in the SSV mAb administration group as compared to the control group.
- Test Example 9 Confirmation test 1 for acute renal failure due to renal ischemia reperfusion injury
- 10 mg / kg of SSV mAb was administered 30 minutes before model creation and immediately after reperfusion.
- IgG manufactured by SIGMA
- BUN urea nitrogen
- Cr creatinine
- the BUN and Cr values were significantly lower than in the control group.
- the SSV mAb administration group was in a better state than the control group.
- Test Example 10 Confirmation test 2 for therapeutic effect on acute renal failure due to renal ischemia reperfusion injury
- the test was conducted in the same manner as in Test Example 10, except that 10 mg / kg of SSV mAb was administered 30 minutes before model creation and 6 hours after reperfusion. went.
- IgG manufactured by SIGMA
- BUN urea nitrogen
- Cr creatinine
- the BUN and Cr values were significantly lower than in the control group.
- the SSV mAb administration group was in a better state than the control group.
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Abstract
Description
したがって、本発明は、炎症疾患に対する新規な治療剤の提供を目的とする。
本発明のハイブリドーマMouse-Mouse hybridoma SSV-C93-3は、原寄託日を2010年8月17日として、独立行政法人製品評価技術基盤機構 特許微生物寄託センター(住所:日本国 千葉県木更津市かずさ鎌足2-5-8バイオテクノロジー本部)において、受託番号NITE BP-972のもと寄託されている。
本発明のモノクローナル抗体またはその抗原結合断片は、SSVLYGGPPSAA(配列番号1)で表わされるアミノ酸配列からなるペプチドまたは該ペプチドと薬学上許容可能な担体との結合体と結合することを一つの特徴としている。かかるモノクローナル抗体またはその抗原結合断片が、炎症疾患に対して優れた治療効果を奏することは意外な事実である。
また、免疫細胞としては、好ましくは脾細胞を使用する。
また、細胞融合温度は、好ましくは25~37℃であり、より好ましくは30~37℃である。
また、ミエローマ細胞と免疫細胞との混合比率は、好ましくは1:1~1:10程度である。
また、所望によりジメチルスルホキシドなどの補助剤を培地に適宜添加することができる。
抗原物質の製造
抗原物質としては、配列番号1で表されるアミノ酸配列からなるペプチドおよびKLHの結合体を使用した。
免疫
PBS中に抗原物質を溶解させた溶液 0.8mL(抗原物質濃度:0.5mg/mL)とフロイトコンプリートアジュバンド(和光純薬株式会社製)0.8mLとを混合し、懸濁液(抗原濃度:0.25mg/mL)を得た。次に、この懸濁液0.2mLをBALB/cマウスに腹腔内投与した。さらに、この懸濁液を2週間毎に同量にてマウスに投与した。そして、投与開始から16週間後、PBS中抗原を溶解させた溶液 0.2mL(抗原濃度:600~1000mg/mL)をマウス腹腔内へ最終投与した。なお、投与の際には、眼底静脈より採血を行ってELISAにより抗体価を測定した。最終投与の4日後、全採血を行い、得られた血液を遠心分離(2000rpm、20分)し、抗血清を得て以下の実験のコントロール抗血清として用いた。また、全採血後、ラットより脾臓を摘出し、得られた脾細胞を以下の細胞融合に用いた。
上記の脾細胞およびミエローマ細胞(P3X63-Ag.8.653)を脾細胞:ミエローマ細胞=10:1~10にて混合して遠心分離(1500rpm,5分)した。遠心分離した後、アスピレーターを用いて上清を除去し、得られた細胞ペレットに37℃のポリエチレングリコール4000(50%PBS溶液)1mLを1分間かけて添加して混合液とした。この混合液を37℃にて1分間静置した後、37℃のIMDM培地(計9mL)を30秒毎に1 mLずつ加えた後、遠心分離(1500rpm、5分)した。遠心分離後、上清を吸引除去し、37℃の15%FCS(JRH BIOSCIENCES製)含有IMDM(GIBCO製)培地を適量添加した。得られた懸濁液を96ウェル培養プレートに100mLずつ分注を行い、37℃/5%CO2インキュベーターにて1日培養した。さらにHAT培地(HAT粉末(HAT MEDIA SUPPLEMENT(×50)、SIGMA製)を無血清IMDM培地10mLに溶かし、10%FCS含有IMDM培地にて50倍希釈したものである。)を100mL添加し、37℃/5%CO2インキュベーターにて培養した。HAT培地の交換は2~3日毎に行い、10日後にはHT培地(HT粉末(HT MEDIA SUPPLEMENT、SIGMA製)を無血清IMDM培地10mLに溶かし、10%FCS含有IMDM培地にて50倍希釈したものである。)に切り替え、3日間、37℃/5%CO2インキュベーターにて培養を行った。以後2~3日ごとに培地(HT培地)の交換を行った。細胞増殖を顕微鏡により確認した後、培養上清(約100 mL)を回収した。この培養上清を用いて、抗体価測定によるハイブリドーマのスクリーニングを行った。
抗体価測定
上記抗原物質(5mg)を含む緩衝液(Baicarbonate buffer:100 mM NaHCO3-NaOH、pH9.2~9.5、ペプチド濃度:1μg/mL) を1ウェル当り50μLずつ96ウェル平底プレートへ添加し、室温にて2時間静置してコーティングした。プレートを洗浄バッファー(PBST)にて3回洗浄し、ブロッキングバッファー(3%スキムミルク1%BSA、PBS)を200~250μL/ウェルにて加え、4℃にて一昼夜反応させた後、3回洗浄した。そして、ハイブリドーマの培養上清を100μL/ウェルにて加え、37℃にて4時間または4℃にて一昼夜反応させた。プレートを3回洗浄した後、希釈バッファー(10 mM Tris-HCl ( pH 8.0 )、0.9 % ( W/V ) NaCl、0.05 % ( W/V ) Tween20)にて10000倍希釈したビオチン標識抗マウスIgG(Biotion-labeled anti-mouse IgG 、SIGMA)を50μL/ウェルにて加え、室温にて2時間反応させた。その後6回洗浄した後、希釈バッファーにて1000倍希釈したアルカリフォスファターゼ標識ストレプタリジン(Streptaridin)を50μL/ウェルにて加え、室温にて1~2時間反応させた。その後6回洗浄を行い、蛍光基質バッファー(Attophos substrate buffer、ロシュダイアグノスティックス社製)を50μL/ウェル加えてプレートを遮光し発色させた。蛍光強度はCytoFluorII(パーセプティブ社製)にて測定した。
上記抗体価測定にて陽性の結果を示したウェル(1×105細胞/mL)に15%FCS10%HCF(Hybridoma cloning factor、オリジン社製)含有IMDM培地を加えて、約200細胞/ウェル となるように96ウェル培養プレートに分注し、37℃5%CO2インキュベーターにて培養を行った。そして、上記と同様に抗体価測定を行い、抗体産生量の多いハイブリドーマを選択した。
ハイブリドーマMouse-Mouse hybridoma SSV-C93-3は、15%FCS含有RPMI培地を用いて培養した(1×106細胞/mL)。次に、ハイブリドーマ培養液を回収し、死細胞片を除くためにフィルターでろ過した。次に、終濃度40%となるように、培養上清に硫酸アンモニウムを加え、40℃で1時間撹拌した。次に、遠心分離(3000g、30分、4℃)を行い、上清を捨て沈殿を回収した。この沈殿を上記培養上清の1/10量のPBSで溶解し、PBSを外液として一晩透析した。
次に、上記沈殿を20mMリン酸ナトリウム緩衝液(pH7.0)で2倍希釈し、1Mトリス-HCl緩衝液とともに、HiTrapNHS活性化カラムに添加した。さらに、0.1Mグリシン-HCl溶液(pH2.7)で抗体の溶出し、フラクションチューブに回収した。
実施例1のモノクローナル抗体(SSVmAb)のアイソタイプを同定するため、Mouos Monoclonal Antibody Isotyping Reagents(シグマ)を用いたアイソタイプ同定試験を行った。
結果は、図1に示される通りであり、IgG1が最も高い値を示した。
図1およびSDS-PAGEの結果から、実施例1のモノクローナル抗体(SSVmAb)のアイソタイプはIgG1であることが確認された。
WO2006/025580号公報には、免疫抑制に用いることができ、配列番号1で表わされるアミノ酸配列からなるペプチドと結合する抗H1モノクローナル抗体として、ハイブリドーマ16G9(受託番号FERM BP-10413)の産生するモノクローナル抗体(16G9mAb)が報告されている。
そこで、WO2006/025580号公報に記載の抗体(16G9mAb)を参考例1とし、実施例1のモノクローナル抗体(SSVmAb)との抗原に対する親和性の比較を行った。
抗原としては、参考例1(16G9mAb)の抗原であるヒストンH1、およびヒストンH1類似抗原であるコアヒストンH2A、H2B、H3およびH4を選択した。
96wellマイクロプレートをヒストンH1、H2A、H2B、H3またはH4でコートした。それぞれのヒストンは、100mM炭酸ナトリウム緩衝液(pH9.3)に溶解させたものを用いた。そのプレートをPBS-tween20(0.05%)で洗浄し、3%スキムミルクと1%BSAで1時間ブロッキングした。SSVmAb 5μg/mLを各wellに添加し、1時間インキュベーションした。結合したSSVmAbをペルオキシダーゼ(HRP)コンジュゲートanti mouse IgG1 Ab(シグマ)を用いて検出し、1時間インキュベーションした。結合したSSVmAbをABTS[2,2’-azino-bis(3-ethylbenzothiazoline-sulfonic acid) ]基質溶液を用いた検出し、Multiskan Ascent(Thermo Fisher Scientific Inc., Waltham, MA)を用いて405nmの吸光度を測定した。
図2に示される通り、実施例1(SSVmAb)では、ヒストンH1に対する親和性よりも、ヒストンH2A、H2B、H3またはH4に対する親和性が高かった。
一方、図3に示される通り、参考例1(16G9)では、ヒストンH2A、H2B、H3およびH4に対する親和性よりも、ヒストンH1に対する親和性の方が高かった。
無処理のDAラット由来の脾臓リンパ球(応答細胞)およびマイトマイシンC(協和発酵工業株式会社製)処理を行ったLEWラット由来の脾臓リンパ球(刺激細胞)を用いた。応答細胞は10%FCS-RPMI培地にて5×105 細胞/mLに調整し、刺激細胞は10%FCS-RPMI培地にて8×106 細胞/mLに調整した。この応答細胞懸濁液および刺激細胞懸濁液をそれぞれ100μLを96穴丸底プレート(Nunc Brand Products社製)に播種した後、混合培養開始時に参考例1のモノクローナル抗体16G9mAb(0.1、2、4または6μg/mL/ウェル)または実施例1のモノクローナル抗体SSVmAb(4μg/mL/ウェル)を添加し、37℃,5%CO2/95% airの条件下にて3.5日以上培養した。また、陽性対照として、免疫抑制剤タクロリムス(FK506:藤沢薬品社製、1nM/ウェル)を添加した。さらに、培養終了15時間前にブロモデオキシウリジン(BrdU)10μLを添加した。そして、BrdUラベリング&ディテクションキットIII(ロシュ・ダイアグノスティクス社製)を用いて、細胞内DNAに取り込まれたBrdU量を指標として、免疫抑制物質により処理された細胞の増殖能を測定し、免疫抑制レベルの指標とした。
実施例1(SSVmAb)では、BrdU量取り込みを示す吸光度は、参考例1(16G9mAb)およびタクロリムス(FK506)のそれより低かった。特に、実施例1(SSV mAb)の吸光度0.552±0.114(平均±S.E.)と、同添加量(4μg/mL/ウェル)の参考例1(16G9mAb)の吸光度1.351±0.389(平均±S.E.)とを比較すると、実施例1の平均値は、参考例1のそれの約41%程度であった。
以下の手法により、C57BL/6マウス(5週齢、メス、日本チャールズリバー社製)より脾臓を摘出し、全脾細胞を調製した。
まず、5 mlのRPMI1640培地(Sigma-Aldrich社製R-8758)を入れた5 ml培養ディッシュ(BD Bioscience社製FALCON 351007)中で脾臓を解剖用ハサミとピンセットにて良くほぐして脾細胞を懸濁後、15 ml遠心チューブ(BD Bioscience社製FALCON 352096)に移した。続いて5 mlディッシュ上をphosphate-buffered saline(PBS, Invitrogen社製20012-027)にて数回洗浄し、これらも先の細胞懸濁液に加えて静置後、上清を別の15 ml遠心チューブに回収した。さらに残渣の不溶性脾臓組織にも再びRPMI1640培地5 mlを加えて静置後上清のみを回収し、これと上記の細胞懸濁液を合わせて1,500 rpm, 5 minの遠心分離を行った。回収した細胞にlysis buffer(150 mM NH4Cl/15 mM NaHCO3/0.1 mM EDTA-Na2, pH 7.3)2 mlを加えてタッピングにより溶血後、PBS 10 mlを加えて1,500 rpm, 5 minにて3回遠心洗浄した細胞を全脾細胞とした。
まず、脾細胞をMACS buffer (0.5% bovine serum albumin (BSA, ナカライテスク社製08777-36)/PBS)にて 5 x 107 cells/200μlの割合で懸濁し、50μl Biotin-antibody cocktail / 5 x 107 cellsを添加して4 ℃, 10 minインキュベートした。これを150 μl MACS buffer / 5 x 107 cellsに懸濁後、100μl anti-biotin micro beads /5 x 107 cellsを添加して4 ℃, 15 minインキュベートした。これにMACS buffer(10 ml)を添加して1500 rpm, 5 min遠心洗浄後、回収細胞をMACS buffer 500μlに懸濁した。MACSカラム (MSカラム、Miltenyi Biotec社製130-042-201) をマグネット(MiniMACS separation Unit、Miltenyi Biotec社製130-090-312)にセットして500μl MACS bufferにてカラムを平衡化後、上述の細胞懸濁液を供した。素通り画分500μlおよびその後のMACS bufferによるカラム洗浄画分(1.5 ml)を回収して精製未刺激T細胞とした(純度約97%)。
まず、各T細胞サンプル(1 x 106 cells)を89μlのFACS buffer (0.5% FBS/PBS/0.02% NaN3)に懸濁後、1μgのanti-mouse CD16/32-blocks Fc binding(eBioscience社製14-0161-85)を加えて4℃, 20 minインキュベートした。これに1次抗体として16G9 mAbまたはSSV mAb (100μg/ml)10μlを加えて4℃, 60 minインキュベートした。FACS bufferにて細胞を2回遠心洗浄後、2次抗体(Biotin-conjugated rat anti-mouse IgM mAb(eBioscience社製13-5780-85)またはBiotin-conjugated rat anti-mouse IgG1 mAb(BD Biosciences社製553441)、各1μg/ml)を100μl加えて4℃, 30 minインキュベートした。再びFACS bufferにて細胞を2回遠心洗浄後、Streptavidin-PE-Cy7(BD Biosciences社製556463、 1μg/ml)を100μl加え、これにFITC-conjugated rat anti-mouse CD3 mAb(BD Biosciences社製553062)を終濃度1 μg/mlになるように添加して4℃, 30 minインキュベートした。FACS bufferにて2回遠心洗浄および40 μmセルストレーナー(BD Bioscience社製FALCON 352340)にてろ過処理後、各サンプルをFACSCaliburフローサイトメーターおよびCellQuestソフトウェア(BD Bioscience社製)に供して16G9 mAbまたはSSV mAb陽性/CD3陽性T細胞数を解析した。
参考例1(16G9 mAb)および実施例1(SSV mAb)を比較すると、CD3陽性T細胞に対する反応性に有意差は認められず、これら抗体は同等の反応性を示した(student t-test、p<0.05)。
実施例1(SSV mAb)によりダウンレギュレーションされる候補タンパク質をプロテオーム解析にて7種同定した。
そして、7種の候補タンパク質うち、ATP合成酵素が実施例1(SSV mAb)のターゲット抗原であることを以下の手法により確認した。
まず、サーモフィッシャー社のAccell siRNAキットを用いて、ミトコンドリアATP合成酵素のノックダウンされたBalb/cマウス由来のT細胞を取得した。
次に、試験例2の手法に準じて、得られたT細胞を用いて、実施例1(SSV mAb) を試験物質としてMLR試験を行った。
ここで、対照試薬としてはIsotype IgG1(eBioscience社)を用いた。また、コントロール試験として、ATP合成酵素をノックダウンしていないマウス由来のT細胞を用いた同様の試験を行った。
図6Aに記載の通り、ATP合成酵素をノックダウンしない場合には、実施例1(SSV mAb)は、Isotype IgG1と比較して有意に細胞増殖を阻害していた。
一方、図6Bに記載の通り、ATP合成酵素をノックダウンした場合には、細胞増殖の阻害に関し、実施例1(SSV mAb)とIsotype IgG1との間には有意差は認められなかった。
図6AおよびBでは、ATP合成酵素のノックダウンによりSSV mAbの免疫抑制活性が低下しており、SSV mAb が、免疫抑制の際に、ATP合成酵素活性をダウンレギュレーションしていることが示唆される。
ハイブリドーマcDNAの合成
FastPure RNA Kit(TaKaRa社製)を使って、試験例1で取得したハイブリドーマ(Mouse-Mouse hybridoma SSV-C93-3)1.6×107cellsからtotal RNAを調製した。Poly (A)+ Isolation Kit from Total RNA(NIPPON GENE製)を使って、240 μgのtotal RNAからmRNAを調製した。Etachinmate(NIPPON GENE社製)を使ってエタノール沈殿を行い、mRNAを沈殿した。75% エタノールで洗浄した後、mRNAを乾燥した。これにRNase free waterを10 μL加え、mRNAを溶解した。得られたmRNA溶液は-80℃で保存した。 SMARTer RACE cDNA Amplification Kit(Clontech社製)を使って、1 μgのSSVハイブリドーマmRNAから5’-RACE用のcDNAを合成した。得られたcDNA溶液は-20℃で保存した。
マウスIgG1 重鎖定常領域の塩基配列をもとに、プライマー 5’- CAC CAT GGA GTT AGT TTG GGC AGC AG -3’ (配列番号12)を作製した。マウス軽鎖κ定常領域の塩基配列をもとにプライマー 5’- CAC GAC TGA GGC ACC TCC AGA TG -3’(配列番号13)を作製した。それぞれのプライマーとUniversal Primer A Mix(SMARTer RACE cDNA Amplification Kit付属プライマー)を用いて、cDNAをテンプレートとした5'-RACEを行った。RACE反応はAdvantage2 PCR Kit(Clontech社製)を用いた。反応液をアガロース電気泳動し、約600 bpの重鎖5’-RACE産物および約550 bpの軽鎖5’-RACE産物を、E.Z.N.A. Gel Extraction Kit(OMEGA bio-tek社製)を用いてゲルから精製した。これをpGEM-T Easy Vector(Promega社製)に連結し、Competent high E.coli DH5α(TOYOBO社製)を形質転換した。得られた形質転換体から、E.Z.N.A. Plasmid Miniprep KitI(OMEGA bio-tek社製)を用いてプラスミドを調製した。調製したプラスミドをテンプレートとして、BigDye Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems社製)を用いてサイクル反応を行った。
その結果、軽鎖可変領域の塩基配列は、配列番号5の第67番~第384番で表されるものであった。
また、重鎖可変領域の塩基配列は、配列番号10の第58番~第423番で表されるものであった。
なお、翻訳開始コドンとKabatらの方法により決定したFR(定常領域)1の位置に基づき、配列番号5の第1番~第66番は軽鎖シグナルペプチドの塩基配列であり、および配列番号10の第1番~第57番は重鎖のシグナルペプチドの塩基配列であると推定した。
また、軽鎖可変領域可変領域のアミノ酸配列のうち、CDR1はRASSSVSYMH(配列番号2)で表わされ、CDR2はATSNLAS(配列番号3)で表わされ、CDR3はQQWSSNPWT(配列番号4)で表わされることを確認した。
また、重鎖可変領域のアミノ酸配列のうち、CDR1はGYNMN(配列番号7)で表わされ、CDR2はNINPYYGSTSYNQKFKG(配列番号8)で表わされ、CDR3はSPYYSNYWRYFDY(配列番号9)で表わされることを確認した。
BALB/cマウス(実験開始時体重20g-30g、n=6)をSPF、恒温恒湿(22±1度、55±5%)にて、プラスティック製ケージ内で12時間明暗サイクルの条件下で飼育した。餌および水は自由摂取とした。実験的敗血症モデルとして一般的に用いられるリポポリサッカライド(LPS)誘発致死的敗血症をLPS40mg/kgの腹腔内投与により誘発した。生理食塩水に溶解したSSV mAbを腹腔内にLPS投与30分前、LPS投与6時間後、12時間後の3回、SSV mAb量として100μg/回で腹腔内に投与した。その後の動物個体の生存を調べた。なお、対照はSSV mAbの代わりに生理食塩水に溶解したIgG(SIGMA社製)を同様に投与した。
その結果を図7に示した。LPS投与(敗血症誘導)後70時間の生存率は対照群(IgG)が20%(累積生存割合0.2)であったのに対しSSV mAb投与群では70%(累積生存割合0.7)であった。LPS投与(敗血症誘導)後70時間の生存率は、SSV mAb投与群(SSV)が対照群(IgG)の約3.5倍であり、SSV mAb投与群の方が有意に高かった(p<0.05)。
8-1
BALB/cマウス(実験開始時体重20g-30g、n=10)をSPF、恒温恒湿(22±1度、55±5%)にて、プラスティック製ケージ内で12時間明暗サイクルの条件下で飼育した。餌および水は自由摂取とした。次に、マウスにLPS40mg/kgの腹腔内投与を行い、炎症疾患として、試験例7の同様にポポリサッカライド(LPS)誘発致死的敗血症を誘発した。生理食塩水に溶解したSSV mAbを腹腔内にLPS投与30分前、LPS投与6時間後、12時間後の3回、SSV mAb量として100μg/回で腹腔内に投与した。その後の動物個体の生存を調べた。なお、対照はSSV mAbの代わりに生理食塩水に溶解したIgG(SIGMA社製)を同様に投与した。
試験例8では、コントロール群およびSSV mAb投与群において、長谷川ら(Surg Res. 2012 May 1;174(1):136-41.)の行った手法に準じ、全身麻酔下にて心臓由来の血液または肺組織を取得した。得られたサンプルについてELISA法を用いた市販の測定キットによってヒストンH1、ヒストンH3およびH4の濃度を測定した。
SSV mAb投与群の血液サンプル中のヒストンH1濃度は試験中増加することなくほぼ一定に維持されていた。一方、コントロール群の血液サンプル中のヒストンH1濃度は、試験期間中増減が認められた。
また、炎症組織(肺)のヒストンH1濃度は、SSV mAb投与群の方が、コントロール群よりも有意に低下していることが確認された。
血液サンプルおよび炎症組織(肺)のヒストンH3濃度は、SSV mAb投与群の方が、コントロール群よりも低い傾向が認められた。
血液サンプルおよび炎症組織(肺)のヒストンH3濃度は、SSV mAb投与群の方が、コントロール群よりも低い傾向が認められた。
その結果、SSV mAbはインビトロにおいてヒストンH1、ヒストンH3およびヒストンH4と結合能を有することを確認した。
試験例8の試験終了後、コントロール群およびSSV mAb投与群のラットからそれぞれ肺組織切片を取得し、長谷川ら(Surg Res. 2012 May 1;174(1):136-41.)の行った手法に準じて、ヘマトキシリン・エオジン染色液(和光純薬製)を用いて染色した。次に、得られたサンプルの顕微鏡写真を撮影した。
参考として、健常ラットについても同様に、肺組織断片の写真を撮影した。
図12A(健常ラット)と、図12B(SSV mAb投与群)とは、肺中の炎症は観察されなかった。一方、図12C(コントロール群)では、肺に炎症が確認された。
試験例8の試験終了後、Murakamiらの手法(Shock, 18 (2002), p. 236)に準じて、鬱血、浮腫、炎症および出血スコアを評価した。具体的には、顕微鏡による24視野の拡大像を観測対象した。そして、SSV mAb投与群およびコントロール群における鬱血、浮腫および炎症の悪化度合いを、スコア0-4の5段階(4が最も悪化度が高い)で評価した。
鬱血、浮腫、炎症および出血のいずれにおいても、コントロール群よりも、SSV mAb投与群の方が有意に低い値を示した。
試験例8では、コントロール群およびSSV mAb投与群において、3時間ごとに静脈血から血清を取得した。そして、得られたサンプルについてELISA法を用いた市販の測定キットによって炎症性サイトカイン(TNF-α、IL-1β、IL-6)と、抑制性サイトカイン(IL-10)の濃度を測定した。
図14A~Cに示される通り、炎症性サイトカインTNF-α、IL-1βおよびIL-6に関しては、SSV mAb投与群の値が、コントロール群と比較して有意に低下した。
一方、図14Dに示される通り、抑制性サイトカインIL-10に関しては、SSV mAb投与群の値が、コントロール群と比較して有意に上昇した。
上記結果からも、SSV mAb投与群では、コントロール群と比較して炎症が抑制されていることが確認された。
Wistar系雄性ラット(n=4)に対して、全身麻酔下に右腎摘出後、左腎を血管クリップにて虚血再灌流させモデルを作成した。次に、SSV mAb投与群では、モデル作成30分前および再灌流直後にSSV mAbを10mg/kg投与した。また、コントロール群ではSSV mAbの代わりIgG(SIGMA社製)を同様に投与した。そして、モデル作成24時間後の血清中の尿素窒素(BUN)およびクレアチニン(Cr)を測定し、腎障害のレベルを評価した。また、試験終了後にラットの剖検を行い、組織学的評価を行った。
Wistar系雄性ラット(n=4~6)を用い、SSV mAb投与群において、モデル作成30分前および再灌流から6時間後にSSV mAbを10mg/kg投与する以外、試験例10と同様に試験を行った。
また、対照はSSV mAbの代わりIgG(SIGMA社製)を同様に投与した。そして、モデル作成24時間後の血清中の尿素窒素(BUN)およびクレアチニン(Cr)濃度を測定し、腎障害のレベルを評価した。また、試験終了後にラットの剖検を行い、組織学的評価を行った。
Claims (18)
- SSVLYGGPPSAA(配列番号1)で表わされるアミノ酸配列からなるペプチドまたは該ペプチドと薬学上許容可能な担体との結合体と結合する、モノクローナル抗体またはその抗原結合断片を含んでなる、炎症疾患治療剤。
- 前記炎症疾患が、ヒストンの関与するものである、請求項1に記載の治療剤。
- 前記炎症疾患が、急性炎症疾患である、請求項1または2に記載の治療剤。
- 前記炎症疾患が、敗血症、腎虚血再灌流傷害および腎不全から選択されるものである、請求項1~3のいずれか一項に記載の治療剤。
- 前記炎症疾患が敗血症である、請求項1~4のいずれか一項に記載の治療剤。
- ヒストンH1、ヒストンH3およびヒストンH4と結合する、請求項1~5のいずれか一項に記載の治療剤。
- RASSSVSYMH(配列番号2)で表わされるアミノ酸配列からなるCDR1、ATSNLAS(配列番号3)で表わされるアミノ酸配列からなるCDR2、およびQQWSSNPWT(配列番号4)で表わされるアミノ酸配列からなるCDR3を含んでなる軽鎖可変領域を含んでなる、請求項1~6のいずれか一項に記載の敗血症治療剤。
- 前記モノクローナル抗体またはその抗原結合断片の軽鎖可変領域が、配列番号6の第23番~第128番で表されるアミノ酸配列を含んでなる、請求項1~7のいずれか一項に記載の治療剤。
- GYNMN(配列番号7)で表わされるアミノ酸配列からなるCDR1、NINPYYGSTSYNQKFKG(配列番号8)で表わされるアミノ酸配列からなるCDR2、およびSPYYSNYWRYFDY(配列番号9)で表わされるアミノ酸配列からなるCDR3を含んでなる重鎖可変領域を含んでなる、請求項1~8のいずれか一項に記載の治療剤。
- 前記モノクローナル抗体またはその抗原結合断片の重鎖可変領域が、配列番号11の第20番~第141番で表されるアミノ酸配列を含んでなる、請求項1~9のいずれか一項に記載の治療剤。
- 前記モノクローナル抗体またはその抗原結合断片が、前記ペプチドまたはペプチドと薬学上許容可能な担体に対するものである、請求項1~10のいずれか一項に記載の治療剤。
- 前記薬学上許容可能な担体が、キーホールリンペットヘモシアニン、オボアルブミンまたはウシ血清アルブミンである、請求項1~11のいずれか一項に記載の治療剤。
- 前記モノクローナル抗体またはその抗原結合断片が、ATP合成酵素活性をダウンレギュレーションしうる、請求項1~12のいずれか一項に記載の治療剤。
- 前記モノクローナル抗体が、キメラ、ヒト化、またはヒト抗体である、請求項1~13のいずれか一項に記載の治療剤。
- 前記モノクローナル抗体が、ハイブリドーマMouse-Mouse hybridoma SSV-C93-3により産生される、請求項1~14のいずれか一項に記載の治療剤。
- 前記抗原結合断片が、Fab、Fab’、(Fab’)2、FvまたはscFvである、請求項1~15のいずれか一項に記載の治療剤。
- 請求項1~16のいずれか一項に記載のモノクローナル抗体またはその抗原結合断片の有効量を被験体に投与することを含んでなる、炎症疾患の治療方法。
- 炎症疾患の治療剤の製造における、請求項1~16のいずれか一項に記載のモノクローナル抗体またはその抗原結合断片の使用。
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP13751092.1A EP2815763B1 (en) | 2012-02-22 | 2013-02-22 | Therapeutic agent for inflammatory disease |
IN7875DEN2014 IN2014DN07875A (ja) | 2012-02-22 | 2013-02-22 | |
KR1020147024978A KR102159412B1 (ko) | 2012-02-22 | 2013-02-22 | 염증 질환 치료제 |
US14/380,292 US20150079090A1 (en) | 2012-02-22 | 2013-02-22 | Therapeutic agent for inflammatory disease |
CN201380020995.1A CN104379165B (zh) | 2012-02-22 | 2013-02-22 | 炎症疾病治疗剂 |
US15/292,450 US20170096478A1 (en) | 2012-02-22 | 2016-10-13 | Therapeutic agent for inflammatory disease |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2012035965A JP2015117181A (ja) | 2012-02-22 | 2012-02-22 | 敗血症治療剤 |
JP2012-035965 | 2012-02-22 |
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US14/380,292 A-371-Of-International US20150079090A1 (en) | 2012-02-22 | 2013-02-22 | Therapeutic agent for inflammatory disease |
US15/292,450 Division US20170096478A1 (en) | 2012-02-22 | 2016-10-13 | Therapeutic agent for inflammatory disease |
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WO2013125687A1 true WO2013125687A1 (ja) | 2013-08-29 |
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PCT/JP2013/054551 WO2013125687A1 (ja) | 2012-02-22 | 2013-02-22 | 炎症疾患治療剤 |
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US (2) | US20150079090A1 (ja) |
EP (1) | EP2815763B1 (ja) |
JP (1) | JP2015117181A (ja) |
KR (1) | KR102159412B1 (ja) |
CN (1) | CN104379165B (ja) |
IN (1) | IN2014DN07875A (ja) |
TW (1) | TWI605059B (ja) |
WO (1) | WO2013125687A1 (ja) |
Cited By (2)
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EP3436482A4 (en) * | 2016-03-29 | 2020-03-11 | OBI Pharma, Inc. | ANTIBODIES, PHARMACEUTICAL COMPOSITIONS AND METHODS |
US10980894B2 (en) | 2016-03-29 | 2021-04-20 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
Families Citing this family (2)
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JP5960596B2 (ja) | 2010-08-20 | 2016-08-02 | 学校法人 城西大学 | 免疫抑制活性を有するモノクローナル抗体またはその抗原結合断片 |
JP6656677B2 (ja) * | 2016-09-23 | 2020-03-04 | ティーエフケイ株式会社 | 化合物又はその塩、抗炎症剤、肺がんに対する抗がん剤、化合物又はその塩の製造方法、炎症性疾患の治療方法及び肺がんの治療方法 |
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WO2006025580A1 (ja) | 2004-09-03 | 2006-03-09 | Amateraspharma Inc. | 抗ヒストンh1モノクローナル抗体およびこれを産生するハイブリドーマ |
JP2011503012A (ja) * | 2007-11-06 | 2011-01-27 | オクラホマ・メディカル・リサーチ・ファウンデーション | 予後に関するバイオマーカーおよび治療の分子標的としての細胞外ヒストン |
WO2012023614A1 (ja) * | 2010-08-20 | 2012-02-23 | 学校法人 城西大学 | 免疫抑制活性を有するモノクローナル抗体またはその抗原結合断片 |
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EP2196212A4 (en) * | 2007-10-04 | 2012-01-25 | Josai University Corp | PREPARATION AND METHOD FOR VACCINE ADMINISTRATION AND IONTOPHORESIS DEVICE USING THE PREPARATION |
-
2012
- 2012-02-22 JP JP2012035965A patent/JP2015117181A/ja active Pending
-
2013
- 2013-02-22 IN IN7875DEN2014 patent/IN2014DN07875A/en unknown
- 2013-02-22 CN CN201380020995.1A patent/CN104379165B/zh not_active Expired - Fee Related
- 2013-02-22 EP EP13751092.1A patent/EP2815763B1/en not_active Not-in-force
- 2013-02-22 WO PCT/JP2013/054551 patent/WO2013125687A1/ja active Application Filing
- 2013-02-22 KR KR1020147024978A patent/KR102159412B1/ko active IP Right Grant
- 2013-02-22 TW TW102106211A patent/TWI605059B/zh active
- 2013-02-22 US US14/380,292 patent/US20150079090A1/en not_active Abandoned
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2016
- 2016-10-13 US US15/292,450 patent/US20170096478A1/en not_active Abandoned
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3436482A4 (en) * | 2016-03-29 | 2020-03-11 | OBI Pharma, Inc. | ANTIBODIES, PHARMACEUTICAL COMPOSITIONS AND METHODS |
US10980894B2 (en) | 2016-03-29 | 2021-04-20 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
US11041017B2 (en) | 2016-03-29 | 2021-06-22 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
TWI780045B (zh) * | 2016-03-29 | 2022-10-11 | 台灣浩鼎生技股份有限公司 | 抗體、醫藥組合物及方法 |
US11833223B2 (en) | 2016-03-29 | 2023-12-05 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
Also Published As
Publication number | Publication date |
---|---|
EP2815763B1 (en) | 2018-06-13 |
CN104379165B (zh) | 2018-08-03 |
IN2014DN07875A (ja) | 2015-04-24 |
US20150079090A1 (en) | 2015-03-19 |
KR102159412B1 (ko) | 2020-09-23 |
KR20140138699A (ko) | 2014-12-04 |
US20170096478A1 (en) | 2017-04-06 |
TW201350502A (zh) | 2013-12-16 |
EP2815763A4 (en) | 2015-07-22 |
EP2815763A1 (en) | 2014-12-24 |
TWI605059B (zh) | 2017-11-11 |
CN104379165A (zh) | 2015-02-25 |
JP2015117181A (ja) | 2015-06-25 |
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