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WO1995009873A1 - Anticorps monoclonaux diriges contre le recepteur de l'il-6 humaine - Google Patents

Anticorps monoclonaux diriges contre le recepteur de l'il-6 humaine Download PDF

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Publication number
WO1995009873A1
WO1995009873A1 PCT/US1994/011305 US9411305W WO9509873A1 WO 1995009873 A1 WO1995009873 A1 WO 1995009873A1 US 9411305 W US9411305 W US 9411305W WO 9509873 A1 WO9509873 A1 WO 9509873A1
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Prior art keywords
antibody
receptor
cells
binding
inhibition
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PCT/US1994/011305
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English (en)
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Ellen S. Vitetta
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Board Of Regents, The University Of Texas System
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Priority to AU79672/94A priority Critical patent/AU7967294A/en
Publication of WO1995009873A1 publication Critical patent/WO1995009873A1/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention generally relates to cytokines and the role which they play in the immune response. More specifically, the invention relates to antibodies directed against IL-6 and methods of using such antibodies.
  • Interleukin-6 is a highly pleiotropic cytokine which plays a critical role in the immune response, in acute phase reactions and in hematopoiesis (1-5) . It is produced by T and B cells, macrophages, bone marrow stromal cells, fibroblasts, macrophages, and endothelial cells (1) . Previous reports suggest that deregulated IL-6 production may be involved in the pathogenesis of many diseases, including multiple myeloma, rheumatoid arthritis, mesangial proliferative glomerulonephritis, Castleman's disease and Kaposi's sarcoma (2) .
  • IL-6 is a major growth factor for myeloma cells and can participate in both autocrine and paracrine growth (3,4) .
  • mAbs monoclonal anti-IL-6 antibodies
  • the IL-6 receptor (IL-6R) has two functionally different chains; a 80 kDa IL-6-binding protein (IL-6R) , and a 130kDa signal-transducing protein (gpl30) .
  • Gpl30 does not bind to IL-6, but it is involved in the formation of high affinity IL-6-binding sites (6) .
  • Monoclonal anti-IL-6R antibodies which neutralize the bioactivity of IL-6 may facilitate the study of the structure-function relationship of the IL-6R and may be good candidates for therapeutic reagents in the treatment of IL-6-related diseases such as multiple myeloma (7,8) .
  • IL-6 plays a central role in the pathogenesis of multiple myeloma and the growth of myeloma cells. Hence, patients with myeloma cells that are responsive to IL-6 might benefit from therapy with anti-IL-6R-based therapy.
  • UV4 monoclonal antibody against the human IL-6 receptor (hIL-6R) was generated by immunizing BALB/c mice with both a human myeloma cell line (U266) and a murine cell line (M12.4/R) transfected with the hIL-6R cDNA.
  • U266 human myeloma cell line
  • M12.4/R murine cell line
  • UV 4 stains the hIL-6R + cell lines U266 and U937, but not the hIL-6R " cell lines Daudi and K562.
  • the inventors Because of the technical difficulty in obtaining large amounts of purified hIL-6R protein, the inventors expressed the hIL-6R on a murine cell line instead of purifying the hIL-6R protein for immunization. To this end, the inventors cloned a truncated hIL-6R gene lacking the cytoplasmic domain from human myeloma U266 cells into eukaryotic expression vector, then transfected the recombinant vector into the IL-6R " murine cell line, M12.4.
  • UV4 specifically reacts with hIL-6R + cell lines U266 and U937, but not with hIL-6R " cell lines, Daudi and K562.
  • UV4 efficiently blocked the binding of IL-6-PE conjugate to its receptor on U266 cells.
  • UV4 inhibited the IL-6-induced proliferation of human bone marrow- derived myeloma cell lines ILKM-2 and ILKM-3, but had no effect on the Burkitt's lymphoma cell line, Daudi or on the IL-6-dependent murine cell lines, as shown in (3H) - thymidine incorporation assays.
  • a direct sandwich RIA further confirmed that UV4 recognized the same molecule as GAsIL-6R. UV4 could sterically interfere with IL-6 binding by binding an epitope adjacent to the IL-6 binding site or to the IL-6 binding-site itself.
  • the present invention deals generally with anti-IL-6 receptor monoclonal antibodies.
  • Such antibodies have the ability to bind to the IL-6 receptor.
  • the antibodies of the invention are able to detectably bind to cells that express the IL-6 receptor on their surface. Examples of such cells include U226, U937, and M12.4/R cell lines.
  • the binding of the anti- IL-6 receptor antibodies allows for the presence of IL-6 receptors to be diagnosed by such methods as, for example, flow cytometric analysis, sandwich assays, and 3 H-Thymidine incorporation assays.
  • Preferred monoclonal antibodies of the invention are capable of inhibiting the binding of IL-6 to an IL-6 receptor. Further, the antibodies of the invention are capable of interfering with proliferative activity of certain IL-6 dependent cells. For example, it has been shown that such antibodies can inhibit the proliferative activity of IL-6 on myeloma cell lines such as, ILKM-2 and ILKM-3 in a dose-dependent manner.
  • a particularly preferred embodiment of the present invention is the UV4 monoclonal antibody, the production and characteristics of which are detailed in the Detailed Description of the Preferred Embodiment Section of this application.
  • the invention involves hybridomas capable of expressing the antibodies of the invention.
  • hybridomas expressing the UV4 antibody are a preferred embodiment of this aspect of the invention.
  • Methods of inhibiting the binding of IL-6 molecules to IL-6 receptors are also included in this invention. These methods involve obtaining antibodies that are made according to the invention, then binding the antibodies to an IL-6 receptor in a manner that inhibits the binding of IL-6 molecules to the receptor. This inhibition can be of IL-6 receptors of intact cells, or on IL-6 receptors which have been more or less purified from cells.
  • the inhibition of binding of IL-6 to the IL-6 receptor can be further used in methods of inhibiting the proliferative activity of IL-6-dependent cells.
  • Such methods are further embodiments of the invention. These methods involve: obtaining an anti-IL-6 receptor antibody; treating IL-6-dependent cells that are susceptible to inhibition by the anti-IL-6 receptor antibody with the antibody; binding the antibody to an IL-6 receptor; and thereby inhibiting the proliferative activity of the cell.
  • the proliferative activity of IL-6-dependent myeloma cells can be inhibited by these methods. For example, proliferation of IL-6-dependent human myeloma cell lines such as ILKM-2 and ILKM-3 can be inhibited in this manner.
  • proliferation of myeloma cells in vivo may be inhibited in this manner.
  • the ability of anti-IL-6 receptor antibodies to inhibit the proliferation of IL-6-dependent cells can be employed to enhance a population of cells that is not susceptible to inhibition by anti-IL-6 receptor antibodies relative to a population of cells that is susceptible to such inhibition.
  • this method can be used to enhance a population of Daudi cells relative to a population of ILKM-2 cells in cell culture.
  • the method can be used to enhance the population of non-myeloma cells relative to a population of myeloma cells. This enhancement of one population of cells relative to another can occur in either in vivo or in vi tro situations.
  • kits for inhibiting the proliferative activity of an IL- 6-dependent cell will comprise an anti-IL-6 receptor antibody.
  • kits for inhibiting the proliferative activity of an IL- 6-dependent cell will comprise an anti-IL-6 receptor antibody.
  • kits may also include a method for introducing the antibody into the location in which inhibition of the IL-6-dependent cells is desired.
  • inventions include methods of detecting an IL-6 receptor. These methods include the steps of: obtaining an anti-IL-6 receptor antibody; treating a sample suspected of having an IL-6 receptor with that antibody; and determining whether the sample binds to an IL-6 receptor in the sample.
  • the IL-6 receptor to be detected can be in either whole cells, or in a more or less purified form.
  • the sample could be a cell lysate, a soluble protein portion of a cell, a membrane fraction of a cell, or in a more purified form of the receptor protein.
  • Various detection methods can be employed, for example, sandwich radioimmunoassays and ELISA assays. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. l Amplification of hIL-6R cDNA by PCR.
  • lane a DNA markers DNA digested with ECOR 1 and Hind 111) ;
  • lane b, lane c PCR products from PCR reaction with 1% formamide (lane b) or without formamide (lane c)
  • FIG. 2 Construction of the hIL-6R expression vector pCMV5/R.
  • FIG. 2A Truncated hIL-6R gene.
  • FIG. 2B Recombinant plasmid pCMV5/R.
  • S signal sequence
  • E extracellular domain
  • T transmembrane domain
  • I intracellular domain
  • hGH human growth hormone transcription terminator
  • CMV human cytomegalovirus promotor
  • AMPr Ampicillin resistance gene.
  • FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D show flow cytometric analysis of the UV4 staining on various human cell lines: (FIG. 3A) U266, myeloma cell line; (FIG. 3B) U937, histiocytic lymphoma cell line; (FIG. 3C) Daudi, Burkitt's lymphoma cell line; and (FIG. 3D) K562, erythromyeloid leukemia cell line.
  • FIG. 4A and FIG. 4B show that UV4 inhibits the binding of PE-IL-6 conjugate to IL-6R: (FIG. 4A) Control antibody MOPC-21; and (FIG. 4B) UV4.
  • FIG. 5A, FIG. 5B and FIG. 5C show that UV4 inhibits the proliferation of human myeloma cells: (FIG. 5A) ILKM2 cells; (FIG. 5B) ILKM3 cells; and (FIG. 5C) Daudi cells.
  • the hIL-6R + cell lines U266 and U937 and the hIL-6R ⁇ cell lines Daudi and K562 were obtained from the American Type Culture Collection (Rockville, MD) .
  • the mouse IL- 6R " cell line, M12.4, was a gift from Dr. Philip Tucker
  • PBS sterile phosphate-buffered saline
  • GIT buffer 4M guanidine isothiocyanate, 0.1 M Tris-HCl pH 7.5, 0.1% ⁇ - mercaptoethanol
  • RNA pellet was resuspended in 300 ⁇ l of 0.3M sodium acetate, pH6.0 and precipitated by the addition of 750 ⁇ l cold ethanol and incubated at -70°C for one hour.
  • the precipitated RNA was collected by centrifugation, washed with 80% cold ethanol, dried under vacuum and dissolved in DEPC
  • RNA (dimethyl pyrocarbonate, Sigma) -treated distilled water. The amount of RNA was determined spectrophotometrically.
  • FIG. 1 demonstrates that PCR products from the reaction with formamide yielded a band of IL-6R cDNA.
  • the IL-6R gene was then inserted into the mammalian expression vector pCMV5.
  • the resulting construct contained a 25bp 5' untranslated region plus the full coding sequence of the extracellular and transmembrane domains and a small portion of the intracellular domain for a total insert size of 1500 bp (FIG. 1, FIG. 2) .
  • the pCMV5/R construct was cotransfected into the mouse lymphoma cell line M12.4 along with the pSV2-Neo plasmid by electroporation. After the initial selection in neomycin (G418) , IL-6R expression on the transfectants was detected by a radioactive binding assay. The expression of hIL-6R in the transfected cells as shown in Table 1. TABLE 1
  • IL-6 As can be seen, the binding of IL-6 to transfected M12.4 cells was much greater than its binding to untransfected cells (M12.4) , suggesting that transfectants express hlL- 6R on their surface and that this IL-6R specifically binds to hIL-6.
  • the pool of resistant transfectants which expressed hIL-6R were subcloned by limiting dilution and the clones with the highest expression were chosen to immunize BALB/c mice.
  • the oligodeoxynucleotide primers for synthesizing hIL-6R first-strand cDNA and PCR amplification were derived from the published sequences of the hIL-6R (11) .
  • the sequences of the primers are as follows: forward primer, 5' -TTATCTAGATAAGCTGGCATGGCAGT-3' (SEQ ID NO:1) ; reverse primer, 5' -ATTATCGATGGAGTGGTAGCCGAGGA-3' (SEQ ID NO:2) .
  • the primers span 1500 bp of the hIL-6R coding region (Nucleotide sequences -25 to 1481) .
  • Coli strain NM 522 cells and eukaryotic expression plasmid vector PCMV5 were a gift from Dr. Yan-Ting Zhou (Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas) .
  • the vector PCMV5 (12) contains the human cytomegalovirus (CMV) promoter, the human growth hormone transcription (hGH) termination and polyadenylation signals, and the SV40 origin of replication and enhancer.
  • CMV human cytomegalovirus
  • hGH human growth hormone transcription
  • First strand hIL-6R cDNA was synthesized from U266 RNA using a first-strand cDNA synthesis kit (Pharmacia, Piscataway, NJ) .
  • the first-strand cDNA was further amplified by the polymerase chain reaction (PCR) using Taq polymerase in the presence of 1% formamide (Gibco, Gaithersburg, MD) and a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) .
  • the amplified cDNA was cloned into the polylinker region of pCMV5.
  • the recombinant plasmid, designated pCMV5/R was then used to transform bacteria, E Coli the NM522.
  • Plasmid DNA was extracted from transformed bacterial clones and the transformed clones which contained plasmid pCMV5/R were verified by restriction enzyme analysis. A large amount of plasmid pCMV5/R from the transformed clone was purified by equilibrium centrifugation in CsCl-ethidium bromide gradients (13) .
  • the IL-6R " murine B cell lymphoma cell line M12.4 was cotransfected with plasmids pCMV5/R and pSV2-Neo by electroporation (Biorad, Hercules, CA) . Cells were grown in complete RPMI-1640 medium. After 48 hours, the cells were selected by the addition of 0.5 mg/ml G418 (Gibco, Gaithersburg, MD) . The G418-resistant transfectants were subcloned by limiting dilution and further analyzed for expression of the hIL-6R. Detection of the cell surface hIL-6R:
  • the rhIL-6 was radioiodinated with Nal 125 using the Iodogen reagent (Pierce, Rockville, IL) (14) .
  • the cells were incubated for one hour on ice with 125 I-rhIL-6 in 2% bovine serum albumin (BSA) in (PBS) containing 0.1% sodium azide and then washed three times with PBS.
  • BSA bovine serum albumin
  • the cell-bound radioactivity was determined on a gamma counter.
  • the U266 and M12.4 cell lines served as positive and negative controls, respectively.
  • the mAb UV4 was generated by immunizing mice with the IL-6R + cell line, U266 and the transfected M12.4/R cells. Initial characterization of primary hybridoma cultures and cloned hybridomas was performed by testing the supernatants against the IL-6R + cell lines U266 and M12.4/R and the IL-6R " cell lines K562 and M12.4 by a cellular ELISA. A mAb UV4 which reacted with U266 and M12.4/R, but not with K562 and M12.4 was selected for further study. Isotyping analysis demonstrated that the UV4 was an IgG 1 k.
  • mice were immunized twice at monthly intervals with 5 X 10 6 U266 cells and then injected i.p. three times with M12.4/R cells. Three days before fusion, the mice were injected intravenously (i.v.) with 1 X 10 7 M12.4/R cells.
  • Splenocytes from immunized mice were fused with mouse myeloma cells SP2/0 using 40% polyethylene glycol (PEG) and grown in hypoxanthine/aminopterin/thymidine (HAT) medium (15) .
  • PEG polyethylene glycol
  • HAT hypoxanthine/aminopterin/thymidine
  • Supernatants from growing clones were screened by a cellular ELISA for antibodies against U266, M12.4/R, K562 and M12.4. Hybridomas reacted with U266 and M12.4/R, but not K562 and M12.4.
  • the specific monoclonal antibodies (mAbs) were further screened by flow
  • the mAbs were purified from culture supernatants by affinity chloromotography with Gamma-binding G-agarose column (Gene Corporation, Gaithersburg, MD) .
  • the 96-well ELISA plates were coated for 30 minutes with 50 ⁇ l poly-L-lysine (0.01% in PBS, Sigma, St. Louis, MO) . After removing the poly-L-lysine, 1 X 10 5 cells per well were centrifuged onto the plates at 2000 RPM for 10 minutes. The cells were fixed for 15 minutes with 0.5% glutaraldehyde, and the plates were washed three times with PBS. 50 ⁇ l of 1% BSA in PBS containing lOOmM glycine was added and incubated for 45 minutes. The plates were then blocked with 150 ⁇ l per well of 2% BSA in PBS containing 0.05% sodium azide and stored at 4°C for use.
  • poly-L-lysine 0.01% in PBS, Sigma, St. Louis, MO
  • PE-hIL-6 (R & D system, Minneapolis, MN) was added for an additional hour on ice. The cells were washed three times to remove unbound PE-IL-6 and antibodies, and resuspended in 0.2 ml of PBS for flow cytometric analysis. The PE-Avidin conjugate (R & D system) served as the control for nonspecific binding.
  • UV4 recognizes the same molecule as the goat anti-hIL-6R antibody:
  • UV4 recognizes the same molecule as GAsIL-6R where the isotype-matched myeloma protein MOPC-21 and the control detecting antibody, 125 I-GAOVA do not. This result demonstrates that the antigen captured by UV4 is the hIL-6R molecule.
  • UV4 was the capturing antibody while 125 I-goat anti-human soluble IL-6R antibody (GAsIL-6R, R & D Systems) was used for detection.
  • GAsIL-6R was iodinated with Na 125 I as described above.
  • 1 x 10 7 cultured Daudi or U266 cells were lysed in 1 ml of digitonin lysis buffer (1% digitonin, 10 mM triethanolamine, PH 7.8, 10 mM iodoacetamide, 0.15M NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/ml of leupeptins, 1 ⁇ g/ml of pepstatin A and 1 ⁇ g/ml of antipain) on ice for 20 minutes, and centrifuged to remove cell debris.
  • digitonin lysis buffer 1% digitonin, 10 mM triethanolamine, PH 7.8, 10 mM iodoacetamide, 0.15M NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/ml of leupeptins, 1 ⁇ g/ml of pepstatin A and 1 ⁇ g/ml of antipain
  • a competitive binding assay was used to determine whether the mAb UV4 blocks the binding of IL-6 to the hIL-6R. The results are shown in FIG. 4. Preincubation of U266 cells with UV4 inhibited the binding of a PE-IL-6 conjugate to U266 cells in a dose-dependent manner.
  • the inventors further examined the effect of the UV4 on the proliferation of the IL-6-dependent human myeloma cell lines ILKM-2 and ILKM-3.
  • the cells were cultured with various amounts of UV4 in the presence of a constant amount (2 ng/ml) of rhIL-6.
  • the cells were then pulsed with (3H) -thymidine.
  • FIG. 5 shows that UV4 inhibits the proliferative activity of IL- 6 on the myeloma cell lines ILKM2 and ILKM3, in a dose- dependent manner.
  • the IC 50 of UV4 on both cell lines was 1.5 X 10 "8 M.
  • UV4 did not interfere with proliferation of IL-6R " human cell line, Daudi. Although the growth of murine cell lines 7TD1 and KD83 is dependent on hIL-6, UV4 did not affect the growth of either line (data not shown) . While the above examples demonstrate various preferred embodiments of the present invention, they are in no way meant to be limiting upon the scope of the invention. The skilled artisan will recognize that many variations of the invention are possible without deviating from the nature and spirit of the claimed invention. All such possibilities are to be considered to be part of the invention.

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Abstract

L'invention concerne des anticorps dirigés contre le récepteur de l'IL-6 ainsi que des hybridomes capables de produire ces anticorps. Dans certains modes de réalisation, ces anticorps sont utilisés pour inhiber la fixation de l'IL-6 à un récepteur de l'IL-6. Dans d'autres modes de réalisation, ils sont utilisés pour inhiber la prolifération de cellules dépendant de l'IL-6. Le matériel utilisé pour inhiber la prolifération des cellules dépendant de l'IL-6 est également décrit.
PCT/US1994/011305 1993-10-06 1994-10-06 Anticorps monoclonaux diriges contre le recepteur de l'il-6 humaine WO1995009873A1 (fr)

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AU79672/94A AU7967294A (en) 1993-10-06 1994-10-06 A monoclonal anti-human il-6 receptor antibody

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US08/132,619 1993-10-06

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WO2009014263A1 (fr) 2007-07-26 2009-01-29 Osaka University Agent pour le traitement de l'ophtalmie contenant un inhibiteur du récepteur de l'interleukine 6 en tant qu'ingrédient actif
WO2009044774A1 (fr) 2007-10-02 2009-04-09 Chugai Seiyaku Kabushiki Kaisha Remède contre une réaction de greffe contre l'hôte comprenant un inhibiteur des récepteurs de l'interleukine-6 en tant qu'ingrédient actif
US7824674B2 (en) 1998-03-17 2010-11-02 Chugai Seiyaku Kabushiki Kaisha Preventive or therapeutic agent for inflammatory bowel disease comprising IL-6 antagonist as an active ingredient
US8034344B2 (en) 2008-05-13 2011-10-11 Novimmune S.A. Anti-IL-6/IL-6R antibodies and methods of use thereof
US8043617B2 (en) 2006-06-02 2011-10-25 Regeneron Pharmaceuticals, Inc. Human antibodies to human IL-6 receptor
US8080248B2 (en) 2006-06-02 2011-12-20 Regeneron Pharmaceuticals, Inc. Method of treating rheumatoid arthritis with an IL-6R antibody
US8562990B2 (en) 2000-10-25 2013-10-22 Chugai Seiyaku Kabushiki Kaisha Method of treating psoriatic arthritis with an IL-6 receptor antibody
US9017678B1 (en) 2014-07-15 2015-04-28 Kymab Limited Method of treating rheumatoid arthritis using antibody to IL6R
US9260516B2 (en) 2006-04-07 2016-02-16 Osaka University Method for promoting muscle regeneration by administering an antibody to the IL-6 receptor
US9539322B2 (en) 2010-05-28 2017-01-10 National University Corporation Hokkaido University Method of enhancing an antitumor T cell response by administering an anti-IL-6 receptor antibody
EP3269738A1 (fr) 2004-03-24 2018-01-17 Chugai Seiyaku Kabushiki Kaisha Sous-types d'anticorps humanisés contre le récepteur de l'interleukine-6
US10717781B2 (en) 2008-06-05 2020-07-21 National Cancer Center Neuroinvasion inhibitor
US10927435B2 (en) 2011-10-11 2021-02-23 Sanofi Biotechnology Compositions for the treatment of rheumatoid arthritis and methods of using same
US11098127B2 (en) 2010-01-08 2021-08-24 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-interleukin-6 receptor (IL-6R) antibodies
US11498969B2 (en) 2019-01-31 2022-11-15 Sanofi Biotechnology Compositions and methods for treating juvenile idiopathic arthritis
US11692037B2 (en) 2017-10-20 2023-07-04 Hyogo College Of Medicine Anti-IL-6 receptor antibody-containing medicinal composition for preventing post-surgical adhesion
US11851486B2 (en) 2017-05-02 2023-12-26 National Center Of Neurology And Psychiatry Method for predicting and evaluating therapeutic effect in diseases related to IL-6 and neutrophils
US12116414B2 (en) 2007-09-26 2024-10-15 Chugai Seiyaku Kabushiki Kaisha Method of modifying isoelectric point of antibody via amino acid substitution in CDR

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