JPH0257195A - Human type monoclonal antibody of anti-tetanus toxin - Google Patents
Human type monoclonal antibody of anti-tetanus toxinInfo
- Publication number
- JPH0257195A JPH0257195A JP63206182A JP20618288A JPH0257195A JP H0257195 A JPH0257195 A JP H0257195A JP 63206182 A JP63206182 A JP 63206182A JP 20618288 A JP20618288 A JP 20618288A JP H0257195 A JPH0257195 A JP H0257195A
- Authority
- JP
- Japan
- Prior art keywords
- human
- monoclonal antibody
- tetanus toxin
- human monoclonal
- mouse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229940118376 tetanus toxin Drugs 0.000 title claims abstract description 24
- 230000002096 anti-tetanic effect Effects 0.000 title description 14
- 210000004027 cell Anatomy 0.000 claims abstract description 37
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 16
- 108010055044 Tetanus Toxin Proteins 0.000 claims abstract description 14
- 230000003472 neutralizing effect Effects 0.000 claims abstract description 12
- 239000012634 fragment Substances 0.000 claims description 17
- 210000000628 antibody-producing cell Anatomy 0.000 claims description 9
- 241000700605 Viruses Species 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 230000009257 reactivity Effects 0.000 claims 4
- 101000597553 Homo sapiens Protein odr-4 homolog Proteins 0.000 claims 2
- 101000687474 Homo sapiens Rhombotin-1 Proteins 0.000 claims 2
- 102100024869 Rhombotin-1 Human genes 0.000 claims 2
- 241000894007 species Species 0.000 claims 1
- 210000004698 lymphocyte Anatomy 0.000 abstract description 13
- 206010043376 Tetanus Diseases 0.000 abstract description 6
- 239000000427 antigen Substances 0.000 abstract description 5
- 108091007433 antigens Proteins 0.000 abstract description 5
- 102000036639 antigens Human genes 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 15
- 239000002609 medium Substances 0.000 description 13
- 230000004927 fusion Effects 0.000 description 8
- 239000012228 culture supernatant Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000006386 neutralization reaction Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 229960000814 tetanus toxoid Drugs 0.000 description 4
- 241000282412 Homo Species 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 238000003113 dilution method Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 101001111742 Homo sapiens Rhombotin-2 Proteins 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100023876 Rhombotin-2 Human genes 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000003163 cell fusion method Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000013341 scale-up Methods 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- SPJOZZSIXXJYBT-UHFFFAOYSA-N Fenson Chemical compound C1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 SPJOZZSIXXJYBT-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108091002531 OF-1 protein Proteins 0.000 description 1
- 108010084214 Peptide PHI Proteins 0.000 description 1
- 239000000132 Peptide PHI Substances 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、破傷風菌の感染による破傷風の発症防止と治
療を行い得るヒト型モノクローナル抗体、又はこれらの
モノクローナル抗体を混合することにより、より効果的
に発症を防止し得るオリゴクローナルなヒト型抗体に関
するものである。Detailed Description of the Invention (Field of Industrial Application) The present invention provides a human monoclonal antibody that can prevent and treat the onset of tetanus caused by Clostridium tetani infection, or a mixture of these monoclonal antibodies that can be more effective. The present invention relates to an oligoclonal human antibody that can prevent the onset of the disease.
（従来の技術）
抗体を、予防、治療又は診断を目的としてヒトに投与す
る場合、ヒト型モノクローナル抗体は、マウスモノクロ
ーナル抗体に比へ抗原性に関する問題が少ないと考えら
れており臨床治療上利用範囲が非常に大きい。破傷風に
ついても、従来の馬抗血清又は供血に頼るヒト抗血清に
代わるものとして期待されている。(Prior art) When antibodies are administered to humans for the purpose of prevention, treatment, or diagnosis, human monoclonal antibodies are thought to have fewer problems with antigenicity than mouse monoclonal antibodies, and their use in clinical treatment is limited. is very large. For tetanus, it also holds promise as an alternative to traditional horse antisera or human antisera that rely on blood donations.
ところで、ヒト型モノクローナル抗体の作製は、マウス
の場合と比べて、融合効率が悪い、抗体産生の安定性が
悪い、抗体を大量に得ることが困難である、免疫された
リンパ球が自由に得られない、ＩｇＧタイプの抗体を得
ることが難しいなどの不利な点があった。これらを克服
するためにヒトの親細胞株の改良、 （ヒト・マウス）
ヘテロミエローマを親細胞として用いる方法〔プロシー
ディング オブ ナショナル アカデミ−オブ サイエ
ンス（Ｐｒｏｃ、 Ｎａｔ、 Ａｃａｄ、 Ｓｃｉ、
ＵＳＡ） ８０．７３０８゜（１９８３）等〕、又はマ
ウスミエローマを親細胞として用いる方法〔ジャーナル
オブ クリニカルインベスティデーション（Ｊ、 Ｃ
ｌ１ｎ、 Ｉｎｖｅｓｔ、） ＴＯ。However, compared to the production of human monoclonal antibodies in mice, the fusion efficiency is poor, the stability of antibody production is poor, it is difficult to obtain antibodies in large quantities, and immunized lymphocytes are freely available. There were disadvantages such as the difficulty in obtaining IgG type antibodies. Improvement of human parent cell lines to overcome these problems (human/mouse)
Method of using heteromyeloma as parent cells [Proceedings of the National Academy of Sciences (Proc. Nat. Acad. Sci.
USA) 80.7308゜(1983), etc.], or a method using mouse myeloma as parent cells [Journal of Clinical Investigation (J, C
l1n, Invest,) TO.
＋３０６． （１９８２）等〕などが考えられている。+306. (1982), etc. are being considered.
その他にｉｎ ｖｉｔｒｏで抗原刺激することにより特
異抗体産生ハイブリドーマを効率よく得る方法〔ヨーロ
ピアン ジャーナル オブ イミュノロジー（Ｅｕｒ。In addition, there is a method for efficiently obtaining specific antibody-producing hybridomas by antigen stimulation in vitro [European Journal of Immunology (Eur.
Ｊ、 １ｍｍｕｎｏ１． 、ｕ＋ ２３． （１９８４
）等）、又は向リンパ性ウィルスであるエプスタイン・
バーウィルス（ＥＢＶ）で抗体産生細胞を形質転換する
方法〔サイエンス （Ｓｃｉｅｎｃｅ） １９９．１４
３９． （１９７８）等〕、更にはＥＢＶ形質転換細胞
を親細胞と融合する方法〔プロシーディング オブ ナ
ショナル アカデミ−オブ サイエンス（Ｐｒｏｃ、
Ｎａｔ、 Ａｃａｄ。J, 1mmuno1. , u+ 23. (1984
), or the lymphotropic virus Epstein.
Method for transforming antibody-producing cells with Barr virus (EBV) [Science 199.14
39. (1978), etc.], and a method for fusing EBV-transformed cells with parent cells [Proceedings of the National Academy of Sciences (Proc.
Nat, Acad.
Ｓｃｉ、 ＬＩＳＡ） ７９．６６５＋、 （１９８２
）等〕などが試みられている。Sci, LISA) 79.665+, (1982
), etc.] have been attempted.
（発明が解決しようとする課題）
従来の技術では、破傷風の予防、治療に有用な、高い中
和能を有するヒト型モノクローナル抗体、特にＩｇＧタ
イプのヒト型モノクローナル抗体を安定にしかも大量に
提供することが困難であフた。(Problems to be Solved by the Invention) Conventional techniques have not been able to provide stably and in large quantities human monoclonal antibodies, particularly IgG type human monoclonal antibodies, which have a high neutralizing ability and are useful for the prevention and treatment of tetanus. It was difficult and ended.
本発明はこのような問題点を解決しようとして行ったも
のである。The present invention has been made in an attempt to solve these problems.
（課題を解決するための手段）
本発明者らは、ＩｇＧタイプの抗破傷風毒素ヒト型モノ
クローナル抗体を安定にしかも大量に産生ずる細胞株の
樹立を目的として検討した結果、抗破傷風毒素中和抗体
価が高く、かつＩｇＧタイプの抗体を産生ずるヒト由来
リンパ球を選択し、ＥＢＶで形質転換する方法又はこの
リンパ球由来のハイブリドーマを作製する方法により、
目的に適合する抗破傷風毒素ヒト型モノクローナル抗体
産生細胞株が得られ、これに基づいて本発明を完成する
に至った。(Means for Solving the Problems) As a result of studies aimed at establishing a cell line that stably and produces large amounts of IgG type anti-tetanus toxin human monoclonal antibodies, the present inventors found that anti-tetanus toxin neutralizing antibodies By selecting human-derived lymphocytes that have high titers and producing IgG-type antibodies and transforming them with EBV, or by producing hybridomas derived from these lymphocytes,
An anti-tetanus toxoid human monoclonal antibody-producing cell line suitable for the purpose was obtained, and the present invention was completed based on this cell line.
以下に本発明について詳細に説明する。The present invention will be explained in detail below.
本発明において用いられたリンパ球は、破傷風トキソイ
ド（Ｔ 、Ｔ 、）によって免疫され、高い抗破傷風毒
素中和抗体を有しているヒトから採取されたものである
。これをｉｎ ｖｉｔｒｏにおいて更にＴ、Ｔ、により
抗原刺激した後に親細胞との融合を行った。リンパ球は
末梢血からフィコールバック（ファルマシア社）を用い
た比重遠心法により分離した。ＥＢＶで形質転換を行う
場合は、上記のリンパ球から更に、羊赤血球を用いたロ
ゼツト法によりＢリンパ球を分離し、これに８９５−８
細胞（マーモセット由来）培養上清から得たＥＢＶを感
染させ、３７℃、５％ＣＯ２下で約２週間培養を行った
。形質転換した細胞は、限界希釈法によりクローニング
を行うか、又は更に親細胞との融合を行った。親細胞と
しては融合効率がよく、得られたバイブリドーマが比較
的安定にＩｇＧタイプの抗体を産生し、しかもヌードマ
ウスで腹水を容易に生産することのできるＳＨＭ Ｄ−
３３（ヒトΦマウスヘテロミエローマ、ＡＴＣＣＣＲＬ
１６６Ｂ）を用いた。親細胞との融合はポリエチレ
ングリコールを用いた公知の方法で行い、特異抗体を産
生ずるハイブリドーマの判定は、Ｔ、Ｔ、をコートした
プレートを用いたＥＩＡ法（酵素免疫測定法）により行
った。クローニングは限界希釈法又は軟寒天法により行
った。抗体はヌードマウスの腹水、又は連続培養装置を
用いた培養上清中から硫安分画法及び／又はＰｒｏｔｅ
ｉｎ−Ａカラムを用いて容易に高純度品を大量に得るこ
とが可能である。The lymphocytes used in the present invention were collected from humans who had been immunized with tetanus toxoid (T , T ) and had high anti-tetanus toxoid neutralizing antibodies. After further antigen stimulation with T and T in vitro, fusion with parent cells was performed. Lymphocytes were separated from peripheral blood by specific gravity centrifugation using Ficollvac (Pharmacia). When transforming with EBV, B lymphocytes are further separated from the above lymphocytes by the rosette method using sheep red blood cells, and then 895-8
The cells were infected with EBV obtained from the culture supernatant of cells (derived from marmoset) and cultured for about 2 weeks at 37°C and 5% CO2. The transformed cells were cloned by the limiting dilution method or further fused with the parent cells. SHM D- has a high fusion efficiency as a parent cell, the resulting hybridoma relatively stably produces IgG type antibodies, and can easily produce ascites in nude mice.
33 (human Φ mouse heteromyeloma, ATCCCRL
166B) was used. Fusion with parent cells was performed by a known method using polyethylene glycol, and hybridomas producing specific antibodies were determined by EIA (enzyme immunoassay) using T, T coated plates. Cloning was performed by the limiting dilution method or the soft agar method. Antibodies can be obtained by ammonium sulfate fractionation and/or Prote from ascites of nude mice or culture supernatant using a continuous culture device.
It is possible to easily obtain a high purity product in large quantities using an in-A column.
以上のような手段を用いることにより、本発明を完成す
るに至った。なお本発明で得られたバイプリドーマＴＴ
ＧＩＨ，ＴＴＧ２、ＴＴＧ３、ＴＴＧ４そして形質転換
細胞ＴＴＧＩは工業技術院微生物工業技術研究所に寄託
しである。寄託番号は、それぞれ微工研菌寄第１０２１
６号、微工研菌寄第１０２１３号、微工研菌寄第１０２
１４号、微工研菌寄第１０２１５号、微工研菌寄第１０
２１２号である。なお上記のハイブリドーマ及び形質転
換細胞の名称は、それらが産生ずるモノクローナル抗体
の名称にも準用する。By using the above-mentioned means, the present invention has been completed. In addition, bilidoma TT obtained by the present invention
GIH, TTG2, TTG3, TTG4 and the transformed cell TTGI have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. The deposit numbers are 1021 and 1021, respectively.
No. 6, Microtech Research Institute No. 10213, Microtechnology Research Institute No. 102
No. 14, Microtech Research Institute No. 10215, Microtechnology Research Institute No. 10
This is No. 212. The names of the above hybridomas and transformed cells also apply to the names of the monoclonal antibodies they produce.
（発明の効果）
本発明により、 ＩｇＧタイプの抗破傷風毒素ヒト型モ
ノクローナル抗体が安定にしかも大量に得られるように
なり、更にモノクローナル抗体を混合することにより実
用化が可能な、高い抗破傷風毒素中和能が得られたこと
から、ヒトの破傷風の予防、治療への臨床応用が大いに
期待される。(Effects of the Invention) According to the present invention, IgG type anti-tetanus toxin human monoclonal antibodies can be obtained stably and in large quantities, and furthermore, by mixing the monoclonal antibodies, a high anti-tetanus toxin human monoclonal antibody can be obtained which can be put to practical use. Since this function was obtained, there are great expectations for its clinical application in the prevention and treatment of tetanus in humans.
（実施例）
（１）ヒトリンパ球の調製
抗破傷風毒素抗体産生ハイブリドーマを効率よく得るた
めに、Ｔ、Ｔ、を追加免疫したボランティアの血清中の
破傷風毒素中和抗体価を調べた。約２０国際単位／ｍｌ
の中和抗体価を示した２人のボランティアから各々２０
ｍ ｌずつ採血し、フィコールバックを用いた比重遠
心法によりリンパ球を分離した。ＥＢＶを感染させ形質
転換細胞を得る場合には、更に羊赤血球を用いたロゼツ
ト法によりＢリンパ球を分離した。(Example) (1) Preparation of human lymphocytes In order to efficiently obtain anti-tetanus toxin antibody-producing hybridomas, the tetanus toxin neutralizing antibody titer in the serum of volunteers boosted with T and T was investigated. Approximately 20 international units/ml
20 each from two volunteers who showed neutralizing antibody titers of
ml of blood was collected, and lymphocytes were separated by specific gravity centrifugation using Ficollvac. When infected with EBV to obtain transformed cells, B lymphocytes were further separated by the rosette method using sheep red blood cells.
（２） ｉｎ ｖｉｔｒｏ抗原刺激
特異抗体産生リンパ球の数を増加させるため、及び後に
行う融合効率を向上させる目的で １ｎｖｉｔｒｏで抗
原刺激を行った。培地はＲＰＭＩ−１６４０、ダルベツ
コＭＥＭ （ＤＭＥＭ）、ハムＦ１２を２：１：１の割
合で混合したＲＤＦ培地８５％と牛胎児血清（Ｆｅ２）
１５％とを混合した培地に芽球化因子（ＰＷＭ）を １
００００倍希釈で添加し、更にＴ、Ｔ、を１０ ｎｇ／
ｉｆの濃度になるように加えた。２４ウェル−マイクロ
プレートの各ウェルに分注した５Ｘ１０５個のリンパ球
を上記の培地で、３７℃、５％ＣＯ２の条件で５−６日
間培養し、以後の融合に用いた。(2) In vitro antigen stimulation In order to increase the number of specific antibody-producing lymphocytes and to improve the efficiency of subsequent fusion, 1 in vitro antigen stimulation was performed. The medium is 85% RDF medium mixed with RPMI-1640, Dulbecco's MEM (DMEM), Ham's F12 at a ratio of 2:1:1, and fetal bovine serum (Fe2).
Add blastization factor (PWM) to a medium mixed with 15%
0,000 times diluted, and further added 10 ng/T,
It was added to the concentration of if. 5×10 5 lymphocytes dispensed into each well of a 24-well microplate were cultured in the above medium at 37° C. and 5% CO 2 for 5-6 days, and used for subsequent fusion.
（３）ＥＢＶ形質転換法
（１）で調製したＢリンパ球 １ｘｌＯ５個に対し、Ｂ
９δ−８細胞の７日目培養上清を ０．４５μｍメンブ
レンフィルターで濾過した濾過液をＥＢＶとして１ ｍ
ｌ加え、３７℃、δ％ＣＯ２で３時間吸着させた。そ
の後、１５％ＦＣ５とＴ、Ｔ、１０ｎ ｇ ／ ｍ ｌ
を加えたＲＤＦ培地に懸濁し、Ｕ底９６ウエルマイクロ
プレートに１ウェル当り１ｘ１０４個の感染細胞を分注
した。１ｏ日から２週間培養することにより細胞が増殖
してくる。これらの増殖したウェルから培養上清を採取
し、ＥＩＡ法により陽性ウェルを判定後、フィーダー細
胞（マイトマイシンＣ処理したリンパ球）を用いたＵ底
９６ウエルマイクロプレートでクローニングを行うか、
又はスケールアップ後、親細胞と更に融合を行った。(3) For 5 1xlO B lymphocytes prepared by EBV transformation method (1),
The 7-day culture supernatant of 9δ-8 cells was filtered through a 0.45 μm membrane filter, and the filtrate was designated as EBV and 1 m
1 was added and adsorption was carried out for 3 hours at 37°C and δ% CO2. Then 15% FC5 and T, 10 ng/ml
The infected cells were suspended in RDF medium supplemented with 1 x 104 infected cells per well in a U-bottom 96-well microplate. By culturing for 2 weeks from day 1, the cells will proliferate. Culture supernatants are collected from these proliferated wells, positive wells are determined by the EIA method, and cloning is performed in a U-bottom 96-well microplate using feeder cells (lymphocytes treated with mitomycin C), or
Alternatively, after scale-up, further fusion with parent cells was performed.
第１表 ＥＢＶ形質転換法による特異抗体産生ウェ
ルの出現率
第１表に示したように、形質転換細胞はすべてのウェル
に出現し、しかもその約７０％が抗破傷風毒素ＩｇＧ抗
体を産生じていた。Table 1: Rate of occurrence of specific antibody-producing wells by EBV transformation method As shown in Table 1, transformed cells appeared in all wells, and about 70% of them produced anti-tetanus toxin IgG antibodies. Ta.
（４）融合操作
親細胞として、 （ヒト・マウス）のヘテロミエローマ
であるＳＨＭ Ｄ−３３を用い、１５％ＦＣ５を含む
ＲＤＦ培地で培養した。ＳＨＭ Ｄ−３３及び上記（２
）又は（３）のヒトリンパ球をＲＤＦ培地で２回洗浄後
、１：２ の割合で混合する。再度遠心後、細胞ペレッ
トをよく分散し、１ ｍ ｌの６０％ポリエチレングリ
コールを１分間にわたって滴下する。更に１分間３７℃
で反応後、５分間で１０ｍ１のＲＤＦ培地を加える。遠
心した後、１５％ＦＣ８培地を含むＲＤＦ培地にＳ濶し
９６ウエルマイクロプレートにｌウェル当り２ｘ１０４
個の親細胞が含まれるように分注する。翌日、ＨＡＴ培
地（０，１ｍＭ ヒボキサンチン、１６μＭチミジン
及び ０．４μＭ アミノプテリン添加１δ％ＦＣ５／
ＲＤＦ培地）に交換し３７℃、６％ＣＯ２の条件下で培
養する。２−３日間隔でＨＡＴ培地を交換する。約２週
問後に、ハイブリドーマの増殖が見られたウェルの培養
上清を採取し、特異抗体産生ウェルをＥＩＡ法で判定す
る。抗破傷風毒素抗体を産生じている細胞は、限界希釈
法又は軟寒天法によりクローニングを行った。ハイブリ
ドーマの抗体産生量は、デイツシュで培養した場合、５
日間の培養で約１０−１５μｇ ／ ｍ １の抗体濃度
が得られ、現在まで６力月以上にわたって安定に生産し
ている。(4) Fusion operation SHM D-33, a human/mouse heteromyeloma, was used as the parent cell and cultured in RDF medium containing 15% FC5. SHM D-33 and the above (2
) or (3) are washed twice with RDF medium and mixed at a ratio of 1:2. After centrifugation again, the cell pellet is well dispersed and 1 ml of 60% polyethylene glycol is added dropwise over 1 minute. 37℃ for another 1 minute
After the reaction, add 10 ml of RDF medium for 5 minutes. After centrifugation, transfer to RDF medium containing 15% FC8 medium and place 2x104 cells per well in a 96-well microplate.
Aliquot the sample to include 500 parent cells. The next day, HAT medium (1δ% FC5/supplemented with 0.1mM hyboxanthin, 16μM thymidine and 0.4μM aminopterin) was added.
RDF medium) and cultured at 37°C and 6% CO2. Change HAT medium every 2-3 days. After about two weeks, culture supernatants from wells in which hybridoma growth was observed are collected, and specific antibody-producing wells are determined by EIA. Cells producing anti-tetanus toxin antibodies were cloned by limiting dilution method or soft agar method. The amount of antibody produced by hybridomas is 5
An antibody concentration of approximately 10-15 μg/m1 was obtained by culturing for one day, and the antibody has been stably produced for more than 6 months to date.
第２表 細胞融合法による特異抗体産生ウェルの出
現率
第２表は、細胞融合法によるハイブリドーマの出現率と
抗破傷風毒素ＩｇＧ抗体産生ハイブリドーマの出現率を
示している。Table 2 Appearance rate of specific antibody-producing wells by cell fusion method Table 2 shows the appearance rate of hybridomas and the appearance rate of anti-tetanus toxin IgG antibody-producing hybridomas by cell fusion method.
（５）抗体が認識する抗原部位〔Ａ・Ｂ−Ｃフラグメン
ト）の決定
抗体が認識する抗原部位の決定は、破傷風毒素をパパイ
ン処理して得られる〔Ａ・Ｂ）フラグメント（Ａフラグ
メントとＢフラグメントの複合物）又はＣフラグメント
をそれぞれコートしたプレートを用いたＥＩＡ法により
行った。 〔Ａ・Ｂ）及びＣフラグメントの精製法は、
まず精製破傷風毒素を２５℃で１６時間パパイン処理を
行った。次いでＧ３０００ＳＷカラム（東ソー社製）を
用いた高速液体クロマトグラフィーによるゲル濾過を行
い、 〔Ａ・Ｂ）フラグメントとＣフラグメントに分離
精製した。更に、 〔Ａ・Ｂ）フラグメント画分は、ハ
イドロキシアパタイトによる吸着クロマトグラフィーを
行い、その後、抗Ｃフラグメント抗体をリガンドとした
カラムを通過させて混在する微量の毒素を除き〔ＡφＢ
〕フラグメントを高度に精製した。これらの精製フラグ
メントを、コーティング緩衝液で１０μｇ／ｍｌに希釈
してプレートにコートした。以後のＥＩＡ実験は常法に
したがった。(5) Determination of the antigenic site [A/B-C fragment) recognized by the antibody The antigenic site recognized by the antibody is determined by [A/B] fragments (A fragment and B fragment) obtained by treating tetanus toxin with papain. The EIA method was performed using a plate coated with a composite of C) or a C fragment, respectively. [A, B) and C fragment purification methods are as follows:
First, purified tetanus toxin was treated with papain at 25°C for 16 hours. Next, gel filtration was performed by high performance liquid chromatography using a G3000SW column (manufactured by Tosoh Corporation) to separate and purify the [A/B] fragment and C fragment. Furthermore, the [A/B) fragment fractions were subjected to adsorption chromatography using hydroxyapatite, and then passed through a column using an anti-C fragment antibody as a ligand to remove a trace amount of toxins [AφB].
] The fragment was highly purified. These purified fragments were diluted to 10 μg/ml in coating buffer and coated onto plates. The subsequent EIA experiments followed the usual method.
く６）マウスを用いた破傷風毒素中和実験マウスを用い
た破傷風毒素中和実験に使用する抗体は以下のようにし
て調製した。 （４）の操作で得られたハイブリドーマ
の培養をスケールアップし、その培養上清を４０％飽和
硫酸アンモニウムで塩析、遠心後、そのペレットをリン
酸緩衝化生理食塩水ＣＰＢＳ（−））で溶解した後Ｐ
Ｂ Ｓ （−）で透析した。遠心後、上清を中和実験に
用いた。6) Tetanus toxin neutralization experiment using mice Antibodies used in tetanus toxin neutralization experiments using mice were prepared as follows. Scale up the hybridoma culture obtained in step (4), salt out the culture supernatant with 40% saturated ammonium sulfate, centrifuge, and dissolve the pellet in phosphate buffered saline CPBS(-)). After doing P
It was dialyzed against B S (-). After centrifugation, the supernatant was used for neutralization experiments.
抗体濃度は、濃度の既知の標準ヒ）ＩｇＧを用いたＥＩ
Ａ法により決定した。Antibody concentration was determined by EI using standard human IgG of known concentration.
Determined by method A.
中和実験は以下のようにして行った。試験毒素とモノク
ローナル抗体を混合し、３７℃で１時間反応させその
０．５ｍｌをマウス（ＯＦ−１株）の後肢に筋肉内注射
を行った。対照として抗破傷風ヒト免疫グロブリン、テ
クノプリン（ミドリ十字社製）、を用い同様の実験を行
った。中和抗体価は９６時間、１週間、２週間マウスを
観察し、症状の変化及び致死で判定した。The neutralization experiment was conducted as follows. Mix the test toxin and monoclonal antibody and let them react at 37°C for 1 hour.
0.5 ml was intramuscularly injected into the hind leg of a mouse (OF-1 strain). A similar experiment was conducted using anti-tetanus human immunoglobulin, Technopurin (manufactured by Midori Juji Co., Ltd.) as a control. The neutralizing antibody titer was determined by observing the mice for 96 hours, 1 week, and 2 weeks, and determining the change in symptoms and mortality.
第３表
モノクローナル抗体の性質
第４表
モノクローナル抗体を混合した
場合の中和抗体価
第３表に結果を示したように、モノクローナル抗体ＴＴ
Ｇ２は〔Ａ・Ｂ）及びＣフラグメントの両方を認識し、
高い抗破傷風毒素中和抗体価を有している。しかも第４
表に示したように、モノクローナル抗体ＴＴＧ２と本発
明のその他のモノクローナル抗体１種又は２種以上とを
混合することにより抗破傷風毒素中和抗体価が３倍以上
上昇した。Table 3 Properties of monoclonal antibodies Table 4 Neutralizing antibody titer when monoclonal antibodies are mixed As shown in Table 3, monoclonal antibodies TT
G2 recognizes both [A and B) and C fragments,
It has a high anti-tetanus toxin neutralizing antibody titer. Moreover, the fourth
As shown in the table, by mixing monoclonal antibody TTG2 with one or more other monoclonal antibodies of the present invention, the anti-tetanus toxoid neutralizing antibody titer increased three times or more.
Claims (1)
ノクローナル抗体。 （２）ヒト由来抗体産生細胞と（ヒト・マウス）ヘテロ
ミエローマ細胞とを融合させて得られたハイブリドーマ
が産生し、以下の性質を有する請求項１記載のヒト型モ
ノクローナル抗体。 Ｉｇクラス：ＩｇＧ 反応性：破傷風毒素の〔Ａ・Ｂ〕フラグメントを認識す
る （３）ヒト由来抗体産生細胞と（ヒト・マウス）ヘテロ
ミエローマ細胞とを融合させて得られたハイブリドーマ
が産生し、以下の性質を有する請求項１記載のヒト型モ
ノクローナル抗体。 Ｉｇクラス：ＩｇＧ 反応性：破傷風毒素の〔Ａ・Ｂ〕及び Ｃフラグメントを認識する （４）向リンパ性ウィルスによって形質転換されたヒト
由来抗体産生細胞が産生し、以下の性質を有する請求項
１記載のヒト型モノクローナル抗体。 Ｉｇクラス：ＩｇＧ 反応性：破傷風毒素の〔Ａ・Ｂ〕フラグメントを認識す
る （５）向リンパ性ウィルスによって形質転換されたヒト
由来抗体産生細胞と（ヒト・マウス）ヘテロミエローマ
細胞とを融合させて得られたハイブリドーマが産生し、
以下の性質を有する請求項１記載のヒト型モノクローナ
ル抗体。 Ｉｇクラス：ＩｇＧ 反応性：破傷風毒素の〔Ａ・Ｂ〕フラグメントを認識す
る （６）請求項３記載のヒト型モノクローナル抗体と、請
求項２記載のヒト型モノクローナル抗体、請求項４記載
のヒト型モノクローナル抗体及び請求項５記載のヒト型
モノクローナル抗体からなる群から選ばれた１種又は２
種以上のヒト型モノクローナル抗体とを混合することに
より破傷風毒素中和能を上昇させる特徴を有するヒト型
モノクローナル抗体。（７）（ヒト・マウス）ヘテロミ
エローマ（ＳＨＭＤ−３３）とヒト由来抗体産生細胞と
のハイブリドーマで、請求項２記載のヒト型モノクロー
ナル抗体を産生するＴＴＧ３。 （８）（ヒト・マウス）ヘテロミエローマ（ＳＨＭＤ−
３３）とヒト由来抗体産生細胞とのハイブリドーマで、
請求項２記載のヒト型モノクローナル抗体を産生するＴ
ＴＧ４。 （９）（ヒト・マウス）ヘテロミエローマ（ＳＨＭＤ−
３３）とヒト由来抗体産生細胞とのハイブリドーマで、
請求項３記載のヒト型モノクローナル抗体を産生するＴ
ＴＧ２。 （１０）請求項４記載のヒト型モノクローナル抗体を産
生する形質転換細胞ＴＴＧ１。 （１１）（ヒト・マウス）ヘテロミエローマ（ＳＨＭＤ
−３３）と形質転換細胞（ＴＴＧ１）とのハイブリドー
マで、請求項５記載のヒト型モノクローナル抗体を産生
するＴＴＧ１Ｈ。[Scope of Claims] (1) A human monoclonal antibody having high neutralizing ability against tetanus toxin. (2) The human monoclonal antibody according to claim 1, which is produced by a hybridoma obtained by fusing human-derived antibody-producing cells and (human/mouse) heteromyeloma cells and has the following properties. Ig class: IgG Reactivity: Recognizes the [A/B] fragment of tetanus toxin (3) A hybridoma obtained by fusing human-derived antibody-producing cells with (human/mouse) heteromyeloma cells is produced, and the following The human monoclonal antibody according to claim 1, which has the following properties. Ig class: IgG Reactivity: Recognizing [A, B] and C fragments of tetanus toxin (4) Produced by human-derived antibody-producing cells transformed by a lymphotropic virus, and having the following properties: Claim 1 The human monoclonal antibody described. Ig class: IgG Reactivity: Recognizes the [A/B] fragment of tetanus toxin (5) By fusing human-derived antibody-producing cells transformed by a lymphotropic virus with (human/mouse) heteromyeloma cells. The obtained hybridoma produces
The human monoclonal antibody according to claim 1, having the following properties. Ig class: IgG Reactivity: Recognizes the [A/B] fragment of tetanus toxin (6) The human monoclonal antibody according to claim 3, the human monoclonal antibody according to claim 2, and the human type according to claim 4 One or two selected from the group consisting of a monoclonal antibody and the human monoclonal antibody according to claim 5.
A human monoclonal antibody that has the characteristic of increasing its ability to neutralize tetanus toxin by mixing it with human monoclonal antibodies of more than one species. (7) TTG3, which is a hybridoma of (human/mouse) heteromyeloma (SHMD-33) and human-derived antibody-producing cells, which produces the human monoclonal antibody according to claim 2. (8) (Human/Mouse) Heteromyeloma (SHMD-
33) and human-derived antibody-producing cells,
T producing the human monoclonal antibody according to claim 2.
TG4. (9) (Human/Mouse) Heteromyeloma (SHMD-
33) and human-derived antibody-producing cells,
T producing the human monoclonal antibody according to claim 3.
TG2. (10) A transformed cell TTG1 that produces the human monoclonal antibody according to claim 4. (11) (Human/Mouse) Heteromyeloma (SHMD)
-33) and a transformed cell (TTG1), TTG1H, which produces the human monoclonal antibody according to claim 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63206182A JP2627076B2 (en) | 1988-08-19 | 1988-08-19 | Anti-tetanus toxin human monoclonal antibody, neutralizing agent for tetanus toxin using the same, and hybridoma producing human monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63206182A JP2627076B2 (en) | 1988-08-19 | 1988-08-19 | Anti-tetanus toxin human monoclonal antibody, neutralizing agent for tetanus toxin using the same, and hybridoma producing human monoclonal antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0257195A true JPH0257195A (en) | 1990-02-26 |
| JP2627076B2 JP2627076B2 (en) | 1997-07-02 |
Family
ID=16519174
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63206182A Expired - Fee Related JP2627076B2 (en) | 1988-08-19 | 1988-08-19 | Anti-tetanus toxin human monoclonal antibody, neutralizing agent for tetanus toxin using the same, and hybridoma producing human monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2627076B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018018123A2 (en) | 2016-07-29 | 2018-02-01 | Fundação Butantan | Human anti-tetanus monoclonal antibodies neutralising infection by c. tetani, method for obtaining said monoclonal antibodies and use thereof in immunotherapy in the case of accidents with possible infection by tetanus bacillus |
| WO2019128120A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Fully human neutralizing antibody combating tetanus toxin |
| WO2019129214A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof |
| WO2019128119A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Fully human monoclonal antibody for neutralizing tetanus toxin, and uses thereof |
| WO2019128121A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Anti-tetanus toxin neutralizing antibody, preparation method therefor, and uses thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60251881A (en) * | 1984-04-26 | 1985-12-12 | シタス コ−ポレイシヨン | Human limphoblastoid cell line and hybridomer induced therefrom |
| JPS61130300A (en) * | 1984-11-27 | 1986-06-18 | ザ ボ−ド オブ トラステイ−ズ オブ ザ レランド スタンフオ−ド ジユニア ユニバ−シテイ | Human monoclonal antibody |
| JPS6263525A (en) * | 1985-09-13 | 1987-03-20 | Green Cross Corp:The | Monoclonal antibody |
-
1988
- 1988-08-19 JP JP63206182A patent/JP2627076B2/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60251881A (en) * | 1984-04-26 | 1985-12-12 | シタス コ−ポレイシヨン | Human limphoblastoid cell line and hybridomer induced therefrom |
| JPS61130300A (en) * | 1984-11-27 | 1986-06-18 | ザ ボ−ド オブ トラステイ−ズ オブ ザ レランド スタンフオ−ド ジユニア ユニバ−シテイ | Human monoclonal antibody |
| JPS6263525A (en) * | 1985-09-13 | 1987-03-20 | Green Cross Corp:The | Monoclonal antibody |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018018123A2 (en) | 2016-07-29 | 2018-02-01 | Fundação Butantan | Human anti-tetanus monoclonal antibodies neutralising infection by c. tetani, method for obtaining said monoclonal antibodies and use thereof in immunotherapy in the case of accidents with possible infection by tetanus bacillus |
| WO2018018123A3 (en) * | 2016-07-29 | 2018-03-22 | Fundação Butantan | Human anti-tetanus monoclonal antibodies neutralising infection by c. tetani, method for obtaining said monoclonal antibodies and use thereof in immunotherapy in the case of accidents with possible infection by tetanus bacillus |
| WO2019128120A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Fully human neutralizing antibody combating tetanus toxin |
| WO2019129214A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof |
| WO2019128119A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Fully human monoclonal antibody for neutralizing tetanus toxin, and uses thereof |
| WO2019128121A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Anti-tetanus toxin neutralizing antibody, preparation method therefor, and uses thereof |
| JP2021508495A (en) * | 2017-12-29 | 2021-03-11 | チューハイ トリノマブ バイオテクノロジー カンパニー，リミティド | Fully natural human neutralizing monoclonal antibody against tetanus toxin and its applications |
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| Publication number | Publication date |
|---|---|
| JP2627076B2 (en) | 1997-07-02 |
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