JPH03183477A - Hybridoma - Google Patents
HybridomaInfo
- Publication number
- JPH03183477A JPH03183477A JP1322118A JP32211889A JPH03183477A JP H03183477 A JPH03183477 A JP H03183477A JP 1322118 A JP1322118 A JP 1322118A JP 32211889 A JP32211889 A JP 32211889A JP H03183477 A JPH03183477 A JP H03183477A
- Authority
- JP
- Japan
- Prior art keywords
- human
- hybridoma
- fusion
- myeloma cell
- antibody
- 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
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- 210000004027 cell Anatomy 0.000 claims abstract description 63
- 230000004927 fusion Effects 0.000 claims abstract description 22
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 17
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 17
- 238000010367 cloning Methods 0.000 claims abstract description 14
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960005508 8-azaguanine Drugs 0.000 claims abstract description 10
- 239000012679 serum free medium Substances 0.000 claims abstract description 10
- 210000004698 lymphocyte Anatomy 0.000 claims description 15
- LPMXVESGRSUGHW-UHFFFAOYSA-N Acolongiflorosid K Natural products OC1C(O)C(O)C(C)OC1OC1CC2(O)CCC3C4(O)CCC(C=5COC(=O)C=5)C4(C)CC(O)C3C2(CO)C(O)C1 LPMXVESGRSUGHW-UHFFFAOYSA-N 0.000 abstract description 10
- LPMXVESGRSUGHW-GHYGWZAOSA-N Ouabain Natural products O([C@@H]1[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O1)[C@H]1C[C@@H](O)[C@@]2(CO)[C@@](O)(C1)CC[C@H]1[C@]3(O)[C@@](C)([C@H](C4=CC(=O)OC4)CC3)C[C@@H](O)[C@H]21 LPMXVESGRSUGHW-GHYGWZAOSA-N 0.000 abstract description 10
- 244000166550 Strophanthus gratus Species 0.000 abstract description 10
- LPMXVESGRSUGHW-HBYQJFLCSA-N ouabain Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1C[C@@]2(O)CC[C@H]3[C@@]4(O)CC[C@H](C=5COC(=O)C=5)[C@@]4(C)C[C@@H](O)[C@@H]3[C@@]2(CO)[C@H](O)C1 LPMXVESGRSUGHW-HBYQJFLCSA-N 0.000 abstract description 10
- 229960003343 ouabain Drugs 0.000 abstract description 10
- 239000002202 Polyethylene glycol Substances 0.000 abstract description 5
- 229920001223 polyethylene glycol Polymers 0.000 abstract description 5
- 238000003113 dilution method Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000001963 growth medium Substances 0.000 abstract description 2
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- 241000699666 Mus <mouse, genus> Species 0.000 description 19
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- 230000016784 immunoglobulin production Effects 0.000 description 9
- 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 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
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- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 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 2
- 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 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 229960003896 aminopterin Drugs 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
Abstract
Description
【発明の詳細な説明】
「産業上の利用分野」
本発明は、ヒト型モノクローナル抗体を産生ずるハイブ
リドーマを高頻度に作製し得る新規親細胞株、（ヒト・
マウス）へテロハイブリドーマに関するものである。DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to a novel parent cell line (human
(mouse) heterohybridoma.
「従来の技術」
抗体を、予防、治療また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
ローナル抗体に比べ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。``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 the range of use for clinical treatment is limited. is very large.
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。By the way, the production of human monoclonal antibodies has lower fusion efficiency, poorer stability of antibody production, and difficulty in obtaining large amounts of antibodies than in the case of mice. Disadvantages include that immunized lymphocytes cannot be obtained freely and that it is difficult to obtain IgG type antibodies.
これらを克服するために、ヒトの親細胞株を改良する方
法、例えば（ヒト・マウス）へ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
、 ｆ１９８３＋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
インベステイゲーション（Ｊ、 Ｃｌ１ｎ。In order to overcome these problems, methods for improving human parent cell lines, such as methods using (human/mouse) heteromyeloma as parent cells [Proceedings of the National Academy of Sciences (Proc. Nat.
, Acad, Sci, USA) 80°7308
, f1983+, etc.], or a method using mouse myeloma as parent cells [Journal of Clinical
Investigation (J, Cl1n.
Ｉｎｖｅｓｔ、１７０．１３０６． １１９８２１等］
などが考えられている６その他に、 ｉｎ ｖｉｔｒｏ
で抗原刺激することにより特異抗体産生バイプリドーマ
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、 Ｊ、 Ｉｍｍｕｎｏｌ、
１４．２３． ｆ１９８４１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（５ｃｉ
ｅｎｃｅｌ １９９゜１４３９、 ｆ１９７８１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 才ブ ナショナル アカデミ−才ブ
サイエンスｆＰｒｏｃ、 Ｎａｔ、 Ａｃａｄ、 Ｓ
ｃｉ、 ＵＳＡＩ ７９．６６５１． １１９８２１等
］などが試みられている。Invest, 170.1306. 119821 etc.]
In addition, in vitro
A method for efficiently obtaining specific antibody-producing bilidoma by antigen stimulation with [European Journal of Immunology (Eur, J. Immunol,
14.23. f19841, etc.], or a method of transforming antibody-producing cells with the lymphotropic virus Epstein-Barr virus (EBV) [Science (5ci
encel 199°1439, f19781, etc.]
, and further a method for fusing EBV-transformed cells with parent cells [
Proceedings National Academy Science fProc, Nat, Acad, S
ci, USAI 79.6651. 119821, etc.] have been attempted.
「発明が解決しようとする課題」
このようにヒト型モノクローナル抗体を産生ずるハイブ
リドーマを高頻度に作製し得る親細胞株を得る試みが種
々なされているが、現在までのところ、以下の諸条件を
満足する親細胞株は得られていない、すなわち、親細胞
株に求められる諸条件とは、ａ、ヒトリンパ球との融合
効率が高い、ｂ、自身では抗体を産生じない、Ｃ９得ら
れたハイブリドーマがヒト型モノクローナル抗体、特に
ＩｇＧタイプの抗体を安定に産生するｄ、得られたハイ
ブリドーマの抗体産生量が多い、ｅ、得られたハイブリ
ドーマが無血清培地中でも充分に増殖する。ｆ、得られ
たハイブリドーマのクローニング効率がよい、などであ
る。``Problems to be Solved by the Invention'' Various attempts have been made to obtain parent cell lines that can frequently produce hybridomas that produce human monoclonal antibodies, but so far, the following conditions have not been met. A satisfactory parent cell line has not been obtained.In other words, the conditions required for a parent cell line are: a) high fusion efficiency with human lymphocytes b) not producing antibodies by itself C9 obtained hybridoma d. The obtained hybridomas stably produce human monoclonal antibodies, especially IgG type antibodies. d. The obtained hybridomas produce a large amount of antibodies. e. The obtained hybridomas proliferate satisfactorily even in serum-free medium. f, the cloning efficiency of the obtained hybridoma is good, etc.
したがって１本発明の目的は、ヒト型モノクローナル抗
体を産生ずるハイブリドーマを作製する親細胞株として
上記のような諸条件を満足する新規な親細胞株を提供す
ることにある。Therefore, one object of the present invention is to provide a new parent cell line that satisfies the above conditions as a parent cell line for producing hybridomas that produce human monoclonal antibodies.
「課題を解決するための手段」
本発明者らは、ヒトミエローマ細胞株ＲＰＭＩ８２２６
とマウスミエローマ細胞株ＦＯをポリエチレングリコー
ルを用いて細胞融合を行い、得られたヘテロハイブリド
ーマを８−アザグアニン耐性化して、ヒトリンパ球との
融合効率が高い親細胞株ＲＦを樹立した。更にこのＲＦ
を限界希釈法又は寒天法によりクローニングを繰り返し
、最も増殖能のよいクローンをＲＦ−５１と命名した。"Means for Solving the Problems" The present inventors have developed the human myeloma cell line RPMI8226.
and mouse myeloma cell line FO were subjected to cell fusion using polyethylene glycol, and the resulting heterohybridoma was made resistant to 8-azaguanine to establish a parent cell line RF that has high fusion efficiency with human lymphocytes. Furthermore, this RF
Cloning was repeated using the limiting dilution method or the agar method, and the clone with the best proliferation ability was named RF-51.
そして、この細胞株がヒト型モノクローナル抗体を産生
ずるハイブリドーマを作製する親細胞株として要求され
る上記諸条件を満足し得ることを確認し、本発明を完成
するに至った。Then, it was confirmed that this cell line could satisfy the above conditions required as a parent cell line for producing hybridomas producing human monoclonal antibodies, and the present invention was completed.
すなわち、本発明は、ヒトミエローマ細胞株（ＲＰＭＩ
８２２６）とマウスミエローマ細胞株（ＦＯｌとを細胞
融合させた後、８−アザグアニン耐性化し、更にクロー
ニングを繰り返して得られ、以下の性質を有することを
特徴とするハイブリドーマを提供するものである。That is, the present invention provides human myeloma cell line (RPMI
8226) and a mouse myeloma cell line (FOI), the cells are made resistant to 8-azaguanine, and cloning is repeated to obtain a hybridoma, which is characterized by having the following properties.
ａ、ヒトリンパ球との融合効率が高い。a, High fusion efficiency with human lymphocytes.
ｂ、自身では抗体を産生じない。b. Does not produce antibodies by itself.
Ｃ，ヒトリンパ球と融合して得られたバイプリドーマは
抗体、特にＩｇＧ抗体を安定に産生する。C. Bipridomas obtained by fusion with human lymphocytes stably produce antibodies, especially IgG antibodies.
ｄ、ヒトリンパ球と融合して得られたハイブリドーマの
抗体産生量が多い。d. Hybridomas obtained by fusion with human lymphocytes produce a large amount of antibodies.
ｅ、ヒトリンパ球と融合して得られたハイブリドーマは
、無血清培地中でも充分に増殖し抗体を産生する
ｆ、ヒトリンパ球と融合して得られたバイプリドーマの
クローニング効率が高い。e. Hybridomas obtained by fusion with human lymphocytes proliferate sufficiently even in serum-free medium and produce antibodies.f. Hybridomas obtained by fusion with human lymphocytes have high cloning efficiency.
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、ヒトミエローマ細胞株は、ＲＰＭＩ８
２２６を用い、マウスミエローマ細胞株としては、抗体
非産生で融合効率の高いマウス親細胞株ＦＯを用いた。In the present invention, the human myeloma cell line is RPMI8
226 was used, and the mouse parent cell line FO, which does not produce antibodies and has high fusion efficiency, was used as the mouse myeloma cell line.
なお、これらの細胞は市販されており、容易に入手可能
である。ＲＰＭＩ８２２６は、ヒボキサンチン、アミノ
プテリン、チミジン（以下ＨＡＴと記す）を加えた培地
で培養可能だがウアバインを加えた培地中では増殖でき
ない、ＦＯは逆に、ＨＡＴ添加培地中では増殖できない
が、ウアバインを添加した培地中でも増殖できる。これ
らの細胞株をポリエチレングリコールを用いた公知の方
法で融合させ、ＨＡＴ及びウアバインを加えた選択培地
にて培養を行い、ＨＡＴ・ウアバイン耐性の（ヒト・マ
ウス）へテロハイブリドーマを得た０次いで、８−アザ
グアニンの濃度を徐々に高くした培地で順次培養するこ
とにより、８−アザグアニン耐性にし、ＨＡＴ感受性の
へテロハイブリドーマＲＦを得た。このヘテロハイブリ
ドーマを限界希釈法又は軟寒天法によりクローン化して
、最も増殖能のよい（ヒト・マウス）ヘテロハイブリド
ーマＲＦ−５ｌを樹立した。このＲＦ−５ｌは、ヒトリ
ンパ球との融合効率が高く、シかもＲＦ−３ｌを用いて
作製したハイブリドーマはクローニング効率がよく抗体
産生も安定していることが確認された。また、ＲＦ−５
１及びＲＦ−５１を用いて作製したバイブリドーマは無
血清培地中でも培養可能である。Note that these cells are commercially available and can be easily obtained. RPMI8226 can be cultured in a medium containing hypoxanthine, aminopterin, and thymidine (hereinafter referred to as HAT), but cannot grow in a medium containing ouabain. FO, on the other hand, cannot grow in a medium containing HAT, but it cannot grow in a medium containing ouabain. It can be grown even in a medium. These cell lines were fused by a known method using polyethylene glycol and cultured in a selective medium containing HAT and ouabain to obtain a HAT/ouabain resistant (human/mouse) heterohybridoma. A heterohybridoma RF made resistant to 8-azaguanine and sensitive to HAT was obtained by sequentially culturing in a medium in which the concentration of 8-azaguanine was gradually increased. This heterohybridoma was cloned by the limiting dilution method or the soft agar method, and the heterohybridoma RF-5l (human/mouse) with the best proliferative ability was established. It was confirmed that this RF-5l has a high fusion efficiency with human lymphocytes, and that hybridomas produced using RF-3l have good cloning efficiency and stable antibody production. Also, RF-5
Hybridomas produced using 1 and RF-51 can be cultured even in serum-free medium.
したがって、ＲＦ−３ｌを用いて作製したハイブリドー
マが産生するＩｇＧタイプのヒト型モノクローナル抗体
は、プロティン−へカラムを用いた常法の手段により容
易に精製できる。Therefore, the IgG type human monoclonal antibody produced by the hybridoma produced using RF-3l can be easily purified by conventional means using a protein column.
「作用及び効果」
本発明により樹立したＲＦ−５ｌは、親細胞株として融
合効率が高く、得られたハイブリドーマは抗体産生が安
定で、しかも無血清培地で培養可能である。この親細胞
株を用いることにより、ヒトに投与可能なヒト型モノク
ローナル抗体の生産及び精製が比較的容易にでき、その
有用性は非常に大きい。"Action and Effect" RF-5l established according to the present invention has a high fusion efficiency as a parent cell line, and the resulting hybridoma has stable antibody production and can be cultured in a serum-free medium. By using this parent cell line, human monoclonal antibodies that can be administered to humans can be produced and purified relatively easily, and are extremely useful.
「実施例」
（１）８−アザグアニン耐性（ヒト・マウス）ヘテロハ
イブリドーマの作製
ヒトミエローマ細胞株ＲＰＭＩ８２２６及びマウスミエ
ローマ細胞株ＦＯはそれぞれ１５％の非動化牛胎児血清
（以下ＦＣ５と記す）を含むＲＤＦ培地（ＲＰＭＩ Ｉ
６４０、ダルベツコＭＥＭ。"Example" (1) Preparation of 8-azaguanine-resistant (human/mouse) heterohybridoma Human myeloma cell line RPMI8226 and mouse myeloma cell line FO each contain 15% inactivated fetal calf serum (hereinafter referred to as FC5). RDF medium (RPMI I
640, Darbetsuko MEM.
ハムＦ１２培地を２：ｌ：１で混合した培地）にて培養
した。これらの細胞はＲＤＦ培地で２回洗浄後、ｌ：１
の割合で混合し、再度遠心する。遠心後、細胞ベレット
をよく分散し、１ｍｌの５０％ポリエチレングリコール
を１分間にわたって滴下した。更に１分間３７℃で反応
後、５分間で１０ｍ１のＲＤＦ培地を加えた。The cells were cultured in a 2:1:1 mixture of Ham's F12 medium. These cells were washed twice with RDF medium, then 1:1
Mix at the same ratio and centrifuge again. After centrifugation, the cell pellet was well dispersed, and 1 ml of 50% polyethylene glycol was added dropwise over 1 minute. After reacting for an additional 1 minute at 37°C, 10 ml of RDF medium was added for 5 minutes.
これを遠心した後、１５％ＦＣ５培地を含むＲＤＦ培地
に懸濁し、９６ウエルマイクロプレートにｌウェル当た
り４ＸｌＯ’個の細胞が含まれるように分注した。翌日
、ＨＡＴ培地（０，１ｍＭヒボキサンチン、１６℃Ｍチ
ミジン及び０．４μＭアミノプテリン添加１５％ＦＣ５
／ＲＤＦ培地）にウアバインｌＯｕＭを添加したＨＡＴ
・ウアバイン培地に交換し、３７℃、５％ＣＯ□の条件
下で培養した。２−３日間隔でＨＡＴ・ウアバイン培地
を交換した。約１週間後にヘテロハイブリドーマの増殖
が確認され、その中で最ら増殖のよいクローンを選択し
、更に２週間ＨＡＴ・ウアバイン培地で培養を続けた。After centrifugation, the cells were suspended in RDF medium containing 15% FC5 medium and dispensed into a 96-well microplate so that each well contained 4X1O' cells. The next day, HAT medium (15% FC5 supplemented with 0.1mM hyboxanthin, 16°C M thymidine and 0.4μM aminopterin)
HAT with ouabain lOuM added to /RDF medium)
- The medium was replaced with ouabain medium and cultured at 37°C and 5% CO□. The HAT/ouabain medium was replaced at intervals of 2-3 days. Approximately one week later, proliferation of heterohybridoma was confirmed, and among them, the clone with the best proliferation was selected and cultured in HAT/ouabain medium for an additional two weeks.
その後１週間１５％ＦＣ５を含むＲＤＦ培地で培養した
後、５μｇ ／ ｍ ｌの８−アザグアニンを加えた１
５％ＦＣ５含有ＲＤＦ培地で培養を始めた。８−アザグ
アニンの濃度を１０．２０゜５０．１１００ｕ／ｍｌに
順次高めた１５％ＦＣ５含有ＲＤＦ培地でそれぞれ１週
間培養を行い、８−アザグアニン耐性ヘテロハイブリド
ーマＲＦを樹立した。After that, they were cultured in RDF medium containing 15% FC5 for 1 week, and then 5 μg/ml of 8-azaguanine was added.
Culture was started in RDF medium containing 5% FC5. Each culture was performed for one week in RDF medium containing 15% FC5 in which the concentration of 8-azaguanine was gradually increased to 10.20.50.1100 u/ml, and 8-azaguanine-resistant heterohybridoma RF was established.
この樹立細胞を限界希釈法又は軟寒天法でクローニング
を行い、最も増殖のよいクローンをＲＦ−３ｌと命名し
た。The established cells were cloned by the limiting dilution method or the soft agar method, and the clone with the best proliferation was named RF-3l.
ＲＦ−３ｌをＨＡＴ培地で培養したところ約５日で死滅
し、ＲＦ−３ｌのＨＡＴ感受性が確認された。ＲＦ−５
ｌのＤＮＡを分離し、ヒトＤＮＡに特異的なａｌｕ遺伝
子の存在を確かめたところ、ＲＦ−３ｌのＤＮＡ中にヒ
トＤＮＡが約ｌＯ％含まれていることが確認された。ま
た、ＲＦ−５ｌは外見上マウスミエローマＦＯと同一で
あり、しかちマウスミエローマ細胞株来の性質である１
０℃Ｍのウアバインに耐性であることから、ＲＦ−３ｌ
はヒト・マウスのへテロハイブリドーマであることが明
らかとなった。また、ＲＦ−５１はヒト及びマウス免疫
グロブリンを分泌していないことら確認した。When RF-3l was cultured in HAT medium, it died in about 5 days, confirming the HAT sensitivity of RF-3l. RF-5
When the DNA of RF-3l was isolated and the presence of the alu gene specific to human DNA was confirmed, it was confirmed that the DNA of RF-3l contained about 10% human DNA. In addition, RF-5l is visually identical to mouse myeloma FO, and has properties inherent to mouse myeloma cell lines.
Since it is resistant to ouabain at 0°C, RF-3l
was revealed to be a human-mouse heterohybridoma. Furthermore, it was confirmed that RF-51 did not secrete human or mouse immunoglobulin.
（２）融合効率の検討
ヒトリンパ球は、ヒト末梢血からフィコールバックを用
いた比重遠心法により分離し、ＲＤＦ培地で２回洗浄後
、同じ＜ ＲＤＦ培地で洗浄したＲＦ−３ｌと２：ｌの
割合で混合する８遠心後、細胞ベレットをよく分散し、
１ｍｌの５０％ポリエチレングリコールを１分間にわた
って滴下した。更に１分間３７℃で反応後、５分間でｌ
０ｔｌのＲＤＦ培地を加えた。これを遠心した後、１５
％ＦＣ５を含むＲＤＦ培地に懸濁し、９６ウエルマイク
ロプレートに１ウエル当たり２ＸｌＯ’個の親細胞が含
まれるように分注した。翌日、ＨＡＴ培地に交換し３７
℃、５％ＣＯ２の条件下で培養した。２−３日間層でＨ
ＡＴ培地を交換した。(2) Examination of fusion efficiency Human lymphocytes were separated from human peripheral blood by specific gravity centrifugation using Ficollvac, washed twice with RDF medium, and then mixed with RF-3l and 2:l of RF-3l washed with RDF medium. After centrifugation, the cell pellet is well dispersed.
1 ml of 50% polyethylene glycol was added dropwise over 1 minute. After reacting for another 1 minute at 37°C, the temperature was increased for 5 minutes.
0 tl RDF medium was added. After centrifuging this, 15
The cells were suspended in RDF medium containing % FC5 and dispensed into a 96-well microplate so that each well contained 2XlO' parental cells. The next day, change to HAT medium 37
The cells were cultured at 5% CO2. H in layer for 2-3 days
AT medium was replaced.
以上の条件でハイブリドーマが増殖してきたウェルの数
を数え、融合効率を計算した。結果を第１表に示した８
この融合効率は、マウスのモノクローナル抗体を作製す
る際のマウス親細胞の融合効率に匹敵する。The number of wells in which hybridomas had proliferated under the above conditions was counted, and the fusion efficiency was calculated. The results are shown in Table 18.
This fusion efficiency is comparable to that of mouse parent cells when producing mouse monoclonal antibodies.
第１表（ヒト末梢血リンパ球を用いた場合の融合効率）
（３）ＲＦ−５ｌの培養上清中の抗体濃度測定ＲＦ−Ｓ
ｌを１５％ＦＣ３を含むＲＤＦ培地中で培養し、培養
上清中の抗体濃度を測定した。測定は酵素免疫測定法（
ＥＩＡ法）により行なった。この結果を第２表に示す。Table 1 (Fusion efficiency when using human peripheral blood lymphocytes)
(3) Measurement of antibody concentration in culture supernatant of RF-5l RF-S
1 was cultured in RDF medium containing 15% FC3, and the antibody concentration in the culture supernatant was measured. Measurement is by enzyme immunoassay (
The test was carried out using the EIA method). The results are shown in Table 2.
第２表（ＲＦ−５ｌ培養土清中の抗体濃度）第２表に示
されるように、ヒトＩｇＧ、ヒトＩｇＭ、マウスＩｇＧ
、マウスＩｇＭ及びヒトカッパ鎖、ヒトラムダ鎖と６に
測定限界以下であり、ＲＦ−Ｓｌは自身では抗体を産生
じないことが明らかとなった。Table 2 (Antibody concentration in RF-5l culture medium) As shown in Table 2, human IgG, human IgM, mouse IgG
, mouse IgM, human kappa chain, and human lambda chain 6 were below the measurement limit, making it clear that RF-Sl does not produce antibodies by itself.
（４）ＲＦ−５ｌを用いて作製したハイブリドーマの抗
体産生の安定性
前記（２）における第１表の実験６で得られたハイブリ
ドーマＧ６を１５％ＦＣ５を含むＲＤＦ培地で培養し、
ハイブリドーマＧ６の抗体産生の安定性を検討した。す
なわち、ハイブリドーマＧ６を、０箇月、４箇月、７箇
月培養した後、ｌＸｌ０’個／＋ａｌの密度で培養を開
始し、５日目の培養上清中に含まれるＩｇＧ濃度を測定
した。(4) Stability of antibody production of hybridoma produced using RF-5l Hybridoma G6 obtained in Experiment 6 in Table 1 in (2) above was cultured in RDF medium containing 15% FC5,
The stability of antibody production by hybridoma G6 was investigated. That is, after culturing hybridoma G6 for 0 months, 4 months, and 7 months, culture was started at a density of 1X10' cells/+al, and the IgG concentration contained in the culture supernatant on the 5th day was measured.
この結果を第３表に示す。The results are shown in Table 3.
第３表（ＲＦ−５ｌを用いて作製したハイブリドーマの
抗体産生の安定性）
第３表から明らかなように、７箇月培養した後でも抗体
産生量はほとんど変化なく安定していることが確認され
た。Table 3 (Stability of antibody production of hybridomas produced using RF-5l) As is clear from Table 3, it was confirmed that the amount of antibody production remained stable with almost no change even after 7 months of culture. Ta.
（５）ＲＦ−５ｌを用いて作製したハイプリド−マのク
ローニング効率
前記（２）の実験で得られた各種ハイブリドーマを用い
てクローニング効率を測定した。すなわち、１５％ＦＣ
５を含むＲＤＦ培地にハイブリドーマを懸濁し、９６ウ
エルプレートに１ウエル当たり１０個、３個、１個、０
．３個の割合で細胞をまき込み、２週間後に細胞が増殖
してきたウェル数を数えた。この結果を第４表に示す。(5) Cloning efficiency of hybridomas produced using RF-5l Cloning efficiency was measured using the various hybridomas obtained in the experiment in (2) above. That is, 15%FC
Hybridomas were suspended in RDF medium containing 5, and placed in a 96-well plate with 10 cells per well, 3 cells, 1 cell, and 0 cells per well.
．． Cells were seeded at a rate of 3, and the number of wells in which cells had proliferated was counted 2 weeks later. The results are shown in Table 4.
第４表（ＲＦ−３ｌを用いて作製したハイブリドーマの
クローニング効率）
（表中のクローニング効率は、「細胞が増殖してきたウ
ェル数」／「細胞をまき込んだウェル数」を表わしてい
る。）
このように、第４表に示されるクローニング効率は、マ
ウスのハイブリドーマの場合に匹敵するものである。Table 4 (Cloning efficiency of hybridomas produced using RF-3l) (The cloning efficiency in the table represents "number of wells in which cells have grown"/"number of wells in which cells have been seeded.") Thus, the cloning efficiency shown in Table 4 is comparable to that for mouse hybridomas.
（６）無血清培地での培養
ＲＦ−５ｌ及び前記（２）における第１表の実験６で得
られたハイブリドーマＧ６を用いて無血清培養を行った
。無血清培地としてはインシュリン５μｇ／ｍ１．
トランスフェリン３５μｇ／ｍｌ、エタノールアミン２
０μＭ、セレニウム２．５ ｎＭ （以下ＩＴＥＳと記
す）を添加したＲＤＦ培地を用いた。それぞれの細胞を
、ｌＸｌ０’個／ｍｌの密度でＩＴＥＳ −ＲＤＦ培地
に懸濁し、３５ｍｍのデイツシュで培養した。そして、
２４時間経過すル毎に、ＲＦ−５ｌについては細胞密度
、ハイブリドーマＧ６については細胞密度とＩｇＧタイ
プの抗体産生量を測定した。なお、抗体産生量は酵素免
疫測定法（Ｅ Ｉ Ａ法）により測定した。この結果を
第１図に示す０図中、折れ線グラフ−・−はＲＦ−５ｌ
の細胞密度を表わし、折れ線グラフ−０−はハイブリド
ーマＧ６の細胞密度を表わし、棒線グラフはＩｇＧ濃度
を表わしている。(6) Culture in serum-free medium Serum-free culture was performed using RF-5l and hybridoma G6 obtained in Experiment 6 in Table 1 in (2) above. Serum-free medium contains insulin 5 μg/ml.
Transferrin 35μg/ml, ethanolamine 2
An RDF medium supplemented with 0 μM and 2.5 nM selenium (hereinafter referred to as ITES) was used. Each cell was suspended in ITES-RDF medium at a density of 1X10' cells/ml and cultured in a 35 mm dateschew. and,
Every 24 hours, the cell density for RF-5l and the cell density and IgG type antibody production for hybridoma G6 were measured. Note that the amount of antibody produced was measured by enzyme immunoassay (EIA method). This result is shown in Figure 1. In Figure 1, the line graph --- indicates RF-5l.
The line graph -0- represents the cell density of hybridoma G6, and the bar graph represents the IgG concentration.
このように、ＲＦ−５Ｉ及びバイブリドーマＧ６ともに
無血清培地中で充分に増殖し、抗体産生量も多いことが
明らかとなった。Thus, it was revealed that both RF-5I and hybridoma G6 proliferated sufficiently in serum-free medium and produced a large amount of antibodies.
第１図は（ヒト・マウス）へテロハイブリドーマＲＦ−
５１と、このＲＦ−５ｔを用いて作製したハイブリドー
マＧ６を、無血清培地で培養したときの培養時間と細胞
密度との関係、並びにハイブリドーマＧ６の培養時間と
抗体産生量との関係を表わしている。Figure 1 shows (human/mouse) heterohybridoma RF-
51 and hybridoma G6 produced using this RF-5t are cultured in a serum-free medium, the relationship between culture time and cell density, and the relationship between culture time and antibody production amount of hybridoma G6 are shown. .
Claims (1)
エローマ細胞株（ＦＯ）とを細胞融合させた後、８−ア
ザグアニン耐性化し、更にクローニングを繰り返して得
られ、以下の性質を有することを特徴とするハイブリド
ーマ。 ａ、ヒトリンパ球との融合効率が高い。 ｂ、自身では抗体を産生しない。 ｃ、ヒトリンパ球と融合して得られたハイブリドーマは
抗体、特にＩｇＧ抗体を安定に産 生する。 ｄ、ヒトリンパ球と融合して得られたハイブリドーマの
抗体産生量が多い。 ｅ、ヒトリンパ球と融合して得られたハイブリドーマは
、無血清培地中でも充分に増殖し 抗体を産生する。 ｆ、ヒトリンパ球と融合して得られたハイブリドーマの
クローニング効率が高い。[Claims] A human myeloma cell line (RPMI8226) and a mouse myeloma cell line (FO) are fused together, made resistant to 8-azaguanine, and then cloned repeatedly. Characteristic hybridoma. a, High fusion efficiency with human lymphocytes. b. Does not produce antibodies by itself. c. Hybridomas obtained by fusion with human lymphocytes stably produce antibodies, especially IgG antibodies. d. Hybridomas obtained by fusion with human lymphocytes produce a large amount of antibodies. e. Hybridomas obtained by fusion with human lymphocytes proliferate sufficiently even in serum-free medium and produce antibodies. f. High cloning efficiency of hybridomas obtained by fusion with human lymphocytes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1322118A JP3043023B2 (en) | 1989-12-12 | 1989-12-12 | Hybridoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1322118A JP3043023B2 (en) | 1989-12-12 | 1989-12-12 | Hybridoma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03183477A true JPH03183477A (en) | 1991-08-09 |
| JP3043023B2 JP3043023B2 (en) | 2000-05-22 |
Family
ID=18140124
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1322118A Expired - Fee Related JP3043023B2 (en) | 1989-12-12 | 1989-12-12 | Hybridoma |
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| Country | Link |
|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0671471A4 (en) * | 1992-11-28 | 1996-09-11 | Chemo Sero Therapeut Res Inst | Process for producing foreign protein. |
| CN111087469A (en) * | 2019-12-18 | 2020-05-01 | 南京泰奥德生物科技有限公司 | Preparation method of fully human monoclonal antibody |
-
1989
- 1989-12-12 JP JP1322118A patent/JP3043023B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0671471A4 (en) * | 1992-11-28 | 1996-09-11 | Chemo Sero Therapeut Res Inst | Process for producing foreign protein. |
| CN111087469A (en) * | 2019-12-18 | 2020-05-01 | 南京泰奥德生物科技有限公司 | Preparation method of fully human monoclonal antibody |
| CN111087469B (en) * | 2019-12-18 | 2024-03-26 | 南京泰奥德生物科技有限公司 | Preparation method of fully human monoclonal antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3043023B2 (en) | 2000-05-22 |
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