JP3554355B2 - IL-6 autocrine proliferating human myeloma cell line - Google Patents

Description

表から明らかなように、ＫＰＭＭ２は形質細胞関連抗原（ＣＤ３８、ＰＣＡ−１およびＢＬ３）、接着分子（ＣＤ４４、ＶＬＡ−β、ＩＣＡＭ−１、ＮＣＡＭ、ＬＦＡ−３およびＶＬＡ−４）ならびにＣＤ４５、ＣＤ６３、ＣＤ７１、ＩｇＧおよびλなどの抗原が陽性であった。
【００３２】
実施例３：免疫グロブリン遺伝子再構成
上記実施例１で樹立した細胞株ＫＰＭＭ２の免疫グロブリン（Ｉｇ）遺伝子再構成をサザンブロット法により分析した。
【００３３】
実施例１に記載の方法により得られたＫＰＭＭ２細胞（１０^７個）から、Ｍａｎｕａｌ ｏｆ Ｃｌｉｎｉｃａｌ Ｉｍｍｕｎｏｌｏｇｙ， ３ｒｄ ｅｄｉｔｉｏｎ， Ａｍｅｒｉｃａｎ Ｓｏｃｉｅｔｙ ｆｏｒ Ｍｉｃｒｏｂｉｏｌｏｇｙ， １９８６の方法に準じてＤＮＡを調製し、３種類の制限酵素ＢａｍＨＩ、ＥｃｏＲＩあるいはＨｉｎｄＩＩＩ（ベーリンガー・マンハイム社製）で別々に処理し、エタノール沈殿としてＤＮＡを回収して、０．８％アガロースゲル（ＳＥＡＫＥＭ ＧＴＧ，ＦＭＣ社製）により２４時間電気泳動を行った。電気泳動したＤＮＡをナイロン膜（ハイボンドＮ^＋，アマシャム社製）に移し、これを乾燥させた。次に、タカラランダムプライマーＤＮＡラベリングキット（宝酒造社製）を用いて、^３２Ｐ標識したヒトＩｇ Ｊ_Ｈ、ＣκおよびＣλプローブ（オンコア社製）を用い、添付の処方に従ってサザンブロット分析を行った。なお、対照として健常人の末梢血単核球から得た再構成を生じていない染色体ＤＮＡを用いた。その結果、ＩｇＨおよびκ鎖遺伝子が再構成していたが、λ鎖遺伝子は再構成していなかった（図３）。以上のことより、ＫＰＭＭ２がモノクローナルな抗体を産生すること、および細胞の単一性が確認された。
【００３４】
実施例４：細胞遺伝学的分析
上記実施例１で樹立した細胞株ＫＰＭＭ２の染色体の構造異常を分析した。
【００３５】
実施例１に記載の方法で得られたＫＰＭＭ２を２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを含むＲＰＭＩ１６４０培養液にて５×１０^５個／ｍｌとなるように培養した。培養４８時間後に、ＫＰＭＭ２に０．０５μｇのコルセミド（ギブコ社製）を１５分間処理し、分裂中期で細胞周期が停止したＫＰＭＭ２細胞を回収した。回収したＫＰＭＭ２細胞を０．０７５ＭのＫＣｌで２０分間処理し、メタノール−酢酸で固定した。次いで、ＫＰＭＭ２細胞の染色体をトリプシン−ギムザバンド染色法によって分析した。
【００３６】
その結果、ＫＰＭＭ２は多くの構造異常をもつ二倍体細胞であることが判明した（図４）。分析した１５細胞すべてが４６、ＸＸ、ｄｅｒ（１；１９）（ｑ１０；ｑ１０）、ｔ（３；１４）（ｑ２１；ｑ３２）、−４、ｔ（６；１１）（ｐ１２；ｐ１５）、ｄｅｒ（１０）ａｄｄ（１０）（ｐ１３）ｄｉｃ（９；１０）（ｑ１０；ｑ２６）、＋１６を示した。
【００３７】
実施例５：ＥＢＶおよびマイコプラズマの検出
実施例１で樹立した細胞株ＫＰＭＭ２のエプスタイン・バールウイルス（ＥＢＶ）およびマイコプラズマ汚染を試験した。
【００３８】
実施例１に記載の方法で得られたＫＰＭＭ２細胞の染色体からエプスタイン・バールウイルス（ＥＢＶ）を検出するために、Ｓｙｓｔｅｍｉｃ Ｇｅｎｅｔｉｃ Ｉｎｓｔｉｔｕｔｅ社から購入したＥＢＶ ＢａｍＷ領域増幅プライマーを用いて、添付の処方に従いＰＣＲ（ポリメレース チェイン リアクション）を行った。マイコプラズマ感染の検出はマイコプラズマＤＮＡ検出用Ｍ．Ｔ．Ｃ．キット（Ｇｅｎ−Ｐｒｏｂｅ Ｉｎｃ．社製）により、添付の処方に従って実施した。
【００３９】
その結果、ＫＰＭＭ２はＥＢＶゲノムおよびマイコプラズマゲノムに対して陰性であった。
【００４０】
実施例６：サイトカインに対する反応性
実施例１で樹立した細胞株ＫＰＭＭ２の各種サイトカインへの反応性を試験した。
【００４１】
実施例１に記載する方法で得られたＫＰＭＭ２を２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを含むＲＰＭＩ１６４０培養液に浮遊させ、容量が２００μｌで９６穴プレート（ファルコン社製）へ１×１０^４個／穴となるように分注した。
【００４２】
９６穴プレートの各穴には、下記の濃度となるように各種サイトカインを別々に加えた。
【００４３】
組換えＩＬ−２、同ＩＬ−３、同腫瘍壊死因子（ＴＮＦ）−α、同顆粒球マクロファージコロニー刺激因子（ＧＭ−ＣＳＦ）、同幹細胞成長因子（ＳＣＦ）（以上、Ｇｅｎｚｙｍｅ社製）、同ＩＬ−４、同ＩＬ−７、同ＩＬ−１０、同ＩＬ−１１、同白血病阻害因子（ＬＩＦ）、同オンコスタチンＭ（ＯＳＭ）（以上、Ｐｅｐｒｏ Ｔｅｃ Ｉｎｃ．社製）、同ＩＬ−９、同トランスフォーミング成長因子（ＴＧＦ）−β（以上、Ｒ＆Ｄ Ｓｙｓｔｅｍ Ｉｎｃ．社製）、同ＩＬ−１α（Ｂｏｅｈｒｉｎｇｅｒ Ｍａｎｈｅｉｍ社製）、同ＩＬ−５（Ｕｐｓｔａｔｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｃ．社製）、同ＩＬ−８（Ａｍｅｒｓｈａｍ社製）、同エリスロポエチン（ＥＰＯ）および同顆粒球コロニー刺激因子（Ｇ−ＣＳＦ）（以上、中外製薬株式会社より提供）は１００ｎｇ／ｍｌ。
【００４４】
組換えインターフェロン（ＩＦＮ）−γ（塩野義製薬株式会社より提供）および天然型ヒトＩＦＮ−α（住友製薬株式会社より提供）は１０００Ｕ／ｍｌ。
【００４５】
組換えＩＬ−６（中外製薬株式会社より提供）は１ｎｇ／ｍｌ。
【００４６】
なお、対照群は、サイトカインを加えない２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを添加したＲＰＭＩ１６４０培養液で培養した。
【００４７】
９６穴プレートの各穴に分注したＫＰＭＭ２を、上記サイトカイン存在下あるいは非存在下で湿潤５％ＣＯ_２中、３７℃で９６時間培養した。その培養終了４時間前に、各穴に^３Ｈ−チミジン（Ａｍｅｒｓｈａｍ社製）を１μＣｉ／穴となるように添加した。ＫＰＭＭ２が取り込んだ^３Ｈ−チミジンの量の測定は、液体シンチレーションカウンター（１２０５ ベータプレート、ファルマシア社製）を用いた。
【００４８】
ＫＰＭＭ２の増殖に対する各種サイトカインの効果を図５に示す。図５から明らかなように、ＫＰＭＭ２細胞はＩＬ−６とともにインキュベートしたときのみに^３Ｈ−チミジンの取り込みが顕著に刺激された。濃度１ｎｇ／ｍｌにおけるＩＬ−６により、^３Ｈ−チミジンの取り込みが２．２倍に上昇した。一方、ＩＦＮ−αおよびＩＦＮ−γはＫＰＭＭ２細胞の増殖を顕著に阻害した。
【００４９】
また、ＫＰＭＭ２のサイトカインに対する反応性を調べるために、ＭＴＴ（３−［４，５−ｄｉｍｅｔｈｙｌｔｈｉａｚｏｌ−２−ｙｌ］−２，５−ｄｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍ ｂｒｏｍｉｄｅ）法（Ｊ． Ｉｍｍｕｎ． Ｍｅｔｈｏｄｓ ６５：５５， １９８３ を参照）および生細胞を直接測定する方法を用いたが、同様の結果が得られた。
【００５０】
実施例７：抗ＩＬ−６ ｍＡｂおよび抗ＩＬ−６Ｒ ｍＡｂによる増殖阻害
実施例１で樹立した細胞株ＫＰＭＭ２に対するマウス抗ヒトＩＬ−６Ｒ ｍＡｂ（モノクローナル抗体）（ＩｇＧ_１クラス：ＰＭ１）およびマウス抗ＩＬ−６ｍＡｂ（ＩｇＧ_１クラス：ＳＫ２）の効果を調べた。ＳＫ２は文献（Ｙ． Ｏｈｅ ｅｔａｌ．， Ｂｒ． Ｊ． Ｃａｎｃｅｒ ６７：９３９， １９９３）に記載されており、またＰＭ１は文献（Ｈｉｒａｔａ ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ． １４３：２９００， １９８９）に記載されている。
【００５１】
実施例１に記載する方法で得たＫＰＭＭ２を２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを含むＲＰＭＩ１６４０培養液に浮遊させ、容量が２００μｌで９６穴プレート（ファルコン社製）へ１×１０^４個／穴となるように分注した。
【００５２】
９６穴プレートの各穴には、各種濃度のＩＬ−６ ｍＡｂ（モノクローナル抗体）（ＳＫ２）および抗ＩＬ−６Ｒ（受容体）ｍＡｂ（ＰＭ１）を別々に加えた。なお、対照群は、抗体を加えない２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを含むＲＰＭＩ１６４０培養液とした。
【００５３】
９６穴プレートの各穴に分注したＫＰＭＭ２を、抗ＩＬ−６ ｍＡｂおよび抗ＩＬ−６Ｒ ｍＡｂ存在下あるいは非存在下で湿潤５％ＣＯ_２中、３７℃で９６時間培養した。その培養終了４時間前に、各穴に^３Ｈ−チミジン（Ａｍｅｒｓｈａｍ社製）を１μＣｉ／穴となるように添加した。ＫＰＭＭ２が取り込んだ^３Ｈ−チミジンの量の測定は、液体シンチレーションカウンター（１２０５ ベータプレート、ファルマシア社製）を用いた。
【００５４】
ＫＰＭＭ２の増殖に対する抗ＩＬ−６ ｍＡｂおよび抗ＩＬ−６Ｒ ｍＡｂの効果を図６に示す。ＳＫ２およびＰＭ１の添加はいずれも用量依存的に細胞増殖を有意に阻害した。特に、ＰＭ１は１μｇ／ｍｌの濃度でＫＰＭＭ２の増殖を完全に阻害した。
【００５５】
また、ＫＰＭＭ２増殖の抗ＩＬ−６ ｍＡｂおよび抗ＩＬ−６Ｒ ｍＡｂに対する効果を調べるために、ＭＴＴ（３−［４，５−ｄｉｍｅｔｈｙｌｔｈｉａｚｏｌ−２−ｙｌ］−２，５−ｄｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍ ｂｒｏｍｉｄｅ）法（Ｊ． Ｉｍｍｕｎ． Ｍｅｔｈｏｄｓ ６５：５５， １９８３ を参照）および生細胞を直接測定する方法を用いたが、同様の結果が得られた。
【００５６】
実施例８：ＥＬＩＳＡによるＩＬ−６産生の測定
上記実施例１で樹立した細胞株ＫＰＭＭ２によるＩＬ−６産生能を試験した。
【００５７】
実施例１に記載する方法で得たＫＰＭＭ２を、２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを含むＲＰＭＩ１６４０培養液に１０^６個／ｍｌとなるように浮遊させ、ヒトＩＬ−６非存在下で湿潤５％ＣＯ_２中、３７℃で培養した。
【００５８】
培養７２時間後、培養上清中に含まれるＫＰＭＭ２が産生したＩＬ−６の濃度を、ヒトＩＬ−６用ＥＬＩＳＡキット（帝人バイオラボラトリー社製）を用いて添付の処方に従い測定した。なお、陰性対照として、２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを添加したＲＰＭＩ１６４０培養液を用いた。培養上清には７９．７±１９．６（平均値±Ｓ．Ｄ．）ｐｇ／ｍｌのＩＬ−６の産生が検出され、対照培養液において検出限界（４．０ｐｇ／ｍｌ）以下であったのに比較すると、培養上清中のＩＬ−６濃度が顕著に増加していることが確認された。
【００５９】
実施例９：フローサイトメトリーによるＩＬ−６Ｒの発現の確認
ＫＰＭＭ２細胞上でのＩＬ−６Ｒ発現を確認するために、ＩＬ−６Ｒに結合し、ＩＬ−６のＩＬ−６Ｒへの結合を阻害しないマウス抗ヒトＩＬ−６Ｒ ｍＡｂであるＭＴ１８抗体（Ｈｉｒａｔａ ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ． １４３：２９００， １９８９）を用いて、間接蛍光抗体法を実施した。
【００６０】
実施例１に記載の方法で得られたＫＰＭＭ２細胞を１０^６個／チューブとなるように、１００μｌの実施例２記載のＦＡＣＳ緩衝液に浮遊させ、１０μｇ／ｍｌのＭＴ１８抗体を加え、４℃にて３時間反応させた後、ＦＡＣＳ緩衝液で２回洗浄し、１００μｌのＦＡＣＳ緩衝液に浮遊させ、さらに、ＦＩＴＣ標識ヤギ抗マウスＩｇＧ抗体（ＴＡＧＯ社製）を５μｇ／ｍｌ添加し、４℃にて３０分間反応させた。ＦＡＣＳ緩衝液で２回洗浄した後、同ＦＡＣＳ緩衝液に浮遊させ、フローサイトメーター（ＥＰＩＣＳ ＰＲＯＦＩＬＥ、コールター社製）で蛍光を測定した。
【００６１】
その結果、図７に示すように、ＫＰＭＭ２細胞上にＩＬ−６Ｒの発現が示された。
【００６２】
実施例１０：ＥＬＩＳＡによるヒトＩｇＧ（Ｍタンパク）産生の測定
ＫＰＭＭ２のヒトＩｇＧ（Ｍタンパク）産生能を試験した。
【００６３】
実施例１に記載の方法で得られたＫＰＭＭ２を１０^６個／ｍｌとなるように、２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを含むＲＰＭＩ１６４０培養液に浮遊させ、容量２ｍｌで１２穴プレート（ファルコン社製）の各穴に分注し、ヒトＩＬ−６非存在下で湿潤５％ＣＯ_２中、３７℃で７２時間培養した。なお、実験は３回行った。その後、培養上清中のヒトＩｇＧ濃度を、ＴＡＧＯ社製ヤギ抗ＩｇＧ抗体（ＮＯ．４１００）およびアルカリフォスファターゼ標識ヤギ抗ＩｇＧガンマ鎖特異的抗体（ＮＯ．２４９０）を用いるＥＬＩＳＡにて測定した。なお、スタンダードとしてカッペル社製ヒトＩｇＧ（ＮＯ．０００１−８６０）を用いた。その結果、培養上清中には１０．１μｇ／ｍｌのヒトＩｇＧが検出され、対照培養液では検出限界以下（５ｎｇ／ｍｌ）であったのに比較して、培養上清中のヒトＩｇＧ濃度が顕著に増加していることが確認された。
【００６４】
実施例１１：ＲＴ−ＰＣＲ（逆転写ポリメレースチェインリアクション）によるＩＬ−６およびＩＬ−６Ｒ ｍＲＮＡの検出
実施例１に記載の方法で得られたＫＰＭＭ２におけるヒトＩＬ−６およびヒトＩＬ−６Ｒの発現を確認するために、ＲＴ−ＰＣＲ（逆転写ポリメレースチェインリアクション）法によりヒトＩＬ−６およびヒトＩＬ−６ＲのメッセンジャーＲＮＡ（ｍＲＮＡ）を検出した。
【００６５】
グアニジウムセシウムクロライド法（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ）によってＫＰＭＭ２細胞（１０^８個）から全ＲＮＡを調製した。陰性対照としてヒトＢ細胞リンパ腫細胞株ＳＫＷ６．４からも同様に全ＲＮＡを調製した。１本鎖ｃＤＮＡ合成はｃＤＮＡ合成キット（Ｉｎｖｉｔｒｏｇｅｎ社製）を用いて、添付の処方に従い全ＲＮＡ５μｇから直接実施した。
【００６６】
陽性対照としてヒトＩＬ−６およびヒトＩＬ−６Ｒ検出用ＰＣＲプライマーを用いた（Ｃｌｏｎｔｅｃｈ Ｉｎｃ．社製）。ＰＣＲ溶液各１００μｌは、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３）、５０ｍＭ 塩化カリウム、Ａｍｐｌｉｔａｑ（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ｃｅｔｕｓ社製）２．５ユニット、１本鎖ｃＤＮＡ合成反応物１μｌ、各プライマー１００ｐｍｏｌを含む。各ＰＣＲ用チューブに鉱物オイル５０μｌを上層してＰＣＲに付した。最初に９４℃で１分メルトし、６０℃で１分と７２℃で１０分のサイクルを３０サイクル繰り返した。最終サイクルの後、最終的に７２℃で１０分伸長を行った。ヒトＩＬ−６およびヒトＩＬ−６Ｒ検出用プライマーを用いた陽性対照群も同様に増幅し、約５０ｐｇの陽性対照ＰＣＲ産物を得た。
【００６７】
各反応チューブから１０μｌを取って１．５％アガロースゲル上で電気泳動を行った。図８に示すように、ＩＬ−６（６２８ｂｐ）およびＩＬ−６Ｒ（２５１ｂｐ）のＰＣＲ産物はいずれもＫＰＭＭ２のＲＮＡから増幅された。これらの結果は、ＫＰＭＭ２がＩＬ−６のオートクライン機構によって増殖することを遺伝子発現の面から支持する。一方、ＳＫＷ６．４細胞はＩＬ−６に応答してＩｇＭを分泌する。ＩＬ−６ＲのＰＣＲ産物はＳＫＷ６．４ ｍＲＮＡから増幅された。しかし、ＩＬ−６のＰＣＲ産物はこの実験で検出されず、このことはＳＫＷ６．４細胞がＩＬ−６を産生していないことを示唆する。
【００６８】
実施例１２：細胞株ＫＰＭＭ２のＳＣＩＤマウスおよびヌードマウスへの移植（１）ＫＰＭＭ２の可移植性およびインビボでの継代
上記実施例１で樹立した細胞株ＫＰＭＭ２の可移植性およびインビボでの継代を以下のように検討した。
【００６９】
ＫＰＭＭ２を２０％ＦＣＳおよび１００μｇ／ｍｌカナマイシンを含むＲＰＭＩ１６４０培養液に１０^８個／ｍｌで懸濁し、０．２ｍｇのウサギ抗アシアロＧＭ１抗体（Ｃｏｄｅ Ｎｏ．０１４−０９８０１、和光純薬工業社製）で処理したＩＬ−６トランスジェニック重症免疫不全マウス（以下ＩＬ−６−ＳＣＩＤ Ｔｍという）（中外製薬製）の腹部皮下に注射針で０．１ｍｌ移植した。その結果、１カ月後には３例全例で移植部位に結節型の腫瘍を形成した。
【００７０】
この腫瘍を無菌的に摘出し、摘出した腫瘍塊を使い捨て注射器のピストンなどでつぶし、ナイロンメッシュ（７０μｍ、ファルコン社製）を通して細胞を回収した。この細胞を上記と同様にＲＰＭＩ１６４０培養液に懸濁し、ＩＬ−６−ＳＣＩＤ Ｔｍ、重症免疫不全マウス（ＳＣＩＤマウスという）（日本クレア社製）、ＢＡＬＢ／ｃ−ｎｕ／ｎｕマウス（以下ヌードマウスという）（日本クレア社製）に皮下移植したところ、同じように移植部位に結節型の腫瘍を形成し、インビボでの継代が可能であった。また腫瘍塊を３ｍｍ角のブロックにし、移植針を用いてＩＬ−６−ＳＣＩＤ Ｔｍ、ＳＣＩＤマウス、ヌードマウスの皮下に移植しても継代可能であった。
（２）ＫＰＭＭ２の異なる移植経路での可移植性
ＫＰＭＭ２の移植経路を皮下（ｓ．ｃ．）、静脈内（ｉ．ｖ．）、腹腔内（ｉ．ｐ．）とした場合の可移植性を以下のように検討した。
【００７１】
（１）においてＳＣＩＤマウスにて継代したＫＰＭＭ２腫瘍を無菌的に摘出し、摘出した腫瘍塊を使い捨て注射器のピストンでつぶし、ナイロンメッシュを通して細胞を回収し、１０^８個／ｍｌの細胞懸濁液を作成した。この懸濁液をＳＣＩＤマウス、あるいは０．２ｍｇのウサギ抗アシアロＧＭ１抗体および５００ＲのＸ線で処理したヌードマウスに０．１ｍｌずつ皮下（ｓ．ｃ．）、静脈内（ｉ．ｖ．）、腹腔内（ｉ．ｐ．）の３経路で移植し、４０日後に生着の判定を行った（表２）。その結果、約４０日後には全例で生着が確認され、ｓ．ｃ．移植では移植部位に、ｉ．ｐ．移植では腹腔内に固形腫瘍を形成した。また、ｉ．ｖ．移植ではＫＰＭＭ２は骨髄での生着が認められた。
【００７２】
【表２】

（３）ＫＰＭＭ２移植動物の血清ヒトＩｇＧ（Ｍタンパク：ミエローマタンパク）の濃度
細胞株ＫＰＭＭ２をＳＣＩＤマウスへ皮下移植し、形成された腫瘍の体積と血清中ヒトＩｇＧ濃度の相関を調べた。上記（２）に記載の方法で細胞株ＫＰＭＭ２をＳＣＩＤマウスへ皮下移植し、ＫＰＭＭ２移植前、移植後２１日目、４２日目の３回、血清サンプルを採取してＥＬＩＳＡ法にてヒトＩｇＧ濃度を測定した。
【００７３】
その結果、マウス血清中のヒトＩｇＧ濃度はＫＰＭＭ２を移植した動物ではいずれも経時的に上昇した。また図９に示すように、皮下移植した動物では皮下に形成された腫瘍の体積と血清ヒトＩｇＧ濃度に相関が見られ、腫瘍の大きな動物ほど血清ヒトＩｇＧ濃度は高値を示した。このことから、血清ヒトＩｇＧ濃度を指標としても抗腫瘍効果の判定ができると考えられた。
（４）ＫＰＭＭ２の可移植性および移植細胞数の検討
ＳＣＩＤマウスとヌードマウスで移植細胞数を変えたときのＫＰＭＭ２の可移植性を検討した。
【００７４】
特別な前処理を行わない雄のＳＣＩＤマウスおよびヌードマウスに、上記（２）記載の方法で各々ＫＰＭＭ２細胞を１０^７、３×１０^６、１０^６個腹部皮下（ｓ．ｃ．）に移植し、またＳＣＩＤマウスには上記（２）の方法で得たＫＰＭＭ２細胞を３×１０^６、１０^６個静脈内（ｉ．ｖ．）でも移植した。
【００７５】
その結果、ＳＣＩＤマウスに皮下移植した場合、３×１０^６個以上移植すると、２１日までに全例で腫瘍結節を形成し、１０^６個移植した場合でも２１日までに２／３、３２日までに全例で腫瘍結節を形成した。ヌードマウスに皮下移植すると、１０^７個移植した場合では２１日までに２／３、３２日までには全例で、また３×１０^６個移植した場合では４２日までには全例で腫瘍結節が形成された。しかし、１０^６個の移植では４２日までに腫瘍の生着は見られなかった。また、ＳＣＩＤマウスに静脈内移植した場合、４２日までに３×１０^６個移植で全例、１０^６個移植で１／３にＫＰＭＭ２が生着した。これらの結果を以下の表３にまとめて示す。
【００７６】
【表３】

（５）ＫＰＭＭ２移植ＳＣＩＤマウスにおけるヒトＣＤ３８抗原の発現の解析
ＫＰＭＭ２を移植したＳＣＩＤマウスにおいて、その生着を確認するために、ＫＰＭＭ２の細胞表面上で特徴的に発現している表面抗原であるヒトＣＤ３８抗原の発現を試験した。
【００７７】
ＫＰＭＭ２を上記（１）に記載する方法でＳＣＩＤマウスへ静脈内移植し、移植後３７日目にマウスを屠殺した。屠殺したマウスの大腿骨より骨髄を回収して懸濁し、ステンレスメッシュ（１００μｍ）を通し、蛍光活性化細胞選択装置（ＦＡＣＳ）用緩衝液（２％ＦＣＳおよび０．１％ＮａＮ_３を含むＰＢＳ（−）溶液）にて骨髄細胞の浮遊液を調製した。なお、陰性対照としてＫＰＭＭ２を移植していないＳＣＩＤマウスの骨髄からも同様の方法にて骨髄細胞浮遊液を調製した。
【００７８】
また、本実施例に記載する方法によりＫＰＭＭ２を皮下移植したＳＣＩＤマウスからは、移植後３７日目に皮下腫瘍塊を外科的に摘出し、摘出した腫瘍塊を２枚のスライドグラスの間にはさんですりつぶし、これをステンレスメッシュ（１００μｍ）を通し、ＦＡＣＳ緩衝液にて骨髄細胞の浮遊液を調製した。
【００７９】
陽性対照として、実施例１に記載した方法でインビトロにて培養したＫＰＭＭ２を用いた。インビトロで４日間培養したＫＰＭＭ２をＦＡＣＳ緩衝液で洗浄した後、同緩衝液中に浮遊させた。
【００８０】
次に、このように調製した細胞浮遊液を用いて、ＦＡＣＳ解析によるヒトＣＤ３８抗原の発現を調べた。
【００８１】
すなわち、１００μｌのＦＡＣＳ緩衝液中にて、各群の浮遊細胞１０^６個に対し、２．５μｇ／ｍｌのフィコエリスリン（ＰＥ）標識抗ヒトＣＤ３８抗体（Ｌｅｕ−１７、ベクトン・ディッキンソン社製）を添加し、氷上で３０分間反応させた。次いで、１ｍｌのＦＡＣＳ緩衝液で２回洗浄後、５００μｌのＦＡＣＳ緩衝液で懸濁し、ＦＡＣＳｃａｎ（ベクトン・ディッキンソン社製）によりＦＡＣＳ解析を行った。
【００８２】
陽性対照群であるインビトロ培養ＫＰＭＭ２のＦＡＣＳ解析の結果から、蛍光強度４０から２０００までの範囲にある細胞をヒトＣＤ３８抗原発現細胞とした（図１０（ａ）参照）。ＫＰＭＭ２を静脈内移植したＳＣＩＤマウスでは、その骨髄細胞のおよそ７１％がヒトＣＤ３８陽性細胞で占められていた（図１０（ｂ）参照）。このことは、ＳＣＩＤマウスに静脈内移植したＫＰＭＭ２がＳＣＩＤマウスの骨髄に生着したことを示している。また、ＫＰＭＭ２をＳＣＩＤマウスに皮下移植して生じた腫瘍塊から得られた細胞は全てヒトＣＤ３８が陽性であり（図１０（ｃ）参照）、ＫＰＭＭ２を皮下移植して生ずる腫瘍塊は、全てＫＰＭＭ２から構成されていることが示された。なお、ＫＰＭＭ２を移植していない陰性対照群のＳＣＩＤマウスの骨髄細胞にはヒトＣＤ３８陽性細胞は全く検出されなかった（図１０（ｄ）参照）。
【００８３】
実施例１３：抗ヒトＩＬ−６抗体ＳＫ２および抗ヒトＩＬ−６Ｒ再構成ヒト型化抗体ＰＭ１の抗腫瘍効果
ＫＰＭＭ２移植動物における抗ヒトＩＬ−６抗体ＳＫ２および抗ヒトＩＬ−６Ｒ再構成ヒト型化抗体ＰＭ１のインビボ抗腫瘍効果を以下のようにして検討した。
【００８４】
上記実施例１２（１）に記載の方法でＳＣＩＤマウスで継代して得たＫＰＭＭ２骨髄腫腫瘍塊をウサギ抗アシアロＧＭ１抗体／Ｘ線処理ヌードマウス（５週齢、雄）に４ｍｍ角ブロックで皮下移植し、翌日に１回だけＳＫ２抗体を１ｍｇ／マウスの１用量、再構成ヒト型化ＰＭ１抗体（国際公開出願ＷＯ９２−１９７５９参照）を０．１２５、０．５および１ｍｇ／マウスの３用量で静脈内投与した。各抗体は０．２ｍｌ／マウスとなるようにＰＢＳ（−）（ニッスイ製）で調製し、陰性対照群には、ＰＢＳ（−）を０．２ｍｌ／マウス投与した。その後腫瘍の大きさを経時的に観察し、対照群の腫瘍が十分大きくなった３５日目に全採血を行ってから腫瘍を摘出して重量を測定した。
【００８５】
その結果を図１１に示す。陰性対照群の平均腫瘍重量が約１ｇであったのに対し、再構成ヒト型化ＰＭ１抗体投与群においては、１ｍｇ／マウス投与した場合で腫瘍増殖抑制率（Ｇｒｏｗｔｈ Ｉｎｈｉｂｉｔｏｒｙ Ｒａｔｉｏ：ＧＩＲ）は７８％、０．５ｍｇ／マウス投与した場合でのＧＩＲは５３％、０．１２５ｍｇ／マウス投与した場合でのＧＩＲは６６％を示し、強い腫瘍増殖抑制効果が見られた。またＳＫ２においても、１ｍｇ／マウス投与群でＧＩＲ６１％と腫瘍増殖を抑制した。
【００８６】
さらに、腫瘍摘出時に採取した血清サンプルのヒトＩｇＧ濃度をＥＬＩＳＡ法にて測定した。その結果を図１２に示す。抗体非投与陰性対照群では血清ヒトＩｇＧ濃度が平均２７．６ｍｇ／ｍｌであったものが、再構成ヒト型化ＰＭ１抗体を１、０．５、０．１２５ｍｇ／マウス投与することでそれぞれ７１％、５５％、７５％抑制された、ＳＫ２ １ｍｇ／マウス投与でも４３％抑制された。腫瘍重量と血清ヒトＩｇＧ濃度は各処理群間でも、個体レベルでもよく相関していた。
【００８７】
実施例１４：ＫＰＭＭ２静脈内移植ＳＣＩＤマウスにおけるイオン化カルシウム濃度の上昇および骨吸収の亢進
ＫＰＭＭ２（１０^７個）を上記実施例１２（２）に記載する方法でＳＣＩＤマウス（日本クレア社製）へ静脈内移植し、移植後経時的に血中イオン化カルシウム濃度および骨吸収の有無を試験した。
【００８８】
ＫＰＭＭ２移植後９日目、２０日目、３０日目および３７日目にＫＰＭＭ２静脈内移植ＳＣＩＤマウスをエーテル麻酔し、マウス眼窩より６０μｌ容量のキャピラリーカラム（チバ・コーニング社製）で採血し、直ちに血中イオン化カルシウム濃度を６３４自動Ｃａ^＋＋／ｐＨアナライザー（チバ・コーニング社製）にて測定した。なお、対照としてＫＰＭＭ２を移植していないＳＣＩＤマウスからも同様の方法で採血し、血中イオン化カルシウム濃度を測定した。
【００８９】
その結果、ＫＰＭＭ２静脈内移植ＳＣＩＤマウスの血中イオン化カルシウム濃度は、移植後３０日目より上昇がみられ、３７日目には対照群のマウスに比べ、約２０％の血中イオン化カルシウム濃度上昇が観察された（図１３）。なお、血中イオン化カルシウム濃度上昇は、ＫＰＭＭ２をＳＣＩＤマウスへ静脈内移植したときの、マウス骨髄におけるＫＰＭＭ２（ヒトＣＤ３８抗原陽性細胞）が占める増加の割合の経時的増加とよく相関していた（図１４参照）。
【００９０】
また、ＫＰＭＭ２静脈内移植３７日目のＳＣＩＤマウス下肢の骨をＸ線撮影して形態的に観察したところ、対照群のマウスと比較し、顕著な骨吸収像が確認された（図１５参照）。以上の結果は、ＫＰＭＭ２静脈内移植ＳＣＩＤマウスの骨病変が、実際の骨髄腫の病変とよく一致していることを示している。
【００９１】
【発明の効果】
本発明によってＩＬ−６オートクライン依存性で増殖する骨髄腫が存在することが明らかとなった。本発明のオートクライン機構によりＩＬ−６依存性で増殖するヒト骨髄腫細胞株は、骨髄腫のＩＬ−６依存性増殖機構モデルとして有用である。また抗ＩＬ−６抗体、抗ＩＬ−６受容体抗体などのＩＬ−６活性阻害剤をはじめとする骨髄腫治療剤の治療モデルとしても使用し得るものである。本発明のオートクライン機構によりＩＬ−６依存性で増殖するヒト骨髄腫細胞株はインビトロおよびインビボで細胞増殖抑制を指標とした骨髄腫治療剤の評価系を作成するのに有用であるのはもちろん、Ｍタンパクを産生し、この産生量が骨髄腫の増殖に極めてよく相関することから、Ｍタンパク産生量の抑制を指標とした骨髄腫治療剤の評価系を作成するのに有用である。
【００９２】
さらに、本発明のヒト骨髄腫細胞株を実験動物に静脈内移植して得られる骨髄腫の骨髄生着モデルでは、多発性骨髄腫の増殖に伴い骨髄腫に特徴的な骨病変が観察され、したがってこの骨病変の抑制を指標とした骨髄腫治療剤の評価系を作成することができる。
【００９３】
これらの点から本発明のヒト骨髄腫細胞株の利用価値は極めて大きい。
【図面の簡単な説明】
【図１】液体培養におけるＫＰＭＭ２の自律的凝集を示す図（生物の形態を表す写真）である。
【図２】ＫＰＭＭ２の形態を示す図（生物の形態を表す写真）である。ライト−ギムザ染色では形質細胞の特徴を有する。
【図３】サザンブロット分析によるＫＰＭＭ２のＪ_ＨおよびＣλ遺伝子の再構成を示す図（電気泳動の写真）である。ＫＰＭＭ２から得たＤＮＡをＢａｍＨＩ、ＥｃｏＲＩおよびＨｉｎｄＩＩＩで消化し、Ｊ_ＨおよびＣλ遺伝子プローブを用いてサザンブロット分析を行った。再構成したバンドを（▲）で示す。
【図４】ＫＰＭＭ２の核型を示す図（生物の形質を表す写真）である。検索した１５細胞はすべて４６、ＸＸ、ｄｅｒ（１；１９）（ｑ１０；ｑ１０）、ｔ（３；１４）（ｑ２１；ｑ３２）、−４、ｔ（６；１１）（ｐ１２；ｐ１５）、ｄｅｒ（１０）ａｄｄ（１０）（ｐ１３）ｄｉｃ（９；１０）（ｑ１０；ｑ２６）、＋１６を示した。
【図５】ＫＰＭＭ２の細胞増殖に対する各種サイトカインの効果を示す図である。使用したサイトカインの濃度は以下の通りである：ＩＬ−６、１ｎｇ／ｍｌ；ＩＦＮ−αおよびＩＦＮ−γ、１０００Ｕ／ｍｌ；その他のサイトカイン、１００ｎｇ／ｍｌ。各数値は３回の試験の平均＋標準偏差（ＳＤ）を表す。
【図６】ＫＰＭＭ２の細胞増殖に対する抗ＩＬ−６ ｍＡｂおよび抗ＩＬ−６Ｒ ｍＡｂの効果を示す図である。ＳＫ２はマウス抗ＩＬ−６ ｍＡｂ（▲）；ＰＭ１はマウス抗ＩＬ−６Ｒ ｍＡｂ（●）。破線は対照を示す。各数値は３回の試験の平均を表す。
【図７】ＫＰＭＭ２細胞におけるＩＬ−６Ｒの発現を示す図である。細胞は抗ＩＬ−６Ｒ ｍＡｂ（ＭＴ１８）で染色した。マウスＩｇＧ２ｂ抗体を対照として用いた。破線はｍＩｇＧ２ｂを、実線はＭＴ１８を表す。
【図８】１．５％アガロースゲル上でのＲＴ−ＰＣＲ（逆転写ＰＣＲ）分析により、ＫＰＭＭ２のＩＬ−６およびＩＬ−６Ｒ ｍＲＮＡの発現を示す図（電気泳動の写真）である。レーン１および４、ＳＫＷ６．４；レーン２および５、ＫＰＭＭ２；レーン３および６、陽性対照。
【図９】ＫＰＭＭ２を皮下移植したマウスにおける腫瘍体積と血清ヒトＩｇＧ濃度の相関を示す図である。
【図１０】（ａ）はインビトロ培養したＫＰＭＭ２におけるヒトＣＤ３８抗原のＦＡＣＳ解析を示す図である。ＫＰＭＭ２は４０から２０００までの範囲の蛍光強度を有する。
（ｂ）はＫＰＭＭ２を静脈内移植したＳＣＩＤマウスの骨髄細胞におけるヒトＣＤ３８抗原のＦＡＣＳ解析を示す図である。７１％の細胞が４０から２０００の範囲の蛍光強度を有する。
（ｃ）はＫＰＭＭ２を皮下移植したＳＣＩＤマウスの腫瘍塊から得た細胞におけるヒトＣＤ３８抗原のＦＡＣＳ解析を示す図である。全ての細胞が４０から２０００の範囲の蛍光強度を有する。
（ｄ）はＫＰＭＭ２を移植していないＳＣＩＤマウスの骨髄細胞におけるヒトＣＤ３８抗原のＦＡＣＳ解析を示す図である。４０から２０００の範囲の蛍光強度を有する細胞は全くみられない。
【図１１】ＫＰＭＭ２に対する抗ＩＬ−６ ｍＡｂおよび抗ＩＬ−６Ｒ ｍＡｂのインビボにおける腫瘍増殖抑制効果を示す図である。ＳＫ２は抗ヒトＩＬ−６ ｍＡｂ；再構成ヒト型化ＰＭ１は抗ヒトＩＬ−６Ｒ ｍＡｂ。
【図１２】ＫＰＭＭ２移植ヌードマウス中の血清ヒトＩｇＧ濃度に対する抗ＩＬ−６ ｍＡｂまたは抗ＩＬ−６Ｒ ｍＡｂの効果を示す図である。
【図１３】ＫＰＭＭ２を静脈内移植したＳＣＩＤマウスの血中イオン化カルシウム濃度（□）の経時的変化を示す図である。（◇）は対照を示す。各数値はＳＣＩＤマウス４匹（３７日目のみ５匹）の平均±Ｓ．Ｄ．を示す。
【図１４】ＫＰＭＭ２を静脈内移植したＳＣＩＤマウスの骨髄中のヒトＣＤ３８抗原陽性細胞の割合（□）の経時的変化を示す図である。（◇）は対照を示す。各数値はＳＣＩＤマウス４匹（３７日目のみ５匹）の平均±Ｓ．Ｄ．を示す。
【図１５】ＫＰＭＭ２を静脈内移植したＳＣＩＤマウスの骨のＸ線撮影像を示す図（生物の形態を示す写真）である。
（ａ）はＫＰＭＭ２を移植していない対照群のＳＣＩＤマウス
（ｂ）はＫＰＭＭ２移植後３７日目のＳＣＩＤマウス[0001]
[Industrial applications]
The present invention relates to a human myeloma cell line, and more particularly to a human myeloma cell line that grows in an IL-6-dependent manner by an autocrine mechanism, an experimental animal transplanted with the cell line, and using the cell line or the experimental animal. The present invention relates to a method for screening a therapeutic agent for myeloma.
[0002]
[Prior art]
Interleukin 6 (IL-6), which is the same factor as B cell stimulating factor 2 (BSF-2) and mouse hybridoma / plasmacytoma growth factor, is multiple myeloma (hereinafter sometimes referred to as MM). It is considered to be a major growth factor for cells (Kawano et al., Nature 332: 83, 1988; Kleine et al., Blood 73: 517, 1989). Multiple myeloma is a tumor in which plasma cells have become malignant, and the bone marrow is used as a growth site, and occurs simultaneously in multiple sites. IL-6 transmits its activity on such cells via two membrane proteins. One is a ligand-binding membrane protein with a molecular weight of 80 kD (IL-6 receptor) to which IL-6 binds, and the other is a membrane protein gp130 involved in non-ligand binding signal transduction (Taga). et al., J. Exp. Med. 196: 967, 1987).
[0003]
In 1988, Kawano et al. Found that fresh human myeloma cells constitutively produced and expressed IL-6 receptor, and that proliferation in vitro was induced by anti-IL-6 receptor (R) antibodies. It was reported that myeloma cells could proliferate by the autocrine mechanism of producing and receiving growth factors by themselves (Kawano et al., Nature 332: 83, 1988), because they are suppressed. On the other hand, Klein et al. Proposed that they do not produce growth factors themselves, but proliferate by a paracrine mechanism that accepts growth factors from the surroundings (Klein et al., Blood 73: 517, 1989). It is also known that serum IL-6 concentration is correlated with myeloma disease (Bataille et al., J. Clin. Invest. 84: 2008, 1989), and IL-6 is associated with myeloma. Is believed to be one of the major growth factors for
[0004]
In the case of freshly isolated myeloma cells, contamination of cells other than myeloma cells is unavoidable, and accurate assays are difficult. It is not clear which of them will proliferate.
[0005]
When human IL-6 cDNA was introduced by transfection into a human myeloma cell line that had an IL-6-dependent growth, it was observed that the human IL-6 cDNA grew autonomously and turned into a tumor. (Okuno et al., Exp. Hematol. 20: 395, 1992). It has also been reported that the human myeloma cell line U266 grows by the IL-6 autocrine mechanism (Jernberg et al., Leukemia 5: 255, 1991; Levy et al., J. Clin. Invest. 88: 696, 1991). However, proliferation of U266 was also induced by exogenous IL-6 (Jernberg et al., Leukemia 5: 255, 1991) and by anti-IL-6 monoclonal antibody (Levy et al., J. Clin. Invest. 88: 696, 1991) There was a report that it was not affected, and the involvement of IL-6 in the proliferation mechanism of U266 is unknown.
[0006]
We found that in one case of fresh myeloma cells obtained from ascites of a patient with multiple myeloma, the tumor cells showed a clear IL-6-dependent growth, and the anti-IL-6 receptor with autonomous growth. It was reported that the antibody was strongly suppressed by a body antibody (Goto et al., Biotherapy 7: 655, 1993).
[0007]
The present inventors further examined the properties of tumor cells obtained by transplanting fresh human myeloma cells into a human IL-6 gene-transfected severe combined immunodeficient mouse (IL-6 transgenic SCID mouse) (Goto et al., No. Proceedings of the 52nd Annual Meeting of the Japanese Cancer Society, 498 pages, October 1993). As a result, three subcutaneously transplanted mice showed plasmacytoma at the transplanted site and metastasis to axillary lymph nodes. No tumor formation was observed by intraperitoneal transplantation. The tumor cells after transplantation showed no change in surface antigens, in vitro IL-6 dependent proliferation in vitro, and the growth inhibitory effect of anti-human IL-6 receptor antibody compared to before transplantation.
[0008]
[Problems to be solved by the invention]
As described above, there are still many unknowns about the proliferation mechanism of myeloma cells, and it is required to elucidate this.
[0009]
An object of the present invention is to establish a myeloma cell line that can serve as a model for the IL-6-dependent growth mechanism of myeloma. The cell lines are useful as in vitro models for the treatment of myeloma with myeloma therapeutics, including IL-6 activity inhibitors such as anti-IL-6 antibodies, anti-IL-6 receptor antibodies.
[0010]
Another object of the present invention is to provide an experimental animal into which the cell line has been transplanted. The above experimental animals are useful as an in vivo model for the treatment of myeloma with myeloma therapeutic agents including IL-6 activity inhibitors such as anti-IL-6 antibodies and anti-IL-6 receptor antibodies.
[0011]
Another object of the present invention is to provide a method for screening a therapeutic agent for myeloma. In the above-mentioned in vitro model, a screening method for a therapeutic agent for myeloma using inhibition of cell growth or inhibition of M protein (myeloma protein) secretion as an index can be used. Further, in the above-mentioned in vivo model, a screening method for a therapeutic agent for myeloma, which uses cell growth suppression, M protein secretion suppression, or bone lesion suppression as an index, can be used. The M protein is an immunoglobulin protein specifically produced by myeloma, and there are five types of myeloma producing IgA, IgM, IgG, IgE and Bence-Jones protein.
[0012]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to achieve the above object, and as a result, succeeded in establishing a cell line that can be a model of myeloma having an IL-6-dependent growth mechanism, and completed the present invention.
[0013]
That is, the present invention provides a human myeloma cell line that grows in an IL-6-dependent manner by an autocrine mechanism.
[0014]
The present invention also provides an experimental animal into which the cell line has been transplanted.
[0015]
Furthermore, the present invention provides an in vitro screening method for a therapeutic agent for myeloma, which comprises adding a therapeutic agent for myeloma to the above cell line and testing for inhibition of myeloma cell growth or inhibition of M protein secretion.
[0016]
Further, the present invention provides an in vivo screening method for a therapeutic agent for myeloma, which comprises administering the therapeutic agent for myeloma to the above-mentioned experimental animal and testing the inhibition of myeloma cell growth, inhibition of M protein secretion, or inhibition of bone lesions.
[0017]
The cell line of the present invention can be established using myeloma cells collected from, for example, ascites of a multiple myeloma patient. In the present invention, in particular, myeloma cells were collected from ascites of IgG and λ-type multiple myeloma patients. One month after the start of the culture, the cells began to proliferate stably and were maintained for one year or more. The cell line thus established is named KPMM2 and has been deposited at the Biotechnology, Industrial Technology Research Institute, Patent Microorganisms Depositary under the accession number FIRM: P-14170 (deposited on February 22, 1994). The cell line KPMM2 has been successfully passaged in vitro and in stable passage in immunodeficient mice such as experimental animals, for example, severe combined immunodeficiency (SCID) mice, IL-6 transgenic SCID mice and nude mice. It is possible.
[0018]
The cell line KPMM2 was confirmed to produce IL-6 in vitro and to express the IL-6 receptor (IL-6R). In addition, the reactivity of KPMM2 to various cytokines³H-thymidine incorporation experiment, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) method (see J. Immun. Methods 65: 55-63, 1983) and live cells. Tests of the measurements showed that only significant stimulation was observed when incubated with IL-6, indicating that KMMM2 proliferates specifically in response to IL-6. Furthermore, the proliferation of KPMM2 was significantly suppressed in a dose-dependent manner by anti-IL-6 mAb (monoclonal antibody) and anti-IL-6R mAb. In addition, RT-PCR (reverse transcription polymerase chain reaction) confirmed that KPMM2 expressed IL-6 and IL-6R mRNA. KPMM2 expresses various adhesion molecules, namely, CD44, VLA-β, ICAM-1, NCAM, LFA-3 and VLA-4, and shows autonomous cell aggregation in vitro.
[0019]
From these various characteristics of KPMM2, it was shown that KPMM2 is a myeloma cell line proliferating by the IL-6 autocrine mechanism. The cell line of the invention is the first cell line whose growth mechanism has been demonstrated to be an IL-6 dependent autocrine mechanism.
[0020]
Therefore, the cell line of the present invention is useful for screening a therapeutic agent for myeloma including an inhibitor of IL-6 activity such as an anti-IL-6 mAb or an anti-IL-6R mAb. For example, an in vitro screening method for a therapeutic agent for myeloma, which comprises adding an anti-IL-6 antibody or an anti-IL-6 receptor antibody to the cell line of the present invention and testing the inhibitory effect on myeloma cell growth, is possible. In addition, the myeloma cell line of the present invention can perform in vitro screening for a therapeutic agent for myeloma using the suppression of M protein secretion as an index because the amount of secreted M protein increases in proportion to the increase in cell number. Furthermore, the cell lines of the present invention are also useful as models for studying the role played by adhesion molecules in myeloma cell proliferation via cell-cell interactions and IL-6 signaling.
[0021]
The present invention also provides a laboratory animal into which the cell line of the present invention has been transplanted. Experimental animals to which the cell line of the present invention is transplanted include, in addition to mice, rats, rabbits, guinea pigs, hamsters, monkeys, and the like. Further, the function of immunocompetent cells such as T cells or B cells is impaired, It is preferable to transplant the cell line of the present invention into an experimental animal in an immunodeficient state. As already mentioned, the cell lines of the present invention are capable of stable passage in immunodeficient mice such as SCID mice, IL-6 transgenic SCID mice and nude mice.
[0022]
Interestingly, the above cell line of the present invention can be transplanted into mice by subcutaneous transplantation or intraperitoneal transplantation, as well as by intravenous transplantation. In the case of subcutaneous and intraperitoneal transplants, solid tumors were observed at the subcutaneous and intraperitoneal sites, respectively.However, in the case of intravenous transplantation, engraftment of tumor cells into the bone marrow was observed. Can be used as a model close to the pathology of myeloma. For example, administering a therapeutic agent for myeloma including an IL-6 activity inhibitor such as an anti-IL-6 antibody or an anti-IL-6 receptor antibody to an experimental animal of the present invention to test the inhibitory effect on myeloma cell proliferation. In vivo screening method for a therapeutic agent for myeloma comprising In experimental animals in which the cell line of the present invention has survived, an increase in serum M protein concentration is observed with the growth of tumors. Screening methods are also possible. Further, when the cell line of the present invention engrafted to the bone marrow of an experimental animal, as the myeloma cells proliferate, an increase in blood ionized calcium concentration, bone destruction, bone lesions such as osteolysis and bone resorption are observed, An in vivo screening method for a therapeutic agent for myeloma using the suppression of these bone lesions as an index is possible.
[0023]
Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.
[0024]
【Example】
Example 1 Establishment and Maintenance of a Myeloma Cell Line
Myeloma cells were collected from ascites of an IgG, λ multiple myeloma patient (76 years old, female, stage IIA). The ascites fluid contained a large number of myeloma cells, with IL-6 levels in the ascites fluid reaching 91.0 pg / ml. The collected ascites was subjected to density gradient centrifugation using Ficoll-Hypaque (manufactured by Pharmacia) to separate mononuclear cells, to remove adherent cells with a plastic Petri dish, and to remove T cells with sheep erythrocytes. Purified to 95% or more. RPMI1640 (manufactured by Gibco) containing 20% fetal calf serum (FCS: manufactured by Xavier Investments, Australia), 4 ng / ml of recombinant human IL-6 (manufactured by Chugai Pharmaceutical) and 100 μg / ml of kanamycin (manufactured by Meiji Seika) 1 × 10 in culture⁶The cells were suspended at a concentration of cells / ml. The cells are then cultured in a 10 ml culture in a 25 ml flask and wet 5% CO 2₂And incubated at 37 ° C. The medium was partially replaced every three days until stable cell growth was observed.
[0025]
One month after the start of the culture, the cells began to proliferate stably and were maintained for one year or more, and were established as cell lines. This cell line was named KPMM2. The doubling times in the presence or absence of IL-6 were 48 hours and 72 hours, respectively.
[0026]
The morphology and Ig secretion of KPMM2 are as follows.
KPMM2 morphology and Ig secretion
KPMM2 cells were observed to proliferate under light microscopy with autonomous cell aggregation (FIG. 1), and Wright-Giemsa staining showed a plasma cell-like morphology (FIG. 2). KPMM2 was positive for acid phosphatase and slightly positive for α-naphthyl butyrate esterase, but negative for peroxidase, AS-D chloroacetate esterase, pass (periodite Schiff reagent) and alkaline phosphatase. Cytoplasmic IgG and λ light chains were detected, but IgA, IgM, and κ light chains were negative by direct immunofluorescence. In addition, cells (10⁶Per ml) for 3 days, IgG and λ light chain secretion was observed in the culture supernatant.
[0027]
Example 2: Analysis of surface antigen
The expression of the surface antigen of the cell line KPMM2 established in Example 1 was determined by direct and indirect immunofluorescence (Fried et al., Flow Cytometry, Boca Raton, CRC Press: 59) using a panel of monoclonal antibodies against various human antigens. −78, 1989).
[0028]
KPMM2 obtained by the method described in Example 1⁶100 μl of a buffer for fluorescence activated cell selection device (FACS) (2% FCS and 0.1% NaN)₃In a phosphate-buffered saline solution (PBS) containing the following (hereinafter referred to as FACS buffer). Then, in the direct fluorescent antibody method, a fluorescin isothiocyanate (FITC) or phycoerythrin (PE) -labeled antibody against various human antigens shown in Table 1 below in a saturating amount is added and incubated at 4 ° C. for 30 minutes. did. After the cells were washed twice with the above FACS buffer, they were analyzed with a flow cytometer (EPICS PROFILE, manufactured by Coulter).
[0029]
On the other hand, in the indirect fluorescent antibody method, unlabeled antibodies against various human antigens shown in Table 1 below were added, incubated at 4 ° C. for 30 minutes, and the cells were washed twice with a FACS buffer. / Ml of FITC or PE-labeled goat anti-mouse IgG antibody F (ab ') 2 fragment (manufactured by TAGO) was added and reacted at 4 ° C for 30 minutes. After washing twice with a FACS buffer, the cells were suspended in the FACS buffer and analyzed with a flow cytometer (EPICS PROFILE, manufactured by Coulter).
[0030]
The surface antigens of KPMM2 are summarized in Table 1 below.
[0031]
[Table 1]

As can be seen from the table, KPMM2 is a plasma cell associated antigen (CD38, PCA-1 and BL3), adhesion molecules (CD44, VLA-β, ICAM-1, NCAM, LFA-3 and VLA-4) and CD45, CD63. , CD71, IgG and λ were positive.
[0032]
Example 3: Immunoglobulin gene rearrangement
The immunoglobulin (Ig) gene rearrangement of the cell line KPMM2 established in Example 1 was analyzed by Southern blotting.
[0033]
KPMM2 cells (10%) obtained by the method described in Example 1⁷), DNA was prepared in accordance with the method of Manual of Clinical Immunology, 3rd edition, American Society for Microbiology, 1986, and treated with three kinds of restriction enzymes BamHI, EcoRI or Hindman separately from BamHI, EcoRI or Hindman Hygerin, respectively. The DNA was recovered as an ethanol precipitate, and electrophoresed on a 0.8% agarose gel (SEAKEM GTG, FMC) for 24 hours. The electrophoresed DNA is transferred to a nylon membrane (Hybond N⁺, Amersham) and dried. Next, using Takara random primer DNA labeling kit (Takara Shuzo),³²P-labeled human Ig J_H, Cκ and Cλ probes (manufactured by Oncore) were used for Southern blot analysis according to the attached instructions. As a control, non-reconstituted chromosomal DNA obtained from peripheral blood mononuclear cells of a healthy person was used. As a result, IgH and κ chain genes were rearranged, but λ chain genes were not rearranged (FIG. 3). From the above, it was confirmed that KPMM2 produced a monoclonal antibody and that the cells were unique.
[0034]
Example 4: Cytogenetic analysis
The chromosomal structural abnormality of the cell line KPMM2 established in Example 1 was analyzed.
[0035]
The KPMM2 obtained by the method described in Example 1 was added to an RPMI1640 culture medium containing 20% FCS and 100 μg / ml kanamycin at 5 × 10 5⁵The cells were cultured so as to obtain the number of cells / ml. Forty-eight hours after culturing, KMPM2 was treated with 0.05 μg of corsemide (manufactured by Gibco) for 15 minutes to collect KMMM2 cells whose cell cycle was stopped in the metaphase. The recovered KMPM2 cells were treated with 0.075 M KCl for 20 minutes and fixed with methanol-acetic acid. The chromosomes of the KPMM2 cells were then analyzed by trypsin-Giemsaband staining.
[0036]
As a result, KPMM2 was found to be a diploid cell having many structural abnormalities (FIG. 4). All 15 cells analyzed were 46, XX, der (1; 19) (q10; q10), t (3; 14) (q21; q32), -4, t (6; 11) (p12; p15), der (10) add (10) (p13) dic (9; 10) (q10; q26), +16.
[0037]
Example 5: Detection of EBV and mycoplasma
The cell line KPMM2 established in Example 1 was tested for Epstein-Barr virus (EBV) and mycoplasma contamination.
[0038]
In order to detect Epstein-Barr virus (EBV) from the chromosome of KPMM2 cells obtained by the method described in Example 1, PCR was carried out using an EBV BamW region amplification primer purchased from Systemic Genetic Institute according to the attached protocol. (Polymer race chain reaction). The detection of mycoplasma infection was carried out by M. for mycoplasma DNA detection. T. C. The test was carried out using a kit (Gen-Probe Inc.) according to the attached prescription.
[0039]
As a result, KPMM2 was negative for the EBV genome and the mycoplasma genome.
[0040]
Example 6: Reactivity to cytokine
The reactivity of the cell line KPMM2 established in Example 1 to various cytokines was tested.
[0041]
KPMM2 obtained by the method described in Example 1 was suspended in an RPMI1640 culture solution containing 20% FCS and 100 μg / ml kanamycin, and 1 × 10 6 was added to a 96-well plate (Falcon) at a volume of 200 μl.⁴The mixture was dispensed so as to obtain individual pieces / holes.
[0042]
Various cytokines were separately added to each well of the 96-well plate so as to have the following concentrations.
[0043]
Recombinant IL-2, IL-3, tumor necrosis factor (TNF) -α, granulocyte macrophage colony stimulating factor (GM-CSF), stem cell growth factor (SCF) (all from Genzyme), IL-4, IL-7, IL-10, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM) (all manufactured by Pepro Tec Inc.), IL-9, The transforming growth factor (TGF) -β (above, manufactured by R & D System Inc.), IL-1α (Boehringer Manheim), IL-5 (Upstate Biotechnology Inc.), IL-8 ( Amersham), erythropoietin (EPO) and granulocyte colony stimulating factor (G-CSF) (above, medium Pharmaceutical provided by Co., Ltd.) is 100ng / ml.
[0044]
Recombinant interferon (IFN) -γ (provided by Shionogi & Co., Ltd.) and natural human IFN-α (supplied by Sumitomo Pharma Co., Ltd.) are 1000 U / ml.
[0045]
Recombinant IL-6 (provided by Chugai Pharmaceutical Co., Ltd.) is 1 ng / ml.
[0046]
The control group was cultured in an RPMI 1640 culture medium supplemented with 20% FCS without added cytokine and 100 μg / ml kanamycin.
[0047]
KPMM2 dispensed into each well of a 96-well plate was added with 5% CO 2 in the presence or absence of the cytokine.₂The medium was cultured at 37 ° C. for 96 hours. Four hours before the end of the culture,³H-thymidine (Amersham) was added at 1 μCi / well. KPMM2 captured³The amount of H-thymidine was measured using a liquid scintillation counter (1205 beta plate, manufactured by Pharmacia).
[0048]
FIG. 5 shows the effects of various cytokines on the growth of KMMM2. As is evident from FIG. 5, KPMM2 cells were only incubated when incubated with IL-6.³H-thymidine incorporation was significantly stimulated. By IL-6 at a concentration of 1 ng / ml,³H-thymidine incorporation increased 2.2-fold. On the other hand, IFN-α and IFN-γ markedly inhibited the growth of KPMM2 cells.
[0049]
In addition, in order to examine the reactivity of KPMM2 to cytokines, see MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) method (J. Immun. Methods 65:55, 1983). ) And a method of directly measuring living cells, but similar results were obtained.
[0050]
Example 7: Growth inhibition by anti-IL-6 and anti-IL-6R mAbs
Mouse anti-human IL-6R mAb (monoclonal antibody) against the cell line KPMM2 established in Example 1 (IgG₁Class: PM1) and mouse anti-IL-6 mAb (IgG₁The effect of class: SK2) was investigated. SK2 is described in the literature (Y. Ohe et al., Br. J. Cancer 67: 939, 1993), and PM1 is described in the literature (Hirata et al., J. Immunol. 143: 2900, 1989). ing.
[0051]
KPMM2 obtained by the method described in Example 1 was suspended in an RPMI1640 culture solution containing 20% FCS and 100 μg / ml kanamycin, and 1 × 10 6 was added to a 96-well plate (manufactured by Falcon) in a volume of 200 μl.⁴The mixture was dispensed so as to obtain individual pieces / holes.
[0052]
Various concentrations of IL-6 mAb (monoclonal antibody) (SK2) and anti-IL-6R (receptor) mAb (PM1) were separately added to each well of the 96-well plate. The control group was an RPMI1640 culture solution containing 20% FCS and 100 μg / ml kanamycin to which no antibody was added.
[0053]
KPMM2 dispensed into each well of a 96-well plate was treated with 5% CO 2 in the presence or absence of anti-IL-6 mAb and anti-IL-6R mAb.₂The medium was cultured at 37 ° C. for 96 hours. Four hours before the end of the culture,³H-thymidine (Amersham) was added at 1 μCi / well. KPMM2 captured³The amount of H-thymidine was measured using a liquid scintillation counter (1205 beta plate, manufactured by Pharmacia).
[0054]
The effect of anti-IL-6 mAb and anti-IL-6R mAb on KPMM2 proliferation is shown in FIG. The addition of both SK2 and PM1 significantly inhibited cell proliferation in a dose-dependent manner. In particular, PM1 completely inhibited the growth of KPMM2 at a concentration of 1 μg / ml.
[0055]
Further, in order to examine the effect of KPMM2 proliferation on anti-IL-6 mAb and anti-IL-6R mAb, the MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) method (J. Immun. Methods 65:55, 1983) and a method of directly measuring live cells, with similar results.
[0056]
Example 8: Measurement of IL-6 production by ELISA
The ability to produce IL-6 by the cell line KPMM2 established in Example 1 was tested.
[0057]
KPMM2 obtained by the method described in Example 1 was added to an RPMI1640 culture medium containing 20% FCS and 100 μg / ml kanamycin in a concentration of 10%.⁶Cells / ml and 5% CO 2 in the absence of human IL-6.₂The medium was cultured at 37 ° C.
[0058]
After 72 hours of culture, the concentration of IL-6 produced by KPMM2 contained in the culture supernatant was measured using an ELISA kit for human IL-6 (manufactured by Teijin Bio-Laboratory) according to the attached instructions. As a negative control, an RPMI1640 culture solution supplemented with 20% FCS and 100 μg / ml kanamycin was used. 79.7 ± 19.6 (mean ± SD) pg / ml of IL-6 was detected in the culture supernatant, which was below the detection limit (4.0 pg / ml) in the control culture. In comparison, it was confirmed that the concentration of IL-6 in the culture supernatant was significantly increased.
[0059]
Example 9: Confirmation of IL-6R expression by flow cytometry
To confirm IL-6R expression on KPMM2 cells, the MT18 antibody (Hirata et al.), A mouse anti-human IL-6R mAb that binds to IL-6R and does not inhibit IL-6 binding to IL-6R. , J. Immunol. 143: 2900, 1989).
[0060]
KPMM2 cells obtained by the method described in⁶The cells were suspended in 100 μl of the FACS buffer described in Example 2 so as to give individual cells / tube, 10 μg / ml of the MT18 antibody was added, the mixture was reacted at 4 ° C. for 3 hours, and washed twice with the FACS buffer. And 100 μl of FACS buffer, and 5 μg / ml of a FITC-labeled goat anti-mouse IgG antibody (manufactured by TAGO) was added, followed by reaction at 4 ° C. for 30 minutes. After washing twice with the FACS buffer, the cells were suspended in the same FACS buffer, and the fluorescence was measured with a flow cytometer (EPICS PROFILE, manufactured by Coulter).
[0061]
As a result, as shown in FIG. 7, the expression of IL-6R was shown on the KPMM2 cells.
[0062]
Example 10: Measurement of human IgG (M protein) production by ELISA
The ability of KMMM2 to produce human IgG (M protein) was tested.
[0063]
KPMM2 obtained by the method described in Example 1⁶Cells / ml in an RPMI1640 culture solution containing 20% FCS and 100 μg / ml kanamycin, dispensed in a volume of 2 ml into each well of a 12-well plate (manufactured by Falcon), and the absence of human IL-6 5% CO wet under₂The medium was cultured at 37 ° C. for 72 hours. The experiment was performed three times. Thereafter, the concentration of human IgG in the culture supernatant was measured by ELISA using a goat anti-IgG antibody (NO. 4100) manufactured by TAGO and an alkaline phosphatase-labeled goat anti-IgG gamma chain specific antibody (NO. 2490). In addition, human IgG (NO.0001-860) manufactured by Kappel was used as a standard. As a result, human IgG at a concentration of 10.1 μg / ml was detected in the culture supernatant, which was below the detection limit (5 ng / ml) in the control culture. Was remarkably increased.
[0064]
Example 11: Detection of IL-6 and IL-6R mRNA by RT-PCR (reverse transcription polymerase chain reaction)
In order to confirm the expression of human IL-6 and human IL-6R in KPMM2 obtained by the method described in Example 1, human IL-6 and human IL-6 were analyzed by RT-PCR (reverse transcription polymerase chain reaction). -6R messenger RNA (mRNA) was detected.
[0065]
KPMM2 cells (10%) were prepared by the guanidium cesium chloride method (Molecular Cloning, Cold Spring Harbor Laboratory Press).⁸) To prepare total RNA. As a negative control, total RNA was similarly prepared from a human B-cell lymphoma cell line SKW6.4. Single-stranded cDNA was synthesized directly from 5 μg of total RNA using a cDNA synthesis kit (manufactured by Invitrogen) according to the attached instructions.
[0066]
As a positive control, PCR primers for detecting human IL-6 and human IL-6R were used (Clontech Inc.). Each 100 μl of the PCR solution contains 10 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 2.5 units of Amplitaq (manufactured by Perkin Elmer Cetus), 1 μl of a single-stranded cDNA synthesis reaction, and 100 pmol of each primer. Each PCR tube was overlaid with 50 μl of mineral oil and subjected to PCR. First, a melt was performed at 94 ° C. for 1 minute, and a cycle of 1 minute at 60 ° C. and 10 minutes at 72 ° C. was repeated 30 times. After the final cycle, a final extension was performed at 72 ° C. for 10 minutes. A positive control group using primers for detection of human IL-6 and human IL-6R was similarly amplified to obtain a positive control PCR product of about 50 pg.
[0067]
10 μl was taken from each reaction tube and electrophoresed on a 1.5% agarose gel. As shown in FIG. 8, the PCR products of IL-6 (628 bp) and IL-6R (251 bp) were both amplified from KPMM2 RNA. These results support that KMMM2 proliferates by the autocrine mechanism of IL-6 in terms of gene expression. On the other hand, SKW6.4 cells secrete IgM in response to IL-6. The IL-6R PCR product was amplified from SKW6.4 mRNA. However, no PCR product of IL-6 was detected in this experiment, suggesting that SKW6.4 cells are not producing IL-6.
[0068]
Example 12: Transplantation of cell line KPMM2 into SCID and nude mice (1) KPMM2 portability and passage in vivo
The transplantability and in vivo passage of the cell line KPMM2 established in Example 1 were examined as follows.
[0069]
KPMM2 was added to RPMI 1640 culture medium containing 20% FCS and 100 μg / ml kanamycin in a concentration of 10%.⁸Cells / ml, and treated with 0.2 mg of rabbit anti-asialo GM1 antibody (Code No. 014-09801, manufactured by Wako Pure Chemical Industries, Ltd.). Tm) (manufactured by Chugai Pharmaceutical Co., Ltd.) was subcutaneously implanted into the abdomen with an injection needle at 0.1 ml. As a result, one month later, all three cases formed a nodular tumor at the transplant site.
[0070]
The tumor was aseptically excised, the excised tumor mass was crushed with a piston of a disposable syringe, and the cells were collected through a nylon mesh (70 μm, Falcon). These cells were suspended in a culture medium of RPMI1640 in the same manner as described above, and IL-6-SCID Tm, severe immunodeficiency mouse (referred to as SCID mouse) (manufactured by CLEA Japan), BALB / c-nu / nu mouse (hereinafter referred to as nude mouse) ) (Manufactured by CLEA Japan), a nodular tumor was similarly formed at the site of implantation, and subculture was possible in vivo. It was also possible to subculture the tumor mass into a block of 3 mm square and transplanted subcutaneously into IL-6-SCID Tm, SCID mouse and nude mouse using a transplant needle.
(2) Portability of KPMM2 in different transplant routes
The transplantability of the KMPM2 when the transplant route was subcutaneous (sc), intravenous (iv), or intraperitoneal (ip) was examined as follows.
[0071]
The KPMM2 tumor subcultured in the SCID mouse in (1) was aseptically excised, the excised tumor mass was crushed with a piston of a disposable syringe, and the cells were collected through a nylon mesh.⁸Cells / ml of cell suspension were prepared. This suspension was subcutaneously (sc), intravenously (iv), 0.1 ml each in SCID mice or nude mice treated with 0.2 mg rabbit anti-asialo GM1 antibody and 500R X-ray. Transplantation was performed by three routes intraperitoneally (ip), and engraftment was determined 40 days later (Table 2). As a result, engraftment was confirmed in all cases after about 40 days, and s. c. In transplantation, i. p. The transplant formed a solid tumor in the abdominal cavity. I. v. In transplantation, KPMM2 was found to engraft in the bone marrow.
[0072]
[Table 2]

(3) Concentration of serum human IgG (M protein: myeloma protein) in KPMM2 transplanted animals
The cell line KPMM2 was implanted subcutaneously into SCID mice, and the correlation between the volume of the formed tumor and the concentration of human IgG in serum was examined. The cell line KMMM2 was subcutaneously transplanted into SCID mice by the method described in the above (2), serum samples were collected three times before transplantation, 21 days and 42 days after transplantation, and human IgG concentration was determined by ELISA. Was measured.
[0073]
As a result, the concentration of human IgG in mouse serum increased with time in all animals transplanted with KPMM2. In addition, as shown in FIG. 9, in the animals implanted subcutaneously, there was a correlation between the volume of the tumor formed subcutaneously and the serum human IgG concentration, and the larger the tumor, the higher the serum human IgG concentration. From this, it was considered that the antitumor effect could be determined using the serum human IgG concentration as an index.
(4) Examination of KPMM2 transplantability and number of transplanted cells
The portability of KPMM2 when the number of transplanted cells was changed between SCID mice and nude mice was examined.
[0074]
10 KPMM2 cells were each added to male SCID mice and nude mice without special pretreatment by the method described in (2) above.⁷, 3 × 10⁶, 10⁶The cells were implanted subcutaneously (sc) into the abdomen, and 3 × 10 5 KPMM2 cells obtained by the method (2) were transplanted into SCID mice.⁶, 10⁶Individuals were also implanted intravenously (iv).
[0075]
As a result, when subcutaneously transplanted into SCID mice, 3 × 10⁶When transplanted, tumor nodules were formed in all cases by day 21 and 10⁶Even in the case of individual transplantation, tumor nodules were formed in 2/3 by day 21 and in all cases by day 32. When implanted subcutaneously in nude mice, 10⁷In the case of individual transplantation, 2/3 by 21 days, all cases by 32 days, and 3 × 10⁶In the case of individual transplantation, tumor nodules were formed in all cases by day 42. However, 10⁶No tumor engraftment was observed by day 42 in the individual transplants. Also, when implanted intravenously in SCID mice, 3 × 10⁶In all cases, 10⁶KPMM2 survived in 1/3 by individual transplantation. The results are summarized in Table 3 below.
[0076]
[Table 3]

(5) Analysis of expression of human CD38 antigen in SCID mice transplanted with KPMM2
In SCID mice transplanted with KMPM2, expression of human CD38 antigen, a surface antigen characteristically expressed on the cell surface of KMMM2, was examined in order to confirm the engraftment.
[0077]
KPMM2 was intravenously transplanted into SCID mice by the method described in (1) above, and the mice were sacrificed 37 days after the transplantation. The bone marrow was recovered from the femur of the sacrificed mouse, suspended, passed through a stainless mesh (100 μm), and subjected to a buffer for fluorescence activated cell selection (FACS) (2% FCS and 0.1% NaN).₃A suspension of bone marrow cells was prepared in a PBS (-) solution containing As a negative control, a bone marrow cell suspension was prepared in the same manner from bone marrow of SCID mice not transplanted with KPMM2.
[0078]
Further, from the SCID mouse to which KMPM2 was subcutaneously transplanted by the method described in this example, a subcutaneous tumor mass was surgically excised on day 37 after implantation, and the excised tumor mass was inserted between two slide glasses. The mixture was ground through a stainless steel mesh (100 μm), and a suspension of bone marrow cells was prepared using a FACS buffer.
[0079]
KPMM2 cultured in vitro by the method described in Example 1 was used as a positive control. KPMM2 cultured in vitro for 4 days was washed with FACS buffer, and then suspended in the same buffer.
[0080]
Next, using the cell suspension thus prepared, the expression of human CD38 antigen was examined by FACS analysis.
[0081]
That is, in 100 μl of FACS buffer, 10⁶2.5 μg / ml of phycoerythrin (PE) -labeled anti-human CD38 antibody (Leu-17, manufactured by Becton Dickinson) was added to each of the cells, and reacted on ice for 30 minutes. Next, the cells were washed twice with 1 ml of FACS buffer, suspended in 500 μl of FACS buffer, and analyzed by FACSScan (manufactured by Becton Dickinson).
[0082]
From the results of FACS analysis of the in vitro cultured KPMM2 as a positive control group, cells having a fluorescence intensity in the range of 40 to 2,000 were determined to be human CD38 antigen-expressing cells (see FIG. 10A). In SCID mice into which KMPM2 had been intravenously transplanted, approximately 71% of the bone marrow cells were occupied by human CD38-positive cells (see FIG. 10 (b)). This indicates that KPMM2 implanted intravenously in SCID mice has engrafted in the bone marrow of SCID mice. In addition, all the cells obtained from the tumor mass generated by subcutaneously transplanting KPMM2 into SCID mice were positive for human CD38 (see FIG. 10 (c)), and all the tumor masses generated by subcutaneously transplanting KPMM2 showed KPMM2. It was shown to be composed of No human CD38-positive cells were detected in the bone marrow cells of the SCID mice in the negative control group not transplanted with KPMM2 (see FIG. 10 (d)).
[0083]
Example 13: Antitumor effect of anti-human IL-6 antibody SK2 and anti-human IL-6R reshaped humanized antibody PM1
The in vivo antitumor effect of the anti-human IL-6 antibody SK2 and the anti-human IL-6R reshaped humanized antibody PM1 in the KMMM2-transplanted animal was examined as follows.
[0084]
A KPMM2 myeloma tumor mass obtained by subculturing SCID mice by the method described in Example 12 (1) above was applied to rabbit anti-asialo GM1 antibody / X-ray-treated nude mice (5 weeks old, male) using a 4 mm square block. Subcutaneous transplantation, one dose of 1 mg / mouse of SK2 antibody and 3 doses of 0.125, 0.5 and 1 mg / mouse of reconstituted humanized PM1 antibody (see International Patent Application WO 92-19759) only once a day Was administered intravenously. Each antibody was prepared with PBS (-) (manufactured by Nissui) at a concentration of 0.2 ml / mouse, and PBS (-) was administered to the negative control group at 0.2 ml / mouse. Thereafter, the size of the tumor was observed over time, and on day 35 when the tumor in the control group became sufficiently large, the whole blood was collected, and the tumor was excised and weighed.
[0085]
The result is shown in FIG. In the negative control group, the average tumor weight was about 1 g, whereas in the group to which the reconstituted humanized PM1 antibody was administered, the tumor growth inhibition rate (Growth Inhibitory Ratio: GIR) was 78% at 1 mg / mouse. The GIR when administering 0.5 mg / mouse was 53%, and the GIR when administering 0.125 mg / mouse was 66%, showing a strong tumor growth inhibitory effect. In addition, SK2 also suppressed tumor growth by GIR 61% in the 1 mg / mouse administration group.
[0086]
Furthermore, the concentration of human IgG in the serum sample collected at the time of tumor removal was measured by ELISA. FIG. 12 shows the result. In the negative control group without antibody administration, the serum human IgG concentration was 27.6 mg / ml on average, but 71% when the reconstituted humanized PM1 antibody was administered at 1, 0.5 and 0.125 mg / mouse, respectively. SK2 1 mg / mouse administration was also suppressed by 43%. Tumor weight and serum human IgG concentration correlated well between treatment groups as well as at the individual level.
[0087]
Example 14: Increased ionized calcium concentration and enhanced bone resorption in KPMM2 intravenously transplanted SCID mice
KPMM2 (10⁷Were implanted intravenously into SCID mice (manufactured by CLEA Japan) according to the method described in Example 12 (2) above, and the blood ionized calcium concentration and the presence or absence of bone resorption were examined with time after transplantation.
[0088]
On days 9, 20, 30, and 37 after KPMM2 transplantation, KPMM2 intravenously transplanted SCID mice were anesthetized with ether, and blood was collected from the orbit of the mouse using a 60 μl volume capillary column (manufactured by Ciba Corning). Medium ionized calcium concentration 634 automatic Ca⁺⁺/ PH analyzer (manufactured by Ciba Corning). As a control, blood was collected from SCID mice not transplanted with KMMM2 in the same manner, and the blood ionized calcium concentration was measured.
[0089]
As a result, the blood ionized calcium concentration of KPMM2 intravenously transplanted SCID mice was increased from day 30 after transplantation, and on day 37, the blood ionized calcium concentration was increased by about 20% compared to the control group of mice. Was observed (FIG. 13). The increase in the ionized calcium concentration in blood correlated well with the time-dependent increase in the rate of increase in KMPM2 (human CD38 antigen-positive cells) in mouse bone marrow when KPMM2 was intravenously transplanted into SCID mice (FIG. 14).
[0090]
Further, when bones of the lower limb of the SCID mouse on the 37th day of KPMM2 intravenous transplantation were X-rayed and observed morphologically, a remarkable bone resorption image was confirmed as compared with the control group of mice (see FIG. 15). . The above results indicate that bone lesions of SCID mice transplanted intravenously with KPMM2 are in good agreement with actual myeloma lesions.
[0091]
【The invention's effect】
The present invention has revealed that there is a myeloma that grows in an IL-6 autocrine-dependent manner. The human myeloma cell line that proliferates in an IL-6-dependent manner by the autocrine mechanism of the present invention is useful as a model of the IL-6-dependent proliferation mechanism of myeloma. It can also be used as a therapeutic model for myeloma therapeutics including IL-6 activity inhibitors such as anti-IL-6 antibodies and anti-IL-6 receptor antibodies. The human myeloma cell line that proliferates in an IL-6-dependent manner by the autocrine mechanism of the present invention is, of course, useful for preparing an evaluation system for a therapeutic agent for myeloma using cell growth inhibition as an index in vitro and in vivo. , M protein is produced, and the production amount correlates very well with the growth of myeloma. Therefore, it is useful for preparing an evaluation system for a therapeutic agent for myeloma using suppression of the production amount of M protein as an index.
[0092]
Further, in a myeloma bone marrow engraftment model obtained by intravenously transplanting the human myeloma cell line of the present invention into an experimental animal, a bone lesion characteristic of myeloma is observed with the proliferation of multiple myeloma, Therefore, an evaluation system for a therapeutic agent for myeloma using the suppression of this bone lesion as an index can be created.
[0093]
From these points, the utility value of the human myeloma cell line of the present invention is extremely large.
[Brief description of the drawings]
FIG. 1 is a view showing autonomous aggregation of KPMM2 in a liquid culture (a photograph showing the form of an organism).
FIG. 2 is a view showing a form of a KPMM2 (a photograph showing a form of an organism). Light-Giemsa staining has the characteristics of plasma cells.
FIG. 3. J of KPMM2 by Southern blot analysis._HFIG. 4 is a diagram (photograph of electrophoresis) showing reconstitution of Cλ gene. DNA obtained from KPMM2 was digested with BamHI, EcoRI and HindIII, and_HAnd Southern blot analysis using Cλ gene probe. The reconstructed band is indicated by (▲).
FIG. 4 is a view showing a karyotype of KPMM2 (a photograph showing a trait of an organism). The 15 cells searched were all 46, XX, der (1; 19) (q10; q10), t (3; 14) (q21; q32), -4, t (6; 11) (p12; p15), der (10) add (10) (p13) dic (9; 10) (q10; q26), +16.
FIG. 5 is a graph showing the effects of various cytokines on the cell proliferation of KPMM2. The concentrations of cytokines used are as follows: IL-6, 1 ng / ml; IFN-α and IFN-γ, 1000 U / ml; other cytokines, 100 ng / ml. Each number represents the mean of three tests + standard deviation (SD).
FIG. 6 shows the effect of anti-IL-6 mAb and anti-IL-6R mAb on cell proliferation of KMMM2. SK2 is a mouse anti-IL-6 mAb (▲); PM1 is a mouse anti-IL-6R mAb (●). Dashed lines indicate controls. Each number represents the average of three tests.
FIG. 7 is a view showing expression of IL-6R in KPMM2 cells. Cells were stained with anti-IL-6R mAb (MT18). Mouse IgG2b antibody was used as a control. The dashed line represents mIgG2b and the solid line represents MT18.
FIG. 8 is a diagram (photograph of electrophoresis) showing the expression of IL-6 and IL-6R mRNA of KPMM2 by RT-PCR (reverse transcription PCR) analysis on a 1.5% agarose gel. Lanes 1 and 4, SKW 6.4; Lanes 2 and 5, KPMM2; Lanes 3 and 6, positive control.
FIG. 9 is a graph showing the correlation between tumor volume and serum human IgG concentration in mice into which KMMM2 has been subcutaneously implanted.
FIG. 10 (a) is a diagram showing FACS analysis of human CD38 antigen on KPMM2 cultured in vitro. KPMM2 has a fluorescence intensity ranging from 40 to 2000.
(B) is a diagram showing FACS analysis of human CD38 antigen in bone marrow cells of SCID mice into which KPMM2 has been implanted intravenously. 71% of the cells have a fluorescence intensity ranging from 40 to 2000.
(C) is a diagram showing FACS analysis of human CD38 antigen in cells obtained from a tumor mass of a SCID mouse into which KMMM2 has been subcutaneously implanted. All cells have a fluorescence intensity ranging from 40 to 2000.
(D) shows FACS analysis of human CD38 antigen in bone marrow cells of SCID mice not transplanted with KMPM2. No cells have a fluorescence intensity in the range of 40 to 2000.
FIG. 11 shows the in vivo tumor growth inhibitory effects of anti-IL-6 mAb and anti-IL-6R mAb on KPMM2. SK2 is an anti-human IL-6 mAb; reshaped humanized PM1 is an anti-human IL-6R mAb.
FIG. 12 shows the effect of anti-IL-6 mAb or anti-IL-6R mAb on serum human IgG concentration in nude mice transplanted with KPMM2.
FIG. 13 is a graph showing the time-dependent change in blood ionized calcium concentration (□) of SCID mice in which KMMM2 was intravenously transplanted. (◇) indicates a control. Each numerical value is the mean ± SEM of four SCID mice (five on day 37 only). D. Is shown.
FIG. 14 is a graph showing the time course of the ratio (□) of human CD38 antigen-positive cells in the bone marrow of SCID mice into which KMMM2 has been intravenously transplanted. (◇) indicates a control. Each numerical value is the mean ± SEM of four SCID mice (five on day 37 only). D. Is shown.
FIG. 15 is a view showing an X-ray image of a bone of a SCID mouse in which KPMM2 has been implanted intravenously (a photograph showing the form of an organism).
(A) SCID mouse of control group not transplanted with KPMM2
(B) SCID mouse on day 37 after KPMM2 transplantation

Claims (14)

A human myeloma cell line that proliferates in an IL-6-dependent manner by an autocrine mechanism, and which is KMMM2 (Biotechnological Institute of Technology, Patent Microorganisms Depositary, Accession No. FIRM: P-14170) . A non-human experimental animal transplanted with the cell line according to claim 1 . The non-human experimental animal according to claim 2 , wherein the experimental animal is immunodeficient. The experimental animal according to claim 2 , wherein the experimental animal is a mouse. The non-human experimental animal according to claim 2 , which is obtained by intravenously transplanting a human myeloma cell line that grows in an IL-6-dependent manner by an autocrine mechanism into the experimental animal. The non-human experimental animal according to claim 2 , which is obtained by subcutaneously transplanting a human myeloma cell line that proliferates in an IL-6-dependent manner by an autocrine mechanism into the experimental animal. The non-human experimental animal according to claim 2 , which is obtained by intraperitoneally transplanting a human myeloma cell line that grows in an IL-6-dependent manner into the experimental animal by an autocrine mechanism. An in vitro screening method for a therapeutic agent for myeloma, which comprises adding the therapeutic agent for myeloma to the cell line according to claim 1 and testing the inhibition of myeloma cell growth. 9. The in vitro screening method according to claim 8 , comprising performing a myeloma cell growth inhibition test using inhibition of M protein secretion as an index. 9. The in vitro screening method according to claim 8 , wherein the therapeutic agent for myeloma is an IL-6 activity inhibitor. 3. An in vivo screening method for a therapeutic agent for myeloma, comprising transplanting the therapeutic agent for myeloma into the non-human experimental animal according to claim 2 and testing for suppression of myeloma cell growth. 12. The in vivo screening method according to claim 11 , comprising performing a myeloma cell growth inhibition test using inhibition of M protein secretion as an index. The in vivo screening method according to claim 11 , comprising performing a myeloma cell growth inhibition test using inhibition of bone lesions as an index. 12. The in vivo screening method according to claim 11 , wherein the therapeutic agent for myeloma is an IL-6 activity inhibitor.

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