JP3587664B2 - Reshaped human anti-HM1.24 antibody - Google Patents

Description

【００８７】
【表２】

【００８８】
【表３】

【００８９】
【表４】

【００９０】
３．キメラ抗体及び再構成ヒト抗体の製造
キメラ抗体あるいは再構成ヒト抗体の製造のためには、前記のようなそれぞれ２種類の発現ベクター、すなわちエンハンサー／プロモーター系のごとき発現制御領域による制御のもとでマウスＨ鎖Ｖ領域及びヒトＨ鎖Ｃ領域をコードするＤＮＡを含んで成る発現ベクター、並びにエンハンサー／プロモーター系のごとき発現制御領域のもとでマウスＬ鎖Ｖ領域及びヒトＬ鎖Ｃ領域をコードするＤＮＡを含んで成る発現ベクター、またはエンハンサー／プロモーター系のごとき発現制御領域による制御のもとでヒト型化Ｈ鎖Ｖ領域及びヒトＨ鎖Ｃ領域をコードするＤＮＡを含んで成る発現ベクター、並びにエンハンサー／プロモーター系のごとき発現制御領域のもとでヒト型化Ｌ鎖Ｖ領域及びヒトＬ鎖Ｃ領域をコードするＤＮＡを含んで成る発現ベクターを作製する。
【００９１】
次に、これらの発現ベクターにより哺乳類細胞のごとき宿主細胞を同時形質転換し、そして形質転換された細胞をインビトロ又はインビボで培養してキメラ抗体あるいは再構成ヒト抗体を製造する（例えば国際特許出願公開番号ＷＯ９１−１６９２８）。また、ヤギなどの哺乳動物に抗体遺伝子を導入してトランスジェニック動物を作製し、その乳汁等からキメラ抗体あるいは再構成ヒト抗体を得ることができる。
【００９２】
また、Ｈ鎖Ｖ領域及びＨ鎖Ｃ領域ならびにＬ鎖Ｖ領域及びＬ鎖Ｃ領域を単一ベクターに連結し、適当な宿主細胞を形質転換し、抗体を産生させることができる。すなわち、キメラ抗体の発現には、クローニングされたｃＤＮＡに存在するマウスリーダー配列及びＨ鎖Ｖ領域及びヒトＨ鎖Ｃ領域をコードするＤＮＡ並びにマウスリーダー配列及びＬ鎖Ｖ領域及びヒトＬ鎖Ｃ領域をコードするＤＮＡを単一の発現ベクターに導入する（国際特許出願公開番号ＷＯ９４−１１５２３参照）。
【００９３】
再構成ヒト抗体の発現には、ヒト型化Ｈ鎖Ｖ領域及びヒトＨ鎖Ｃ領域をコードするＤＮＡ並びにヒト型化Ｌ鎖Ｖ領域及びヒトＬ鎖Ｃ領域をコードするＤＮＡを単一の発現ベクターに導入する（国際特許出願公開番号ＷＯ９４−１１５２３参照）。そして該ベクターを用いて宿主細胞を形質転換し、次にこの形質転換された宿主をインビボ又はインビトロで培養して目的とするキメラ抗体または再構成ヒト抗体を生産させる。
以上のようにして目的とするキメラ抗体あるいは再構成ヒト抗体をコードする遺伝子で形質転換した形質転換体を培養し、産生したキメラ抗体あるいは再構成ヒト抗体は、細胞内または細胞外から分離し均一にまで精製することができる。
【００９４】
なお、本発明の目的蛋白質であるキメラ抗体あるいは再構成ヒト抗体の分離・精製を、アフィニティーカラムを用いて行うことができる。例えば、プロテインＡを用いたカラムとして、ＨｙｐｅｒＤ，ＰＯＲＯＳ，ＳｅｐｈａｒｏｓｅＦ．Ｆ．等が挙げられる。また、その他に、通常の蛋白質で用いられる分離・精製方法を使用すればよく、何ら限定されるものではない。例えば各種クロマトグラフィー、限外濾過、塩折、透析等を適宜選択、組合せれば、キメラ抗体あるいは再構成ヒト抗体は分離・精製することができる。
【００９５】
本発明のキメラ抗ＨＭ１．２４抗体又は再構成ヒト抗ＨＭ１．２４抗体の製造のために任意の発現系、例えば真核細胞、例えば動物細胞、例えば樹立された哺乳類細胞系、昆虫細胞系、真糸状菌細胞、及び酵母細胞、並びに原核細胞、例えば細菌細胞、例えば大腸菌細胞等を使用することができる。好ましくは、本発明のキメラ抗体又は再構成ヒト抗体は哺乳類細胞、例えばＣＯＳ細胞、ＣＨＯ細胞Ｈｅｌａ細胞、Ｖｅｒｏ細胞、ミエローマ細胞又はＢＨＫ細胞中で発現される。
【００９６】
これらの場合、哺乳類細胞での発現のために有用な常用のプロモーターを用いることができる。例えば、ヒト・サイトメガロウィルス前期（ｈｕｍａｎ ｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ ｉｍｍｅｄｉａｔｅ ｅａｒｌｙ；ＨＣＭＶ）プロモーターを使用するのが好ましい。ＨＣＭＶプロモーターを含有する発現ベクターの例には、ＨＣＭＶ−ＶＨ−ＨＣγ１，ＨＣＭＶ−ＶＬ−ＨＣκ等であって、ｐＳＶ２ｎｅｏに由来するもの（国際特許出願公開番号ＷＯ９２−１９７５９）が含まれる。
【００９７】
また、その他に、本発明のために用いることのできる哺乳動物細胞における遺伝子発現のプロモーターとしてはレトロウイルス、ポリオーマウイルス、アデノウイルス、シミアンウイルス４０（ＳＶ４０）などのウイルスプロモーターやヒト・ポリペプチド・チェーン・エロンゲーション・ファクター１α（ＨＥＦ−１α）などの哺乳動物細胞由来のプロモーターを用いればよい。例えばＳＶ４０のプロモーターを使用する場合は、Ｍｕｌｌｉｇａｎらの方法（Ｎａｔｕｒｅ ２７７ １０８（１９７９））、また、ＨＥＦ−１αプロモーターを使用する場合は、Ｍｉｚｕｓｈｉｍａ，Ｓ．らの方法（Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，１８，５３２２，１９９０）に従えば容易に実施することができる。
【００９８】
複製起原としては、ＳＶ４０、ポリオーマウイルス、アデノウイルス、牛パピローマウイルス（ＢＰＶ）等の由来のものを用いることができ、さらに宿主細胞系中での遺伝子コピー数増幅のため、発現ベクターは選択マーカーとして、アミノグリコシドホスホトランスフェラーゼ（ＡＰＨ）遺伝子、チミジンキナーゼ（ＴＫ）遺伝子、大腸菌キサンチン−グアニンホスホリボシルトランスフェラーゼ（Ｅｃｏｇｐｔ）遺伝子、ジヒドロ葉酸還元酵素（ＤＨＦＲ）遺伝子等を含むことができる。
【００９９】
４．キメラ抗体及びヒト型化抗体の結合阻害活性
（１）抗体の濃度測定
精製抗体の濃度の測定は、ＥＬＩＳＡ または吸光度の測定により行う。
抗体濃度測定のためのＥＬＩＳＡプレートを次のようにして調製する。ＥＬＩＳＡ用９６穴プレート（例えばＭａｘｉｓｏｒｐ， ＮＵＮＣ ）の各穴を例えば１μｇ／ｍｌの濃度に調製したヤギ抗ヒトＩｇＧ抗体１００μｌを固相化する。
【０１００】
１００μｌの希釈バッファー（例えば５０ｍＭ Ｔｒｉｓ−ＨＣｌ、１ｍＭ ＭｇＣｌ_２ 、０．１５Ｍ ＮａＣｌ、０．０５％ Ｔｗｅｅｎ２０、０．０２％ＮａＮ_３ 、１％ 牛血清アルブミン（ＢＳＡ）、ｐＨ８．１）でブロッキングの後、キメラ抗体、ハイブリッド抗体または再構成ヒト抗体を発現させた細胞の培養上清、例えばＣＯＳ細胞又はＣＨＯ細胞の培養上清あるいは精製キメラ抗体、ハイブリッド抗体または再構成ヒト抗体を段階希釈して各穴に加え、次にアルカリフォスファターゼ結合ヤギ抗ヒトＩｇＧ抗体１００μｌを加え、１ｍｇ／ｍｌの基質溶液（Ｓｉｇｍａ１０４、ｐ−ニトロフェニルリン酸、ＳＩＧＭＡ）を加え、次に４０５ｎｍでの吸光度をｍｉｃｒｏｐｌａｔｅ ｒｅａｄｅｒ（Ｂｉｏ Ｒａｄ）で測定する。濃度測定のスタンダードとして、ヒトＩｇＧ１κ（Ｔｈｅ Ｂｉｎｄｉｎｇ Ｓｉｔｅ）を用いることができる。精製抗体の濃度は、２８０ｎｍの吸光度を測定し、１ｍｇ／ｍｌを１．３５ＯＤとして算出する。
【０１０１】
（２）抗原結合活性
抗原結合活性の測定は、ヒト羊膜細胞株ＷＩＳＨ（ＡＴＣＣＣＣＬ２５）を用いたＣｅｌｌ−ＥＬＩＳＡで行うことができる。Ｃｅｌｌ−ＥＬＩＳＡプレートは次のようにして調製する。９６穴プレートに１０％ウシ胎児血清を含有するＰＲＭＩ１６４０培地により適切な濃度に調製したＷＩＳＨ細胞を加え、一晩培養した後、ＰＢＳ（−）で２回洗浄後０．１％グルタルアルデヒド（ナカライテスク社製）にて固定する。
【０１０２】
ブロッキングの後、キメラ抗ＨＭ１．２４抗体、ハイブリッド抗ＨＭ１．２４抗体または再構成ヒト抗ＨＭ１．２４抗体を発現させた細胞、例えばＣＯＳ細胞やＣＨＯ細胞の培養上清、あるいは精製したキメラ抗ＨＭ１．２４抗体、ハイブリッド抗ＨＭ１．２４抗体または再構成ヒト抗ＨＭ１．２４抗体を段階希釈して各穴に１００μｌ加え、室温にて２時間インキュベーションおよび洗浄の後、ペルオキシダーゼ標識ウサギ抗ヒトＩｇＧ抗体（ＤＡＫＯ社製）を加える。
室温にて１時間インキュベーションおよび洗浄の後、基質溶液を加えインキュベーションする。次いで、６Ｎ硫酸５０μｌで反応を停止させ、ＭＩＣＲＯＰＬＡＴＥ ＲＥＡＤＥＲ Ｍｏｄｅｌ ３５５０（Ｂｉｏ−Ｒａｄ社製）を用いて４９０ｎｍでの吸光度を測定する。
【０１０３】
（３）結合阻害活性の測定
ビオチン標識マウス抗ＨＭ１．２４抗体による結合阻害活性は、ヒト羊膜細胞株ＷＩＳＨ（ＡＴＣＣＣＣＬ２５）を用いたＣｅｌｌ−ＥＬＩＳＡで行うことができる。Ｃｅｌｌ−ＥＬＩＳＡプレートは上記（２）に従い調製できる。９６穴プレートに１０％ウシ胎児血清を含有するＲＰＭＩ１６４０培地により適切な濃度に調製したＷＩＳＨ細胞を加え、一晩培養した後、ＰＢＳ（−）で２回洗浄後０．１％グルタルアルデヒド（ナカライテスク社製）にて固定する。
【０１０４】
ブロッキングの後、キメラ抗ＨＭ１．２４抗体、ハイブリッド抗ＨＭ１．２４抗体または再構成ヒト抗ＨＭ１．２４抗体を発現させた細胞、例えばＣＯＳ細胞やＣＨＯ細胞の培養上清、あるいは精製したキメラ抗ＨＭ１．２４抗体、ハイブリッド抗ＨＭ１．２４抗体または再構成ヒト抗ＨＭ１．２４抗体を段階希釈して各穴に５０μｌ加え、同時に２μｇ／ｍｌのビチオン標識マウス抗ＨＭ１．２４抗体５０μｌを添加し、室温にて２時間インキュベーションおよび洗浄の後、ペルオキシダーゼ標識ウサギ抗ヒトＩｇＧ抗体（ＤＡＫＯ社製）を加える。
室温にて１時間インキュベーションした後洗浄し、基質溶液を加えインキュベーションした後６Ｎ硫酸５０μｌで反応を停止させ、ＭＩＣＲＯＰＬＡＴＥ ＲＥＡＤＥＲ Ｍｏｄｅｌ ３５５０（Ｂｉｏ−Ｒａｄ社製）用いて４９０ｎｍでの吸光度を測定する。
【０１０５】
ＡＤＣＣ活性の測定
本発明のキメラ抗体あるいは再構成ヒト抗体のＡＤＣＣ活性は、次のようにして測定することができる。まず、ヒトの抹消血や骨髄より比重遠心法で単核球を分離し、エフェクター細胞として調製する。また、ヒト骨髄腫細胞、例えば、ＲＰＭＩ ８２２６細胞（ＡＴＣＣ ＣＣＬ １５５）を^５１Ｃｒにより標識して、標的細胞として調製する。次いで、標識した標的細胞にＡＤＣＣ活性を測定するキメラ抗体あるいは再構成ヒト抗体を加えインキュベートし、その後、標的細胞に対し適切な比のエフェクター細胞を加えインキュベートする。
【０１０６】
インキュベートした後上清をとり、ガンマカウンターで放射活性を測定する。その際、最大遊離放射能測定用に１％のＮＰ−４０を用いることができる。細胞障害活性（％）は、（Ａ−Ｃ）／（Ｂ−Ｃ）×１００で計算することができる。なお、Ａは抗体存在下において遊離された放射活性（ｃｐｍ）、ＢはＮＰ−４０による遊離された放射活性（ｃｐｍ）、およびＣは抗体を含まず培養液のみで遊離された放射活性（ｃｐｍ）である。
また、抗体Ｃ領域にＡＤＣＣ活性あるいはＣＤＣ活性を期待する場合、抗体Ｃ領域としてヒトＣγ１、ヒトＣγ３を用いることができる。さらに、抗体Ｃ領域のアミノ酸を一部付加、改変、修飾することにより、より強力なＡＤＣＣ活性あるいはＣＤＣ活性を誘導することができる。
【０１０７】
例えば、アミノ酸置換によるＩｇＧのＩｇＭ様ポリマー化（Ｓｍｉｔｈ， Ｒ．Ｉ． Ｆ． ＆ Ｍｏｒｒｉｓｏｎ， Ｓ．Ｌ， ＢＩＯ／ＴＥＣＨＮＯＬＯＧＹ（１９９４）１２， ６８３−６８８）、アミノ酸付加によるＩｇＧのＩｇＭ様ポリマー化（Ｓｍｉｔｈ， Ｒ． Ｉ． Ｆ． ｅｔ ａｌ．， Ｊ．Ｉｍｍｕｎｏｌ．（１９９５）１５４， ２２２６−２２３６） 、Ｌ鎖をコードする遺伝子の直列連結での発現（Ｓｈｕｆｏｒｄ， Ｗ．ｅｔ ａｌ．， Ｓｃｉｅｎｃｅ（１９９１）２５２，７２４−７２７）、アミノ酸置換によるＩｇＧの二量体化（Ｃａｒｏｎ， Ｐ．Ｃ．ｅｔ ａｌ．， Ｊ．Ｅｘｐ．Ｍｅｄ．（１９９２）１７６， １１９１−１１９５ 、Ｓｈｏｐｅｓ， Ｂ．Ｊ．Ｉｍｍｕｎｏｌｏｇｙ（１９９２）１４８， ２９１８−２９２２） 、化学的修飾によるＩｇＧの二量体化（Ｗｏｌｆｆ， Ｅ．Ａ．ｅｔ ａｌ．， Ｃａｎｃｅｒ Ｒｅｓ．（１９９３）５３， ２５６０−２５６５）および抗体ヒンジ領域のアミノ酸改変によるエフェクター機能の導入（Ｎｏｒｄｅｒｈａｕｇ， Ｌ．ｅｔ ａｌ．， Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏｌ．（１９９１）２１， ２３７９−２３８４） が挙げられる。これらは、プラマーを使用したオリゴマー部位特異的変異導入法、制限酵素切断部位を利用した塩基配列の付加、共有結合をもたらす化学修飾剤を使用することによって達成される。
【０１０８】
骨髄腫体内診断薬
本発明のキメラ抗ＨＭ１．２４抗体あるいは再構成ヒト抗ＨＭ１．２４抗体は、ラジオアイソトープ等の標識化合物と結合させることにより、骨髄腫体内診断薬として用いることができる。
さらには、キメラ抗ＨＭ１．２４抗体あるいは再構成ヒト抗ＨＭ１．２４抗体の断片、例えばＦａｂ、Ｆ（ａｂ′）_２ 、ＦｖまたはＨ鎖とＬ鎖のＦｖを適当なリンカーで連結させたシングルチェインＦｖ（ｓｃＦｖ）とラジオアイソトープ等の標識化合物を結合させたものも、同様に骨髄腫体内診断薬として用いることができる。
【０１０９】
具体的には、これら抗体の断片は、これら抗体の断片をコードする遺伝子を構築し、これを発現ベクターに導入した後、適当な宿主細胞で発現させるか、あるいはキメラ抗ＨＭ１．２４抗体または再構成ヒト抗ＨＭ１．２４抗体を適当な酵素を用いて消化することで得られる。
上記の骨髄腫体内診断薬は、非経口的に全身に投与することができる。
医薬組成物および骨髄腫治療剤
本発明のキメラ抗体ＨＭ１．２４抗体あるいはヒト型化抗ＨＭ１．２４抗体の治療効果を確認するには、前記抗体を骨髄腫細胞を移植された動物に投与し、抗腫瘍効果を評価することにより行われる。
【０１１０】
動物に移植する骨髄腫細胞としては、ヒト骨髄腫細胞が好ましく、例えば、ＫＰＭＭ２（特許出願公開番号特開平７−２３６４７５）、ＲＰＭＩ８２２６（ＡＴＣＣ ＣＣＬ １５５）、ＡＲＨ７７（ＡＴＣＣ ＣＲＬ １６２１）、Ｓ６Ｂ４５（Ｓｕｚｕｋｉ， Ｈ．ら、 Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏｌ．（１９９２）２２， １９８９−１９９３）が挙げられる。移植される動物としては、免疫機能が低下または欠失した動物が好ましく、ヌードマウス、ＳＣＩＤマウス、ベージュマウス、ヌードラットが挙げられる。
また、評価する抗腫瘍効果の確認は、血清中のヒトイムノグロブリン量の変化、腫瘍体積・重量の測定、尿中のヒトベンズジョーンズタンパク質量の変化あるいは動物の生存期間等に従い行うことができる。
【０１１１】
本発明のキメラ抗ＨＭ１．２４抗体あるいは再構成ヒト抗ＨＭ１．２４抗体を有効成分として含む医薬組成物および骨髄腫治療剤は、非経口的に全身あるいは局所的に投与することができる。例えば、点滴などの静脈内注射、筋肉内注射、腹腔内注射、皮下注射を選択することができ、患者の年齢、症状により適宜投与方法を選択することができる。
有効投与量は、一回につき体重１ｋｇ あたり０．０１ｍｇから１０００ｍｇの範囲で選ばれる。あるいは、患者あたり５ｍｇ／ｂｏｄｙ、好ましくは５０−１００ｍｇ／ｂｏｄｙ の投与量を選ぶことができる。
本発明のキメラ抗ＨＭ１．２４抗体あるいは再構成ヒト抗ＨＭ１．２４抗体を有効成分として含む医薬組成物および骨髄腫治療剤は、投与経路次第で医薬的に許容される担体や添加物を共に含むものであってもよい。
【０１１２】
このような担体および添加物の例として、水、医薬的に許容される有機溶剤、コラーゲン、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、カルボキシメチルセルロースナトリウム、ポリアクリル酸ナトリウム、アルギン酸ナトリウム、水溶性デキストラン、カルボキシメチルスターチナトリウム、ペクチン、メチルセルロース、エチルセルロース、キサンタンガム、アラビアゴム、カゼイン、ゼラチン、寒天、ジグリセリン、グリセリン、プロピレングリコール、ポリエチレングリコール、ワセリン、パラフィン、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン（ＨＳＡ ）、マンニトール、ソルビトール、ラクトース、医薬添加物として許容される界面活性剤などが挙げられる。使用される添加物は、本発明の剤形に応じて上記の中から適宜あるいは組み合わせて選択されるが、これらに限定されるものではない。
【０１１３】
【実施例】
次に、本発明を実施例によりさらに具体的に説明する。
実施例１ ． マウス抗 ＨＭ１．２４ 抗体可変領域をコードする ｃＤＮＡ のクローニング
１． メッセンジャーＲＮＡ （ｍＲＮＡ）の単離
マウス抗ＨＭ１．２４抗体を産生する２ ｘ １０^８ 個のハイブリドーマ細胞（ＦＥＲＭ ＢＰ−５２３３）からＦａｓｔ Ｔｒａｃｋ ｍＲＮＡ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ Ｖｅｒｓｉｏｎ ３．２ （Ｉｎｖｉｔｒｏｇｅｎ社製）を用いてキット添付の指示書に従い、ｍＲＮＡの単離を行った。
【０１１４】
２． 抗体可変領域をコードする遺伝子のＰＣＲ 法による増幅
Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ｃｅｔｕｓ社製）を用いてＰＣＲ を行った。
２−１． マウスＬ 鎖Ｖ 領域をコードする遺伝子の増幅および断片化
単離したｍＲＮＡよりＡＭＶ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ Ｆｉｒｓｔ−ｓｔｒａｎｄ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ （Ｌｉｆｅ Ｓｃｉｅｎｃｅ社製）を用いて一本鎖ｃＤＮＡを合成し、ＰＣＲ に用いた。また、ＰＣＲ 法に使用するプライマーは、マウスカッパ型Ｌ 鎖リーダー配列とハイブリダイズする配列番号：２９〜３９に示すＭＫＶ （Ｍｏｕｓｅ Ｋａｐｐａ Ｖａｒｉａｂｌｅ）プライマー（Ｊｏｎｅｓ， Ｓ． Ｔ． ら、Ｂｉｏ／Ｔｅｃｈｎｏｌｏｇｙ， ９， ８８−８９， （１９９１））を用いた。
【０１１５】
ＰＣＲ 溶液１００ μｌ は、１０ ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ８．３）、５０ ｍＭ ＫＣｌ 、０．１ ｍＭ ｄＮＴＰｓ （ｄＡＴＰ， ｄＧＴＰ， ｄＣＴＰ， ｄＴＴＰ） 、１．５ ｍＭ ＭｇＣｌ_２、５ユニットのＤＮＡ ポリメラーゼＡｍｐｌｉ Ｔａｑ （Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ｃｅｔｕｓ社製）、０．２５ ｍＭ の配列番号：２９〜３９に示すＭＫＶ プライマーと３ ｍＭの配列番号：４０に示すＭＫＣ プライマーおよび一本鎖ｃＤＮＡ １００ ｎｇ を含有し、これを５０μｌ の鉱油で覆った後、９４℃の初期温度にて３分間そして次に９４℃にて１分間、５５℃にて１分間および７２℃にて１分間、この順序で加熱した。この温度サイクルを３０回反復した後、反応混合物をさらに７２℃にて１０分間インキュベートした。増幅したＤＮＡ 断片を低融点アガロース（Ｓｉｇｍａ 社製）にて精製し、ＸｍａＩ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ 社製）およびＳａｌＩ（宝酒造製）により３７℃にて消化した。
【０１１６】
２−２． マウスＨ 鎖Ｖ 領域をコードするｃＤＮＡの増幅および断片化
マウスＨ 鎖Ｖ 領域をコードする遺伝子は５’−ＲＡＣＥ 法（Ｒａｐｉｄ Ａｍｐｌｉｆｉｃａｔｉｏｎ ｏｆ ｃＤＮＡ ｅｎｄｓ； Ｆｒｏｈｍａｎ， Ｍ．Ａ． ら、Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ， ８５， ８９９８−９００２， （１９８８） 、Ｅｄｗａｒｄｓ， Ｊ．Ｂ．Ｄ．Ｍ．，ら、Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．， １９， ５２２７−５２３２， （１９９１） ）により増幅した。マウスＩｇＧ２ａ 定常領域に特異的にハイブリダイズするプライマーＰ１（配列番号；４１）を用いてｃＤＮＡを合成した後、５’−ＡｍｐｌｉＦＩＮＤＥＲ ＲＡＣＥ ＫＩＴ （ＣＬＯＮＥＴＥＣＨ 社製）を用いてマウスＨ 鎖Ｖ 領域をコードするｃＤＮＡの増幅をマウスＩｇＧ２ａ 定常領域に特異的にハイブリダイズするプライマーＭＨＣ２ａ （配列番号：４２）およびキット添付のアンカープライマー（配列番号：７７）を用いて行った。増幅したＤＮＡ 断片を低融点アガロース（Ｓｉｇｍａ 社製）にて精製し、そしてＥｃｏＲＩ （宝酒造社製）およびＸｍａＩ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ 社製）により３７℃にて消化した。
【０１１７】
３． 連結および形質転換
上記のようにして調製したマウスカッパ型Ｌ 鎖Ｖ 領域をコードする遺伝子を含んで成るＤＮＡ 断片を、ＳａｌＩおよびＸｍａＩで消化することにより調製したｐＵＣ１９ ベクターと、５０ ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ７．６）、１０ ｍＭ ＭｇＣｌ_２ 、１０ ｍＭ ジチオスレイトール、１ ｍＭ ＡＴＰ、５０ ｍｇ／ｍｌのポリエチレングリコール（８０００）および１ユニットＴ４ ＤＮＡリガーゼ（ＧＩＢＣＯ−ＢＲＬ 社製）を含有する反応混合物中で、１６℃にて２．５ 時間反応させ連結した。同様にマウスＨ 鎖Ｖ 領域をコードする遺伝子を含んで成るＤＮＡ 断片を、ＥｃｏＲＩ およびＸｍａＩで消化することにより調製したｐＵＣ１９ ベクターと１６℃にて３ 時間反応させ連結した。
【０１１８】
次に、１０μｌ の上記連結混合物を大腸菌ＤＨ５ αのコンピテント細胞５０μｌ に加え、そしてこの細胞を氷上で３０分間、４２℃にて１分間そして再び氷上で１分間静置した。次いで４００ μｌ の２ｘＹＴ培地（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， Ｓａｍｂｒｏｏｋ ら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ， （１９８９） ）を加え、３７℃にて１時間インキュベートした後、５０μｇ／ｍｌ のアンピシリンを含有する２ｘＹＴ寒天培地（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， Ｓａｍｂｒｏｏｋ ら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ， （１９８９） ）上にこの大腸菌をまき、３７℃にて一夜インキュベートして大腸菌形質転換体を得た。
【０１１９】
この形質転換体を、５０μｇ／ｍｌのアンピシリンを含有する２ｘＹＴ培地１０ ｍｌ 中で３７℃にて一夜培養し、そしてこの培養物から、アルカリ法（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， Ｓａｍｂｒｏｏｋ ら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ， （１９８９） ）に従ってプラスミドＤＮＡ を調製した。
こうして得られた、抗ＨＭ１．２４抗体を産生するハイブリドーマに由来するマウスカッパ型Ｌ 鎖Ｖ 領域をコードする遺伝子を含有するプラスミドをｐＵＣＨＭＶＬ９と命名した。上記の方法に従って得られた、抗ＨＭ１．２４抗体を産生するハイブリドーマに由来するマウスＨ 鎖Ｖ 領域をコードする遺伝子を含有するプラスミドをｐＵＣＨＭＶＨＲ１６と命名した。
【０１２０】
実施例２ ． ＤＮＡ の塩基配列の決定
前記のプラスミド中のｃＤＮＡコード領域の塩基配列を、自動ＤＮＡ シークエンサー（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍ Ｉｎｃ．製）およびＴａｑ Ｄｙｅ Ｄｅｏｘｙ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ （Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍ Ｉｎｃ． 製）を用いて、メーカー指定のプロトコールに従って塩基配列を決定した。
プラスミドｐＵＣＨＭＶＬ９に含まれるマウス抗ＨＭ１．２４抗体のＬ 鎖Ｖ 領域をコードする遺伝子の塩基配列を配列番号：１に示す。また、プラスミドｐＵＣＨＭＶＨＲ１６に含まれるマウス抗ＨＭ１．２４抗体Ｈ 鎖Ｖ 領域をコードする遺伝子の塩基配列を配列番号：２に示す。
【０１２１】
実施例３ ． ＣＤＲ の決定
Ｌ 鎖およびＨ 鎖のＶ 領域の全般的構造は、互いに類似性を有しており、それぞれ４つのフレームワーク部分が３つの超可変領域、すなわち相補性決定領域（ＣＤＲ ）により連結されている。フレームワークのアミノ酸配列は、比較的良く保存されているが、一方ＣＤＲ 領域のアミノ酸配列の変異性は極めて高い（Ｋａｂａｔ， Ｅ．Ａ．， ら、「Ｓｅｑｕｅｎｃｅｓ ｏｆ Ｐｒｏｔｅｉｎｓ ｏｆ Ｉｍｍｕｎｏｌｏｇｉｃａｌ Ｉｎｔｅｒｅｓｔ 」ＵＳ Ｄｅｐｔ． Ｈｅａｌｔｈ ａｎｄ Ｈｕｍａｎ Ｓｅｒｖｉｃｅｓ， １９８３）。
このような事実に基づき、抗ＨＭ１．２４抗体の可変領域のアミノ酸配列をＫａｂａｔ らにより作製された抗体のアミノ酸配列のデータベースに当てはめて、相同性を調べることによりＣＤＲ 領域を表５に示すごとく決定した。
【０１２２】
【表５】

【０１２３】
実施例４ ． クローニングした ｃＤＮＡ の発現の確認（キメラ抗 ＨＭ１．２４ 抗体の作製）
１．発現ベクターの作製
キメラ抗ＨＭ１．２４抗体を発現するベクターを作製するため、それぞれのマウス抗ＨＭ１．２４抗体Ｌ 鎖およびＨ 鎖Ｖ 領域をコードするｃＤＮＡクローンｐＵＣＨＭＶＬ９およびｐＵＣＨＭＶＨＲ１６をＰＣＲ 法により修飾した。そしてＨＥＦ 発現ベクター（国際特許出願公開番号ＷＯ９２−１９７５９参照）に導入した。
【０１２４】
Ｌ 鎖Ｖ 領域のための後方プライマーＯＮＳ−Ｌ７２２Ｓ （配列番号：４３）およびＨ 鎖Ｖ 領域のための後方プライマーＶＨＲ１６Ｓ（配列番号：４４）は、各々のＶ 領域のリーダー配列の最初をコードするＤＮＡ にハイブリダイズし且つＫｏｚａｋ コンセンサス配列（Ｋｏｚａｋ， Ｍ， ら、Ｊ． Ｍｏｌ． Ｂｉｏｌ．， １９６， ９４７−９５０， （１９８７） ）およびＨｉｎｄＩＩＩ 制限酵素認識部位を有するように設計した。Ｌ 鎖Ｖ 領域のための前方プライマーＶＬ９Ａ（配列番号：４５）およびＨ 鎖Ｖ 領域のための前方プライマーＶＨＲ１６Ａ（配列番号：４６）は、Ｊ 領域の末端をコードするＤＮＡ 配列にハイブリダイズし且つスプライスドナー配列およびＢａｍＨＩ 制限酵素認識部位を有するように設計した。
【０１２５】
１０ ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ８．３）、５０ ｍＭ ＫＣｌ 、０．１ ｍＭ ｄＮＴＰｓ、１．５ ｍＭ ＭｇＣｌ_２、１００ ｐｍｏｌｅ ずつの各プライマー、１００ ｎｇの鋳型ＤＮＡ （ｐＵＣＨＭＶＬ９又はｐＵＣＨＭＶＨＲ１６）、および５ ｕｎｉｔのＡｍｐｌｉ Ｔａｑ 酵素を含有する１００ μｌ のＰＣＲ 反応混合物を５０μｌ の鉱油で覆い、９４℃にて最初の変性の後、９４℃にて１分間、５５℃にて１分間、７２℃にて１分間のサイクルを３０回行い、最後に７２℃にて１０分間インキュベートした。
ＰＣＲ 生成物を１．５％低融点アガロースゲルを用いて精製し、ＨｉｎｄＩＩＩ およびＢａｍＨＩ で消化し、そしてＬ 鎖Ｖ 領域についてはＨＥＦ−ＶＬ−ｇκに、Ｈ 鎖Ｖ 領域についてはＨＥＦ−ＶＨ−ｇγ１ にそれぞれクローニングした。ＤＮＡ 配列決定の後、正しいＤＮＡ 配列を有するＤＮＡ 断片を含むプラスミドをそれぞれＨＥＦ−１．２４Ｌ−ｇκ及びＨＥＦ−１．２４Ｈ−ｇγ１ と命名した。
【０１２６】
前記プラスミドＨＥＦ−１．２４Ｌ−ｇκ及びＨＥＦ−１．２４Ｈ−ｇγ１ からそれぞれの可変領域をコードする領域を制限酵素Ｈｉｎｄ ＩＩＩおよびＢａｍＨＩ により制限断片とし、これらをプラスミドベクターｐＵＣ １９のＨｉｎｄ ＩＩＩおよびＢａｍＨＩ 部位に挿入し、各々、ｐＵＣ１９−１．２４Ｌ−ｇκ及びｐＵＣ１９−１．２４Ｈ−ｇγ１ と命名した。
なお、それぞれのプラスミドｐＵＣ１９−１．２４Ｌ−ｇκ又はｐＵＣ１９−１．２４Ｈ−ｇγ１ を含有する大腸菌は、それぞれ、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ｐＵＣ１９−１．２４Ｌ−ｇκ）およびＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ｐＵＣ１９−１．２４Ｈ−ｇγ１ ）と称し、それぞれＦＥＲＭ ＢＰ−５６４６及びＦＥＲＭ ＢＰ−５６４４として、工業技術院生命工学工業技術研究所（茨城県つくば市東１丁目１番３号）に平成８年８月２９日にブダペスト条約に基づき国際寄託された。
【０１２７】
２．ＣＯＳ−７細胞へのトランスフェクション
キメラ抗ＨＭ１．２４抗体の一過性発現を観察するため、前記発現ベクターをＣＯＳ−７（ＡＴＣＣ ＣＲＬ−１６５１ ）細胞において試験した。ＨＥＦ−１．２４Ｌ−ｇκ及びＨＥＦ−１．２４Ｈ−ｇγ１ をＧｅｎｅ Ｐｕｌｓｅｒ 装置（ＢｉｏＲａｄ社製）を用いてエレクトロポレーションによりＣＯＳ−７細胞に同時形質転換した。各ＤＮＡ （１０μｇ ）を、ＰＢＳ 中１ ｘ １０^７ 細胞／ｍｌ の０．８ｍｌ のアリコートに加え、１５００Ｖ、２５μＦの容量にてパルスを与えた。
室温にて１０分間の回復期間の後、エレクトロポレーション処理された細胞を、１０％のγ−グロブリンフリーウシ胎児血清を含有するＤＭＥＭ培養液（ＧＩＢＣＯ 社製）３０ ｍｌ に加えた。ＣＯ_２ インキュベーターＢＮＡ１２０Ｄ （ＴＡＢＡＩ 社製）中で７２時間のインキュベーションの後、培養上清を集め、遠心分離により細胞破片を除去し、これを以下の実験に用いた。
【０１２８】
３． ＦＣＭ 解析
キメラ抗ＨＭ１．２４抗体の抗原結合活性は、ＫＰＭＭ２細胞を用いたＦＣＭ （フローサイトメトリー）解析で行った。４．７ ｘ １０^５ 個のＫＰＭＭ２細胞（特許出願公開番号 特開平７−２３６４７５）をＰＢＳ（−） で洗浄した後、上記キメラ抗ＨＭ１．２４抗体産生ＣＯＳ−７細胞培養液５０μｌおよびＦＡＣＳ緩衝液（２ ％ウシ胎児血清、０．１ ％アジ化ナトリウム含有ＰＢＳ（−））５０μｌ 、または５００ μｇ／ｍｌの精製マウス抗ＨＭ１．２４抗体 ５μｌ およびＦＡＣＳ緩衝液９５μｌ を加え、氷温下１時間インキュベートした。
【０１２９】
コントロールとしてキメラ抗ＨＭ１．２４抗体産生ＣＯＳ 細胞培養液の代わりに２ μｇ／ｍｌのキメラＳＫ２ （国際特許出願公開番号ＷＯ９４−２８１５９）５０μｌ およびＦＡＣＳ緩衝液５０μｌ 、または精製マウス抗ＨＭ１．２４抗体の代わりに５００ μｇ／ｍｌの精製マウス ＩｇＧ２ａκ（ＵＰＣ１０ ）（ＣＡＰＰＥＬ社製）５ μｌ およびＦＡＣＳ緩衝液９５μｌ を加え、同様にインキュベートした。ＦＡＣＳ緩衝液で洗浄した後、２５μｇ／ｍｌのＦＩＴＣ標識ヤギ抗ヒト抗体（ＧＡＨ ）（ＣＡＰＰＥＬ社製）、または１０μｇ／ｍｌのＦＩＴＣ標識ヤギ抗マウス抗体（ＧＡＭ ）（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ社製）１００ μｌ を加え、氷温下３０分間インキュベートした。ＦＡＣＳ緩衝液で洗浄した後、１ ｍｌ のＦＡＣＳ緩衝液に懸濁し、ＦＡＣＳｃａｎ（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ社製）で各細胞の蛍光強度を測定した。
【０１３０】
図１に示す通り、キメラ抗ＨＭ１．２４抗体を添加した細胞では、マウス抗ＨＭ１．２４抗体を添加した場合同様、コントロールと比較して蛍光強度のピークが右側にシフトしたことから、キメラ抗ＨＭ１．２４抗体がＫＰＭＭ２細胞と結合したことが明らかになった。このことより、クローニングしたｃＤＮＡはマウス抗ＨＭ１．２４抗体のＶ領域をコードしていることが確認された。
【０１３１】
実施例５ ． キメラ抗 ＨＭ１．２４ 抗体安定産生 ＣＨＯ 細胞株の樹立
１． キメラＨ 鎖発現ベクターの作製
前記プラスミドＨＥＦ−１．２４Ｈ−ｇγ１ を制限酵素ＰｖｕＩおよびＢａｍＨＩ にて消化し、ＥＦ１ プロモーターおよびマウス抗ＨＭ１．２４抗体Ｈ 鎖Ｖ 領域をコードするＤＮＡ を含む約２．８ｋｂｐの断片を１．５％低融点アガロースゲルを用いて精製した。次に、ＤＨＦＲ遺伝子およびヒトＨ 鎖定常領域をコードする遺伝子を含むヒトＨ 鎖発現ベクターＤＨＦＲ− △Ｅ−ＲＶｈ−ＰＭ１ｆ（国際特許出願公開番号ＷＯ９２／１９７５９参照）に使用されている発現ベクターをＰｖｕＩおよびＢａｍＨＩ にて消化することにより調製した約６ｋｂｐの断片内に上記ＤＮＡ 断片を挿入し、キメラ抗ＨＭ１．２４抗体Ｈ 鎖発現ベクター ＤＨＦＲ−△Ｅ−ＨＥＦ−１．２４Ｈ−ｇγ１ を構築した。
【０１３２】
２． ＣＨＯ細胞への遺伝子導入
キメラ抗ＨＭ１．２４抗体安定産生系を樹立するために、ＰｖｕＩで消化して直鎖状にした前記発現ベクターＨＥＦ−１．２４Ｌ−ｇκおよび ＤＨＦＲ−△Ｅ−ＨＥＦ−１．２４Ｈ−ｇγ１ をエレクトロポレーション法により前述と同様（前記ＣＯＳ−７ 細胞へのトランスフェクション）の条件下で同時にＣＨＯ 細胞ＤＸＢ１１ （Ｍｅｄｉｃａｌ Ｒｅｓｅａｒｃｈ Ｃｏｕｎｃｉｌ Ｃｏｌｌａｂｏｒａｔｉｏｎ Ｃｅｎｔｅｒ より供与）に遺伝子導入した。
【０１３３】
３． ＭＴＸによる遺伝子増幅
遺伝子導入したＣＨＯ 細胞は５００ μｇ／ｍｌのＧ４１８（ＧＩＢＣＯ−ＢＲＬ 社製）および１０％ のウシ胎児血清を添加したヌクレオシド不含α−ＭＥＭ培養液（ＧＩＢＣＯ−ＢＲＬ 社製）中ではＬ 鎖およびＨ 鎖発現ベクターが共に導入されたＣＨＯ 細胞のみが生存でき、それらを選別した。次に、上記培養液中に１０ ｎＭ のＭＴＸ （Ｓｉｇｍａ 社製）を加え、増殖したクローンの内、キメラ抗ＨＭ１．２４抗体の産生量が高いものを選択した結果、約２０μｇ／ｍｌのキメラ抗体産生効率を示すクローン＃８−１３ を得、キメラ抗ＨＭ１．２４抗体産生細胞株とした。
【０１３４】
実施例６ ． キメラ抗 ＨＭ１．２４ 抗体の作製
キメラ抗ＨＭ１．２４抗体の作製は以下の方法で行った。上記キメラ抗ＨＭ１．２４抗体産生ＣＨＯ 細胞を、培地として５％γ−グロブリンフリー新生仔ウシ血清（ＧＩＢＣＯ−ＢＲＬ 社製）含有 Ｉｓｃｏｖｅ’ｓ Ｍｏｄｉｆｉｅｄ Ｄｕｌｂｅｃｃｏ’ｓ Ｍｅｄｉｕｍ（ＧＩＢＣＯ−ＢＲＬ 社製）を用い、高密度細胞培養装置Ｖｅｒａｘ ｓｙｓｔｅｍ ２０ （ＣＥＬＬＥＸ ＢＩＯＳＣＩＥＮＣＥ Ｉｎｃ．社製）で３０日間連続培養した。
【０１３５】
培養開始後１３、２０、２３、２６及び３０日目に培養液を回収し、加圧式ろ過フィルターユニットＳＡＲＴＯＢＲＡＮ （Ｓａｒｔｏｒｉｕｓ 社製）を用いてろ過した後、抗体大量分取システムＡｆｉ−Ｐｒｅｐ Ｓｙｓｔｅｍ （日本ガイシ社製）およびＳｕｐｅｒ Ｐｒｏｔｅｉｎ Ａ ｃｏｌｕｍｎ（ｂｅｄ ｖｏｌｕｍｅ ： １００ ｍｌ 、日本ガイシ社製）を用いて、付属の説明書に基づき吸着／ 洗浄緩衝液としてＰＢＳ（−）、溶出緩衝液として０．１Ｍ クエン酸ナトリウム緩衝液（ｐＨ３ ）を用いてキメラ抗ＨＭ１．２４抗体をアフィニティー精製した。溶出画分は直ちに１Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０ ）を添加して、ｐＨ７．４ 付近に調整した。抗体濃度は、２８０ｎｍの吸光度を測定し、１ｍｇ／ｍｌ を１．３５ＯＤとして算出した。
【０１３６】
実施例７ ． キメラ抗 ＨＭ１．２４ 抗体の活性測定
キメラ抗ＨＭ１．２４抗体は下記の結合阻害活性にて評価を行った。
１． 結合阻害活性の測定
１−１． ビオチン標識抗ＨＭ１．２４抗体の作製
マウス抗ＨＭ１．２４ 抗体を０．１ Ｍ 重炭酸緩衝液で４ ｍｇ／ｍｌ に希釈した後、５０ ｍｇ／ｍｌのＢｉｏｔｉｎ−Ｎ− ｈｙｄｒｏｘｙ ｓｕｃｃｉｎｉｍｉｄｅ（ＥＹ ＬＡＢＳ Ｉｎｃ．社製）４ μｌ を添加し、室温で３ 時間反応させた。その後、０．２ Ｍ グリシン溶液１．５ ｍｌを加え室温で３０分間インキュベートし反応を停止させ、ＰＤ−１０ カラム（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ 社製）を用いてビオチン化ＩｇＧ 画分を分取した。
【０１３７】
１−２． 結合阻害活性の測定
ビオチン標識マウス抗ＨＭ１．２４抗体による結合阻害活性は、ヒト羊膜細胞株ＷＩＳＨ細胞（ＡＴＣＣ ＣＣＬ ２５ ）を用いたＣｅｌｌ−ＥＬＩＳＡで行った。Ｃｅｌｌ−ＥＬＩＳＡ プレートは次のようにして調製した。９６穴プレートに１０％ウシ胎児血清を含有するＲＰＭＩ１６４０培地により４ ｘ １０^５ 細胞／ｍｌ に調製したＷＩＳＨ細胞懸濁液１００ μｌ を加え、一晩培養した後、ＰＢＳ（−）で２回洗浄後０．１ ％グルタルアルデヒド（ナカライテスク社製）にて固定した。
【０１３８】
ブロッキングの後、アフィニティー精製により得られたキメラ抗ＨＭ１．２４抗体あるいはマウス抗ＨＭ１．２４抗体を段階希釈して各穴に５０μｌ 加え、同時に２ μｇ／ｍｌのビオチン標識マウス抗ＨＭ１．２４抗体５０μｌ を添加し、室温にて２時間インキュベーションおよび洗浄の後、ペルオキシダーゼ標識ストレプトアビジン（ＤＡＫＯ社製）を加えた。室温にて１時間インキュベーションした後洗浄し、基質溶液を加えインキュベーションの後、６Ｎ 硫酸５０μｌ で反応を停止させ、ＭＩＣＲＯＰＬＡＴＥ ＲＥＡＤＥＲ Ｍｏｄｅｌ ３５５０（Ｂｉｏ−Ｒａｄ 社製）を用いて４９０ ｎｍでの吸光度を測定した。
その結果、図２に示す通り、ビオチン標識マウス抗ＨＭ１．２４抗体に対してキメラ抗ＨＭ１．２４抗体はマウス抗ＨＭ１．２４抗体と同等の結合阻害活性を示した。このことより、キメラ抗体はマウス抗ＨＭ１．２４抗体と同じＶ 領域を有することが示された。
【０１３９】
実施例８ ． キメラ抗 ＨＭ１．２４ 抗体の ＡＤＣＣ 活性の測定
ＡＤＣＣ（Ａｎｔｉｂｏｄｙ−ｄｅｐｅｎｄｅｎｔ Ｃｅｌｌｕｌａｒ Ｃｙｔｏｔｏｘｉｃｉｔｙ）活性の測定はＣｕｒｒｅｎｔ ｐｒｏｔｏｃｏｌｓ ｉｎ Ｉｍｍｕｎｏｌｏｇｙ， Ｃｈａｐｔｅｒ ７． Ｉｍｍｕｎｏｌｏｇｉｃ ｓｔｕｄｉｅｓ ｉｎ ｈｕｍａｎｓ， Ｅｄｉｔｏｒ， Ｊｏｈｎ Ｅ， Ｃｏｌｉｇａｎ ｅｔ ａｌ．， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｉｎｃ．， １９９３の方法に従った。
１． エフェクター細胞の調製
健常人および多発性骨髄腫患者の末梢血および骨髄より比重遠心法で単核球を分離した。すなわち健常人および多発性骨髄腫患者の末梢血および骨髄に等量のＰＢＳ（−）を加え、Ｆｉｃｏｌｌ（Ｐｈａｒｍａｃｉａ 社製）− Ｃｏｎｒｅｙ（第一製薬社製）（比重１．０７７ ）に積層し、４００ ｇ で３０分間遠心した。単核球層を分取し、１０％ ウシ胎児血清（Ｗｉｔａｋｅｒ 社製）を含むＲＰＭＩ１６４０（Ｓｉｇｍａ 社製）で２回洗浄後、同培養液で細胞数が５ ｘ １０^６／ｍｌになるように調製した。
【０１４０】
２． 標的細胞の調製
ヒト骨髄腫細胞株ＲＰＭＩ ８２２６ （ＡＴＣＣ ＣＣＬ １５５）を０．１ｍＣｉの^５１Ｃｒ−ｓｏｄｉｕｍ ｃｈｒｏｍａｔｅとともに１０％ ウシ胎児血清（Ｗｉｔａｋｅｒ 社製）を含むＲＰＭＩ１６４０（Ｓｉｇｍａ 社製）中で３７℃にて６０分インキュベートすることにより放射性標識した。放射性標識の後、細胞をＨａｎｋｓ ｂａｌａｎｃｅｄ ｓａｌｔ ｓｏｌｕｔｉｏｎ （ＨＢＳＳ） で３回洗浄し、２ ｘ １０^５／ｍｌに調製した。
【０１４１】
３． ＡＤＣＣアッセイ
９６ウエルＵ 底プレート（Ｃｏｒｎｉｎｇ 社製）に放射性標識した２ ｘ １０^５／ｍｌの標的細胞を５０μｌ と、アフィニティー精製によって得られた１ μｇ／ｍｌのキメラ抗ＨＭ１．２４抗体、マウス抗ＨＭ１．２４抗体、あるいはコントロールヒトＩｇＧ１（Ｓｅｒｏｔｅｃ 社製）５０μｌ 加え、４ ℃で１５分間反応させた。
その後、５ ｘ １０^６／ｍｌのエフェクター細胞を１００ μｌ を加え、炭酸ガス培養器内で４ 時間培養した。その際、エフェクター細胞（Ｅ ）と標的細胞（Ｔ ）の比（Ｅ：Ｔ ）を ０：１、：５：１、２０：１又は５０：１とした。
【０１４２】
１００ μｌ の上清をとり、ガンマカウンター（ＡＲＣ３６１， Ａｌｏｋａ 社製）で培養上清中に遊離された放射活性を測定した。最大遊離放射能測定用には１ ％ＮＰ−４０ （ＢＲＬ 社製）を用いた。細胞障害活性（％）は（Ａ−Ｃ ）／（Ｂ−Ｃ ）ｘ１００で計算した。なおＡ は抗体存在下において遊離された放射活性（ｃｐｍ ）、Ｂ はＮＰ−４０ により遊離された放射活性（ｃｐｍ ）および Ｃは抗体を含まず培養液のみで遊離された放射活性（ｃｐｍ ）を示す。
図３に示す通り、ヒトコントロールＩｇＧ１と比較してキメラ抗ＨＭ１．２４抗体を添加した場合、Ｅ：Ｔ 比の上昇に従い細胞障害活性が上昇したことから、このキメラ抗ＨＭ１．２４抗体がＡＤＣＣ活性を有することが示された。さらに、マウス抗ＨＭ１．２４抗体を添加しても細胞障害活性は全く見られないことから、エフェクター細胞がヒト由来の細胞の場合、ＡＤＣＣ活性を得るためにはヒト抗体のＦｃ部分が必要であることが示された。
【０１４３】
実施例９ ． 再構成ヒト抗ＨＭ １．２４ 抗体の作製
１． 再構成ヒト抗ＨＭ１．２４抗体Ｖ領域の設計
マウスモノクローナル抗体のＣＤＲ がヒト抗体に移植されている再構成ヒト抗体を作製するためには、マウスモノクローナル抗体のＦＲとヒト抗体のＦＲとの間に高い相同性が存在することが望ましい。従って、マウス抗ＨＭ１．２４抗体のＬ鎖及びＨ鎖のＶ領域を、Ｐｒｏｔｅｉｎ Ｄａｔａ Ｂａｎｋ を用いて構造が解明されているすべての既知抗体のＶ領域と比較した。
【０１４４】
マウス抗ＨＭ１．２４抗体のＬ鎖Ｖ領域はヒトＬ鎖Ｖ領域のサブグループＩＶ（ＨＳＧＩＶ ）のコンセンサス配列に最も類似しており、６６．４％の相同性が存在する。一方、ＨＳＧＩ、ＨＳＧＩＩ 及びＨＳＧ ＩＩＩ とはそれぞれ５６．９％、５５．８％及び６１．５％の相同性を示した。
マウス抗ＨＭ１．２４抗体のＬ鎖Ｖ領域は既知ヒト抗体Ｌ鎖Ｖ領域との比較において、ヒトＬ鎖Ｖ領域のサブグループＩの一つであるヒトＬ鎖Ｖ領域ＲＥＩ に６７．０％の相同性を示した。従って、再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖Ｖ領域の作製のための出発材料としてＲＥＩ のＦＲを使用した。
【０１４５】
再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖Ｖ領域のバージョンａを設計した。このバージョンにおいては、ヒトＦＲは再構成ヒトＣＡＭＰＡＴＨ−１Ｈ抗体中に存在するＲＥＩ に基くＦＲ（Ｒｉｅｃｈｍａｎｎ， Ｌ． ら、Ｎａｔｕｒｅ ３２２， ２１−２５，（１９８８）を参照、国際特許出願公開番号ＷＯ９２−１９７５９に記載の再構成ヒトＰＭ−１のＬ鎖Ｖ領域のバージョンａに含まれるＦＲ）と同一であり、そしてマウスＣＤＲ はマウス抗ＨＭ１．２４抗体のＬ鎖Ｖ領域中のＣＤＲ と同一とした。
【０１４６】
マウス抗ＨＭ１．２４抗体のＨ鎖Ｖ領域はヒトＨ鎖Ｖ領域のＨＳＧ Ｉのコンセンサス配列に最も類似しており、５４．７％の相同性が存在する。一方、ＨＳＧＩＩ 及びＨＳＧＩＩＩとはそれぞれ３４．６％及び４８．１％の相同性を示した。マウス抗ＨＭ１．２４抗体のＨ鎖Ｖ領域は既知のヒト抗体Ｈ鎖Ｖ領域との比較において、ＦＲ１ からＦＲ３ までは、ヒトＨ鎖Ｖ領域のサブグループＩの一つであるヒト抗体ＨＧ３ のＨ鎖Ｖ領域（Ｒｅｃｈａｖｉ， Ｇ． ら、Ｐｒｏｃ． Ｎａｔ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８０， ８５５−８５９ ）に非常に類似しており、その相同性は６７．３％であった。
【０１４７】
このため、ヒト抗体ＨＧ３ のＦＲを、再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域の作製のための出発材料として用いた。しかしながら、ヒト抗体ＨＧ３ のＦＲ４ のアミノ酸配列は記述されていないために、今回ＦＲ４ に関してはマウス抗ＨＭ１．２４抗体のＨ鎖のＦＲ４ と最も高い相同性を示すヒト抗体ＪＨ６ （Ｒａｖｅｔｃｈ， Ｊ． Ｖ．ら、Ｃｅｌｌ， ２７， ５８３−５９１ ）のＦＲ４ のアミノ酸配列を用いた。ＪＨ６ のＦＲ４ は一つのアミノ酸を除いてマウス抗ＨＭ１．２４抗体のＨ鎖のＦＲ４ と同一のアミノ酸配列を有する。
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域の第一のバージョンａにおいて、ヒトＦＲ１ 中の３０位およびヒトＦＲ３ 中の７１位のアミノ酸をマウス抗ＨＭ１．２４抗体のアミノ酸と同一とした以外、ＦＲ１ からＦＲ３ まではヒトＨＧ３ のＦＲ１ からＦＲ３ と同一であり、そしてＣＤＲ はマウス抗ＨＭ１．２４抗体のＨ鎖Ｖ領域中のＣＤＲ と同一とした。
【０１４８】
２． 再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖Ｖ領域の作製
再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖を、ＰＣＲ 法によるＣＤＲ グラフティングにより作製した。この方法を図４に模式的に示す。ヒト抗体ＲＥＩ 由来のＦＲを有する再構成ヒト抗ＨＭ１．２４抗体（バージョンａ）の作製のために８個のＰＣＲ プライマーを使用した。外部プライマーＡ（配列番号：４７）及びＨ（配列番号：４８）は、ＨＥＦ 発現ベクターＨＥＦ−ＶＬ−ｇκのＤＮＡ 配列とハイブリダイズするように設計された。
ＣＤＲ −グラフティングプライマーＬ１Ｓ （配列番号：４９）、Ｌ２Ｓ （配列番号：５０）及びＬ３Ｓ （配列番号：５１）はセンスＤＮＡ 配列を有し、そしてＣＤＲ −グラフティングプライマーＬ１Ａ （配列番号：５２）、Ｌ２Ａ （配列番号：５３）及びＬ３Ａ （配列番号：５４）はアンチ−センスＤＮＡ 配列を有しそしてそれぞれプライマーＬ１Ｓ 、Ｌ２Ｓ 及びＬ３Ｓ の５′−末端のＤＮＡ 配列に対する相補的ＤＮＡ 配列（２０〜２３ｂｐ）を有する。
【０１４９】
第一ＰＣＲ 段階において４つの反応Ａ−Ｌ１Ａ 、Ｌ１Ｓ−Ｌ２Ａ 、Ｌ２Ｓ−Ｌ３Ａ 、及びＬ３Ｓ−Ｈ を行い、そして各ＰＣＲ 生成物を精製した。第一ＰＣＲ からの４つのＰＣＲ 生成物をそれら自体の相補性によりアッセンブリさせた（国際特許出願公開番号ＷＯ９２−１９７５９参照）。次に、外部プライマーＡ及びＨを加えて、再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖Ｖ領域をコードする全長ＤＮＡ を増幅した（第２ＰＣＲ ）。前記ＰＣＲ においては、ヒト抗体ＲＥＩ からのＦＲに基く再構成ヒトＯＮＳ−Ｍ２１ 抗体Ｌ鎖Ｖ領域バージョンａをコードするプラスミドＨＥＦ−ＲＶＬ−Ｍ２１ａ（国際特許出願公開番号ＷＯ９５−１４０４１を参照）を鋳型として用いた。
【０１５０】
第一ＰＣＲ 段階においては、１０ ｍＭ Ｔｒｉ−ＨＣｌ （ｐＨ８．３ ）、５０ ｍＭ ＫＣｌ 、０．１ｍＭ ｄＮＴＰｓ 、１．５ ｍＭ ＭｇＣｌ_２、１００ ｎｇの鋳型ＤＮＡ 、１００ ｐｍｏｌｅ の各プライマー及び５ｕのＡｍｐｌｉ Ｔａｑ を含有するのＰＣＲ 混合物を用いた。各ＰＣＲ チューブは５０μｌ の鉱油で覆膜した。最初に９４℃で変性した後、９４℃にて１分間、５５℃にて１分間及び７２℃にて１分間の反応サイクルを行い、次に７２℃にて１０分間インキュベートした。
ＰＣＲ 生成物Ａ−Ｌ１Ａ （２１５ｂｐ ）、Ｌ１Ｓ−Ｌ２Ａ （９８ｂｐ）、Ｌ２Ｓ−Ｌ３Ａ （１４０ｂｐ ）及びＬ３Ｓ−Ｈ （１５１ｂｐ ）を１．５ ％低融点アガロースゲルを用いて精製し、第二ＰＣＲ でアッセンブリした。第二ＰＣＲ においては、１μｇ の各第一ＰＣＲ の生成物、及び５ｕのＡｍｐｌｉ Ｔａｑ を含有する９８μｌ のＰＣＲ 混合物を、９４℃にて２分間、５５℃にて２分間及び７２℃にて２分間で２サイクルインキュベートし、そして次に１００ ｐｍｏｌｅ の各外部プライマー（Ａ及びＨ）を加えた。ＰＣＲ チューブを５０μｌ の鉱油で覆い、そして前記と同一の条件で３０サイクルのＰＣＲ を行った。
【０１５１】
第二ＰＣＲ により生じた５１６ｂｐ のＤＮＡ 断片を１．５ ％低融点アガロースゲルで精製し、ＢａｍＨＩ 及びＨｉｎｄＩＩＩ で消化し、得られたＤＮＡ 断片をＨＥＦ 発現ベクターＨＥＦ−ＶＬ−ｇκにクローニングした。ＤＮＡ 配列決定の後、再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖Ｖ領域の正しいアミノ酸配列をコードするＤＮＡ 断片を含むプラスミドをＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκと命名した。本プラスミドＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκに含まれるＬ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：９に示す。
再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖Ｖ領域のバージョンｂを、ＰＣＲ を用いる変異誘発法によって作製した。変異原プライマーＦＴＹ−１ （配列番号：５５）およびＦＴＹ−２ （配列番号：５６）は、７１位のフェニルアラニンがチロシンに変異するように設計した。
【０１５２】
プラスミドＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκを鋳型とし、上記プライマーを用いて増幅した後、最終生成物を精製し、ＢａｍＨＩ およびＨｉｎｄＩＩＩ で消化し、得られたＤＮＡ 断片をＨＥＦ 発現ベクターＨＥＦ−ＶＬ−ｇκにクローニングし、プラスミドＨＥＦ−ＲＶＬｂ−ＡＨＭ−ｇκを得た。本プラスミドＨＥＦ−ＲＶＬｂ−ＡＨＭ−ｇκに含まれるＬ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１０に示す。
【０１５３】
３． 再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域の作製
３−１． 再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域バージョンａ− ｅの作製
再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域をコードするＤＮＡ を次の様にして設計した。ヒト抗体ＨＧ３ のＦＲ１ 〜３ およびヒト抗体ＪＨ６ のＦＲ４ をコードするＤＮＡ 配列を、マウス抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域のＣＤＲ をコードするＤＮＡ 配列とつなげることにより、再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域をコードする全長ＤＮＡ を設計した。
次に、このＤＮＡ 配列のそれぞれ５′−側及び３′−側にＨｉｎｄＩＩＩ 認識部位／Ｋｏｚａｋ コンセンサス配列及びＢａｍＨＩ 認識部位／スプライスドナー配列を付加して、ＨＥＦ 発現ベクターに挿入できるようにした。
【０１５４】
こうして設計したＤＮＡ 配列を４個のオリゴヌクレオチドに分け、そして次に、これらのオリゴヌクレオチドのアセンブリーを妨害する可能性のあるオリゴヌクレオチド中の二次構造についてコンピューター解析した。４個のオリゴヌクレオチド配列ＲＶＨ１〜ＲＶＨ４を配列番号：５７〜６０に示す。これらのオリゴヌクレオチドは１１９ 〜１４４ 塩基の長さを有し、２５〜２６ｂｐのオーバラップ領域を有する。オリゴヌクレオチドの内のＲＶＨ２（配列番号：５８）、ＲＶＨ４（配列番号：６０）はセンスＤＮＡ 配列を有し、そして他のＲＶＨ１（配列番号：５７）、ＲＶＨ３（配列番号：５９）はアンチセンスＤＮＡ 配列を有する。これら４個のオリゴヌクレオチドのＰＣＲ 法によるアセンブリーの方法を図に示す（図５参照）。
【０１５５】
１００ ｎｇずつの４種のオリゴヌクレオチド及び５ｕのＡｍｐｌｉ Ｔａｑ を含有する９８μｌ のＰＣＲ 混合物を、９４℃にて２分間の最初の変性の後、９４℃にて２分間、５５℃にて２分間及び７２℃にて２分間のから成る２サイクルのインキュベーションを行った。１００ ｐｍｏｌｅ ずつのＲＨＰ１（配列番号：６１）及びＲＨＰ２（配列番号：６２）を外部プライマーとして添加した後、ＰＣＲ チューブを５０μｌ の鉱油で覆い、そして９４℃にて１分間の最初の変性の後、９４℃にて１分間、５５℃にて１分間及び７２℃にて１分間の３８サイクルを行い、そして次に７２℃にて１０分間インキュベートした。
【０１５６】
４３８ｂｐ のＤＮＡ 断片を１．５ ％低融点アガロースゲルを用いて精製し、ＨｉｎｄＩＩＩ 及びＢａｍＨＩ により消化し、そして次にＨＥＦ 発現ベクターＨＥＦ−ＶＨ−ｇγ１ にクローニングした。ＤＮＡ 配列決定の後、正しいＨ鎖Ｖ領域のアミノ酸配列をコードするＤＮＡ 断片を含むプラスミドをＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ と命名した。本プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１１に示す。
再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域の各バージョンｂ、ｃ、ｄ及びｅを以下のようにして作製した。
【０１５７】
バージョンｂは、変異原プライマーとして６６位のアルギニンがリジンに変異するように設計したＢＳ（配列番号：６３）およびＢＡ（配列番号：６４）を用い、プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として、ＰＣＲ 法により増幅し、プラスミドＨＥＦ−ＲＶＨｂ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｂ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１２に示す。
バージョンｃは、変異原プライマーとして７３位のトレオニンがリジンに変異するように設計したＣＳ（配列番号：６５）およびＣＡ（配列番号：６６）を用い、プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として、ＰＣＲ 法により増幅し、プラスミドＨＥＦ−ＲＶＨｃ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｃ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１３に示す。
【０１５８】
バージョンｄは、変異原プライマーとして６６位のアルギニンがリジンに、７３位のトレオニンがリジンに変異するように設計したＤＳ（配列番号：６７）およびＤＡ（配列番号：６８）を用い、プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ としてプラスミドＨＥＦ−ＲＶＨｄ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｄ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１４に示す。
バージョンｅは、変異原プライマーとして６７位のバリンがアラニンに、６９位のメチオニンがロイシンに変異するように設計したＥＳ（配列番号：６９）およびＥＡ（配列番号：７０）を用い、プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として増幅し、プラスミドＨＥＦ−ＲＶＨｅ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｅ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域に含まれるアミノ酸配列および塩基配列を配列番号：１５に示す。
【０１５９】
３−２． Ｈ鎖ハイブリッドＶ領域の作製
Ｈ鎖ハイブリッドＶ領域を２種構築した。１つはＦＲ１ とＦＲ２ のアミノ酸配列がマウス抗ＨＭ１．２４ 抗体由来であり、ＦＲ３ とＦＲ４ のアミノ酸配列が再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域のバージョンａ由来となるマウス・ヒトハイブリッド抗ＨＭ１．２４抗体、もう１つはＦＲ１ とＦＲ２ のアミノ酸配列が再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域のバージョンａ由来であり、ＦＲ３ とＦＲ４ のアミノ酸配列がマウス抗ＨＭ１．２４抗体由来となるヒト・マウスハイブリッド抗ＨＭ１．２４抗体である。ＣＤＲ 領域のアミノ酸配列はすべてマウス抗ＨＭ１．２４抗体由来である。
【０１６０】
２種のＨ鎖ハイブリッドＶ領域はＰＣＲ 法により作製した。この方法を図６及び７に模式的に示す。２種のＨ鎖ハイブリッドＶ領域作製のために４種のプライマーを使用した。外部プライマーａ（配列番号：７１）及びｈ（配列番号：７２）は、ＨＥＦ 発現ベクターＨＥＦ−ＶＨ−ｇγ１ のＤＮＡ 配列とハイブリダイズするように設計された。Ｈ鎖ハイブリッド作製プライマーＨＹＳ （配列番号：７３）はセンスＤＮＡ 配列を有し、Ｈ鎖ハイブリッドプライマーＨＹＡ （配列番号：７４）はアンチセンスＤＮＡ 配列を有しそしてたがいに相補的なＤＮＡ 配列となるよう設計された。
【０１６１】
ＦＲ１ とＦＲ２ のアミノ酸配列がマウス抗ＨＭ１．２４抗体由来であり、ＦＲ３ とＦＲ４ のアミノ酸配列が再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域のバージョンａ由来となるＨ鎖ハイブリッドＶ領域の作製のために、第一ＰＣＲ 段階においてプラスミド ＨＥＦ−１．２４Ｈ−ｇγ１ を鋳型とし外部プライマーａとＨ鎖ハイブリッドプライマーＨＹＡ を用いたＰＣＲ と、プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型としＨ鎖ハイブリッドプライマーＨＹＳ （配列番号：７３）と外部プライマーｈ（配列番号：７２）を用いたＰＣＲ を行い、そして各ＰＣＲ 産物を精製した。第一ＰＣＲ からの２つのＰＣＲ 生成物をそれら自体の相補性によりアッセンブリさせた（国際特許出願公開番号ＷＯ９２−１９７５９参照）。
【０１６２】
次に、外部プライマーａ（配列番号：７１）及びｈ（配列番号：７２）を加えて、ＦＲ１ とＦＲ２ のアミノ酸配列がマウス抗ＨＭ１．２４抗体由来であり、ＦＲ３ とＦＲ４ のアミノ酸配列が再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域のバージョンａ由来となるＨ鎖ハイブリッドＶ領域をコードする全長ＤＮＡ を第二ＰＣＲ 段階で増幅した。
ＦＲ１ とＦＲ２ のアミノ酸配列が再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域のバージョンａ由来であり、ＦＲ３ とＦＲ４ のアミノ酸配列がマウス抗ＨＭ１．２４抗体由来となるＨ鎖ハイブリッドＶ領域の作製のために、第一ＰＣＲ 段階においてプラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型とし外部プライマーａとＨ鎖ハイブリッドプライマーＨＹＡ を用いたＰＣＲ と、プラスミド ＨＥＦ−１．２４Ｈ−ｇγ１ を鋳型としＨ鎖ハイブリッドプライマーＨＹＳ と外部プライマーｈを用いたＰＣＲ を行い、そして各ＰＣＲ 産物を精製した。第一ＰＣＲ からの２つのＰＣＲ 生成物をそれら自体の相補性によりアッセンブリさせた（国際特許出願公開番号ＷＯ９２−１９７５９参照）。
【０１６３】
次に、外部プライマーａ及びｈを加えて、ＦＲ１ とＦＲ２ のアミノ酸配列が再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域のバージョンａ由来であり、ＦＲ３ とＦＲ４ のアミノ酸配列がマウス抗ＨＭ１．２４ 抗体由来となるＨ鎖ハイブリッドＶ領域をコードする全長ＤＮＡ を第二ＰＣＲ 段階で増幅した。
第一ＰＣＲ 、ＰＣＲ 産物の精製、アッセンブリ、第二ＰＣＲ 、及びＨＥＦ 発現ベクターＨＥＦ−ＶＨ−ｇγ１ へのクローニングの方法は実施例 ９． 再構成ヒトＨＭ１．２４抗体Ｌ鎖Ｖ領域の作製に示す方法に準じた。
【０１６４】
ＤＮＡ 配列決定の後、ＦＲ１ とＦＲ２ のアミノ酸配列がマウス抗ＨＭ１．２４抗体由来であり、ＦＲ３ とＦＲ４ のアミノ酸配列が再構成ヒト抗ＨＭ１．２４抗体のＨ鎖Ｖ領域のバージョンａ由来となるＨ鎖ハイブリッドＶ領域の正しいアミノ酸配列をコードするＤＮＡ 断片を含むプラスミドをＨＥＦ−ＭＨ−ＲＶＨ−ＡＨＭ−ｇγ１ と命名した。本プラスミドＨＥＦ−ＭＨ−ＲＶＨ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列及び塩基配列を配列番号：７５に示す。また、ＦＲ１ とＦＲ２ のアミノ酸配列が再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域のバージョンａ由来であり、ＦＲ３ とＦＲ４ のアミノ酸配列がマウス抗体ＨＭ１．２４ 抗体由来となるＨ鎖ハイブリッドＶ領域の正しいアミノ酸配列をコードするＤＮＡ 断片を含むプラスミドをＨＥＦ−ＨＭ−ＲＶＨ−ＡＨＭ−ｇγ１ と命名した。本プラスミドＨＥＦ−ＨＭ−ＲＶＨ−ＡＨＭ−ｇγ１ に 含まれるＨ鎖Ｖ領域のアミノ酸配列及び塩基配列を配列番号：７６に示す。
【０１６５】
３−３． 再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域バージョンｆ〜ｓの作製
再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域の各バージョンｆ、ｇ、ｈ、ｉ、ｊ、ｋ、ｌ、ｍ、ｎ、ｏ、ｐ、ｑ、ｒ及びｓを以下のようにして作製した。
バージョンｆは、変異原プライマーとして７５位のトレオニンがセリンに、７８位のバリンがアラニンに変異するように設計したＦＳ（配列番号：７８）およびＦＡ（配列番号：７９）を用い、プラスミドＨＥＦ−ＲＶＨｅ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として、ＰＣＲ 法により増幅し、プラスミドＨＥＦ−ＲＶＨｆ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｆ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１６に示す。
【０１６６】
バージョンｇは、変異原プライマーとして４０位のアラニンがアルギニンに変異するように設計したＧＳ（配列番号：８０）およびＧＡ（配列番号：８１）を用い、プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として増幅し、プラスミドＨＥＦ−ＲＶＨｇ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｇ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１７に示す。
バージョンｈは、変異原プライマーとしてＦＳ（配列番号：７８）およびＦＡ（配列番号：７９）を用い、プラスミドＨＥＦ−ＲＶＨｂ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として増幅し、プラスミドＨＥＦ−ＲＶＨｈ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｈ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１８に示す。
【０１６７】
バージョンｉは、変異原プライマーとして８３位のアルギニンがアラニンに、８４位のセリンがフェニルアラニンに変異するように設計したＩＳ（配列番号：８２）およびＩＡ（配列番号：８３）を用い、プラスミドＨＥＦ−ＲＶＨｈ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡとして増幅し、プラスミドＨＥＦ−ＲＶＨｉ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｉ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１９に示す。
バージョンｊは、変異原プライマーとして６６位のアルギニンがリジンに変異するように設計したＪＳ（配列番号：８４）とＪＡ（配列番号：８５）を用い、プラスミドＨＥＦ−ＲＶＨｆ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として増幅し、プラスミドＨＥＦ−ＲＶＨｊ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｊ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２０に示す。
【０１６８】
バージョンｋは、変異原プライマーとして８１位のグルタミン酸がグルタミンに変異するように設計したＫＳ（配列番号：８６）およびＫＡ（配列番号：８７）を用い、プラスミドＨＥＦ−ＲＶＨｈ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として増幅し、プラスミドＨＥＦ−ＲＶＨｋ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｋ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２１に示す。
バージョンｌは、変異原プライマーとして８１位のグルタミン酸がグルタミンに、８２Ｂ 位のセリンがイソロイシンに変異するように設計したＬＳ（配列番号：８８）およびＬＡ（配列番号：８９）を用い、プラスミドＨＥＦ−ＲＶＨｈ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として増幅し、プラスミドＨＥＦ−ＲＶＨｌ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｌ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２２に示す。
【０１６９】
バージョンｍは、変異原プライマーとして８１位のグルタミン酸がグルタミンに、８２ｂ 位のセリンがイソロイシンに、８７位のトレオニンがセリンに変異するように設計したＭＳ（配列番号：９０）とＭＡ（配列番号：９１）を用い、プラスミドＨＥＦ−ＲＶＨｈ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として増幅し、プラスミドＨＥＦ−ＲＶＨｍ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｍ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２３に示す。
バージョンｎは、変異原プライマーとして８２Ｂ 位のセリンがイソロイシンに変異するように設計したＮＳ（配列番号：９２）およびＮＡ（配列番号：９３）を用い、プラスミドＨＥＦ−ＲＶＨｈ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ としてプラスミドＨＥＦ−ＲＶＨｎ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｎ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２４に示す。
【０１７０】
バージョンｏは、変異原プライマーとして８７位のトレオニンがセリンに変異するように設計したＯＳ（配列番号：９４）およびＯＡ（配列番号：９５）を用い、プラスミドＨＥＦ−ＲＶＨｈ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ としてプラスミドＨＥＦ−ＲＶＨｏ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｏ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２５に示す。
バージョンｐは、変異原プライマーとして７８位のバリンがアラニンに変異するように設計したＰＳ（配列番号：９６）およびＰＡ（配列番号：９７）を用い、プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として、ＰＣＲ 法により増幅し、プラスミドＨＥＦ−ＲＶＨｐ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｐ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２６に示す。
【０１７１】
バージョンｑは、変異原プライマーとして７５位のトレオニンがセリンに変異するように設計したＱＳ（配列番号：９８）およびＱＡ（配列番号：９９）を用い、プラスミドＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として、ＰＣＲ 法により増幅し、プラスミドＨＥＦ−ＲＶＨｑ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｑ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２７に示す。
バージョンｒは、変異原プライマーとしてＣＳ（配列番号：６５）およびＣＡ（配列番号：６６）を用い、プラスミドＨＥＦ−ＲＶＨｐ−ＡＨＭ−ｇγ１ を鋳型ＤＮＡ として、ＰＣＲ 法により増幅し、プラスミドＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：２８に示す。
【０１７２】
再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域のバージョンｓを、ＰＣＲ を用いる変異誘発法によって作製した。変異原プライマーＳＳ（配列番号：１００）およびＳＡ（配列番号：１０１）は、６９位のメチオニンがイソロイシンに変異するように設計した。
プラスミドＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１ を鋳型とし、上記プライマーを用いて増幅した後、最終生成物を精製し、ＢａｍＨＩ およびＨｉｎｄＩＩＩ で消化し、得られたＤＮＡ 断片をＨＥＦ 発現ベクターＨＥＦ−ＶＨ−ｇγ１ にクローニングし、プラスミドＨＥＦ−ＲＶＨｓ−ＡＨＭ−ｇγ１ を得た。本プラスミドＨＥＦ−ＲＶＨｓ−ＡＨＭ−ｇγ１ に含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：１０２ に示す。
【０１７３】
なお、前記プラスミドＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκ及びＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１ からそれぞれの可変領域をコードする領域を制限酵素ＨｉｎｄＩＩＩ およびＢａｍＨＩ により制限断片とし、これらをプラスミドベクターｐＵＣ１９ のＨｉｎｄＩＩＩ およびＢａｍＨＩ 部位に挿入した。それぞれのプラスミドはｐＵＣ１９−ＲＶＬａ−ＡＨＭ−ｇκ及びｐＵＣ１９−ＲＶＨｒ−ＡＨＭ−ｇγ１ と命名した。
なお、それぞれのプラスミドｐＵＣ１９−ＲＶＬａ−ＡＨＭ−ｇκおよびｐＵＣ１９−ＲＶＨｒ−ＡＨＭ−ｇγ１ を含有する大腸菌は、それぞれ、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ｐＵＣ１９−ＲＶＬａ−ＡＨＭ−ｇκ）およびＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ｐＵＣ１９−ＲＶＨｒ−ＡＨＭ−ｇγ１ ）と称し、工業技術院生命工学工業技術研究所（茨城県つくば市東１丁目１番３号）に平成８年８月２９日に、各々ＦＥＲＭ ＢＰ−５６４５およびＦＥＲＭ ＢＰ−５６４３としてブダペスト条約に基づき国際寄託された。
【０１７４】
なお、前記プラスミドＨＥＦ−ＲＶＨｓ−ＡＨＭ−ｇγ１ からの可変領域をコードする領域を制限酵素ＨｉｎｄＩＩＩ およびＢａｍＨＩ により制限断片とし、これをプラスミドベクターｐＵＣ１９ のＢａｍＨＩ およびＨｉｎｄＩＩＩ 部位に挿入した。得られたプラスミドをｐＵＣ１９−ＲＶＨｓ−ＡＨＭ−ｇγ１ と命名した。
プラスミドｐＵＣ１９−ＲＶＨｓ−ＡＨＭ−ｇγ１ を含有する大腸菌は、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ｐＵＣ１９−ＲＶＨｓ−ＡＨＭ−ｇγ１ ）と称し、工業技術院生命工学工業技術研究所（茨城県つくば市東１丁目１番３号）に平成９年（１９９７年）９月２９日にＦＥＲＭ ＢＰ−６１２７としてブダペスト条約に基づき国際寄託された。
【０１７５】
４． 再構成ヒト抗ＨＭ１．２４抗体、キメラ抗ＨＭ１．２４抗体、及びＨ鎖ハイブリッド抗
体の作製
再構成ヒト抗ＨＭ１．２４抗体の各鎖を評価するために再構成ヒト抗ＨＭ１．２４抗体とポジティブコントロール抗体としてキメラ抗ＨＭ１．２４抗体を発現させた。そして再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域のバージョンｂ以降の各バージョンを作製する際、どのＦＲ内のアミノ酸残基を置換すべきかを検討するためにＨ鎖ハイブリッド抗体を発現させた。また、再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖バージョンａの評価のためにキメラＨ鎖との組合せで発現させた。
【０１７６】
４−１． 再構成ヒト抗ＨＭ１．２４抗体の発現（１）
再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖のための発現ベクター（ＨＥＦ−ＲＶＨａ−ＡＨＭ−ｇγ１ 〜ＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１ ）と再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖のための発現ベクター（ＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκあるいはＨＥＦ−ＲＶＬｂ−ＡＨＭ−ｇκ）各１０μｇ をＧｅｎｅ Ｐｕｌｓｅｒ 装置（ＢｉｏＲａｄ社製）を用いてエレクトロポレーションによりＣＯＳ−７細胞に同時形質転換した。各ＤＮＡ （１０μｇ ）を、ＰＢＳ 中１ ｘ １０^７ 細胞／ｍｌ の０．８ｍｌ のアリコートに加え、１５００Ｖ、２５μＦの容量にてパルスを与えた。
【０１７７】
室温にて１０分間の回復期間の後、エレクトロポレーション処理された細胞を、１０％のγ−グロブリンフリーウシ胎児血清を含有するＤＭＥＭ培養液３０ ｍｌ （ＧＩＢＣＯ 社製）に加えた。３７℃、５％ＣＯ_２ の条件下で７２時間の培養をＣＯ_２ インキュベーターＢＮＡ１２０Ｄ （ＴＡＢＡＩ 社製）を用いて行った後、培養上清を集め、遠心ローター０３（ＨＩＴＡＣＨＩ 社製）を装着した遠心機１５ＰＲ−２２（ＨＩＴＡＣＨＩ 社製）により１０００ ｒｐｍ、５分間の遠心分離を行い細胞破片を除去し、マイクロコンセントレーター（Ｃｅｎｔｒｉｃｏｎ １００ 、Ａｍｉｃｏｎ社製）を遠心ローターＪＡ−２０ ・１ （ＢＥＣＫＭＡＮ 社製）を装着した遠心器Ｊ２−２１ （ＢＥＣＫＭＡＮ 社製）により２０００ ｒｐｍの条件下で限外濾過濃縮をおこない、Ｃｅｌｌ−ＥＬＩＳＡに用いた。
【０１７８】
再構成ヒト抗ＨＭ１．２４抗体の発現（２）
再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖バージョンｓのための発現ベクター（ＨＥＦ−ＲＶＨｓ−ＡＨＭ−ｇγ１）と再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖のための発現ベクター（ＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκ） 各１０μｇ をＧｅｎｅ Ｐｕｌｓｅｒ 装置（ＢｉｏＲａｄ社製）を用いてエレクトロポレーションによりＣＯＳ 細胞に同時形質転換した。各ＤＮＡ（１０μｇ）を、ＰＢＳ 中１ ｘ １０^７ 細胞／ｍｌ の０．８ｍｌ のアリコートに加え、１，５００Ｖ、２５μＦ の容量にてパルスを与えた。
【０１７９】
室温にて１０分間の回復期間の後、エレクトロポレーション処理された細胞を、１０％のγ−グロブリンフリーウシ胎児血清を含有するＤＭＥＭ培養液３０ｍｌ（ＧＩＢＣＯ社製）に加えた。３７℃、５％ ＣＯ_２の条件下で７２時間の培養をＣＯ_２ インキュベーターＢＮＡ１２０Ｄ （ＴＡＢＡＩ社製）を用いて行った後、培養上清を集め、遠心ローター０３（ＨＩＴＡＣＨＩ社製）を装着した遠心機０５ＰＲ−２２ （ＨＩＴＡＣＨＩ社製）により１０００ｒｐｍ 、５分間の遠心分離を行い細胞破片を除去し、マイクロコンセントレーター（Ｃｅｎｔｒｉｃｏｎ １００、Ａｍｉｃｏｎ社製）を遠心ローターＪＡ−２０ ・１ （ＢＥＣＫＭＡＮ社製）を装着した遠心器Ｊ２−２１ （ＢＥＣＫＭＡＮ社製）により２０００ｒｐｍ の条件下で限外濾過濃縮をおこない、濾過フィルター、マイレクスＧＶ１３ｍｍ（ミリポア社製）用いて濾過滅菌したものをＣｅｌｌ−ＥＬＩＳＡに用いた。
【０１８０】
４−２． キメラ抗ＨＭ１．２４抗体の発現
キメラ抗ＨＭ１．２４抗体Ｈ鎖のための発現ベクター ＨＥＦ−１．２４Ｈ−ｇγ１ とキメラ抗ＨＭ１．２４抗体Ｌ鎖のための発現ベクター ＨＥＦ−１．２４Ｌ−ｇκ各１０μｇ を用い、上記再構成ヒト抗ＨＭ１．２４抗体の発現の方法にしたがってＣｅｌｌ−ＥＬＩＳＡに用いるためのキメラ抗ＨＭ１．２４抗体を調製した。
【０１８１】
４−３． ヒト型化Ｌ鎖バージョンａとキメラＨ鎖からなる抗ＨＭ１．２４抗体の発現
キメラ抗ＨＭ１．２４抗体Ｈ鎖のための発現ベクターＨＥＦ−１．２４Ｈ−ｇ γ１ と再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖バージョンａのための発現ベクターＨＥＦ−ＲＶＬａ−ＡＨＭ−Ｇκ各１０μｇを用い、上記再構成ヒト抗ＨＭ１．２４抗体の発現の方法に従って、Ｃｅｌｌ−ＥＬＩＳＡに用いるためのヒト型化Ｌ鎖バージョンａとキメラＨ鎖からなる抗ＨＭ１．２４抗体を調製した。
【０１８２】
４−４． Ｈ鎖ハイブリッド抗体の発現
Ｈ鎖ハイブリッドＶ領域のための発現ベクター（ＨＥＦ−ＭＨ−ＲＶＨ−ＡＨＭ−ｇγ１ 或いはＨＥＦ−ＨＭ−ＲＶＨ−ＡＨＭ−ｇγ１ ）と再構成ヒト抗ＨＭ１．２４抗体Ｌ鎖のための発現ベクターＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκ各１０μｇ を用い、上記再構成ヒト抗ＨＭ１．２４抗体の発現の方法にしたがってＣｅｌｌ−ＥＬＩＳＡに用いるためのＨ鎖ハイブリッド抗体を調製した。
【０１８３】
４−５． 抗体濃度の測定
得られた抗体の濃度測定はＥＬＩＳＡ により行った。ＥＬＩＳＡ 用９６穴プレート（Ｍａｘｉｓｏｒｐ， ＮＵＮＣ社製）の各穴にコーティングバッファー（０．１Ｍ ＮａＨＣＯ_３， ０．０２％ ＮａＮ_３，ｐＨ９．６ ）により１μｇ／ｍｌの濃度に調製したヤギ抗ヒトＩｇＧ 抗体（ＢＩＯ ＳＯＵＲＣＥ社製）１００μｌを加え、室温で１時間のインキュベーションを行い固相化した。１００ μｌの希釈バッファー（５０ｍＭ Ｔｒｉｓ−ＨＣｌ， １ｍＭ ＭｇＣｌ_２， ０．１５Ｍ ＮａＣｌ，０．０５％Ｔｗｅｅｎ２０， ０．０２％ＮａＮ_３， １％牛血清アルブミン（ＢＳＡ），ｐＨ８．１）でブロッキングした後、限外濾過濃縮を行った再構成ヒト抗ＨＭ１．２４抗体、キメラ抗ＨＭ１．２４抗体、及びＨ鎖ハイブリッド抗体を順次段階希釈して各穴に１００ μｌずつ加え室温で１時間のインキュベーションおよび洗浄の後、アルカリフォスファターゼ標識ヤギ抗ヒトＩｇＧ 抗体（ＤＡＫＯ社製）１００ μｌを加えた。
【０１８４】
室温にて１時間のインキュベーションおよび洗浄の後、基質バッファー（５０ｍＭ ＮａＨＣＯ_３， １０ｍＭ ＭｇＣｌ_２（ｐＨ９．８）） に溶解した１ｍｇ／ｍｌ の基質溶液（Ｓｉｇｍａ１０４， ｐ−ニトロフェニルリン酸、ＳＩＧＭＡ 社製）１００ μｌを加え、４０５ｎｍ での吸光度をＭＩＣＲＯＰＬＡＴＥ ＲＥＡＤＥＲ Ｍｏｄｅｌ ３５５０（Ｂｉｏ−Ｒａｄ社製）を用いて測定した。濃度測定の標品としてヒトＩｇＧ１κ（Ｔｈｅ Ｂｉｎｄｉｎｇ Ｓｉｔｅ社製）を用いた。
【０１８５】
５． 再構成ヒト抗ＨＭ１．２４抗体安定産生ＣＨＯ細胞株の樹立
５−１． 再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖発現ベクターの作製
プラスミドＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１ を制限酵素ＰｖｕＩ及びＢａｍＨＩ にて消化し、ＥＦ１ プロモーター及び再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖Ｖ領域をコードするＤＮＡ を含む約 ２．８ ｋｂｐの断片を １．５％低融点アガロースゲルを用いて精製した。次に、ＤＨＦＲ遺伝子およびヒトＨ鎖定常領域をコードする遺伝子を含むヒトＨ鎖発現ベクターＤＨＦＲ− ΔＥ−ＲＶｈ− ＰＭ１ｆ（国際特許出願公開番号ＷＯ９２−１９７５９）に使用されている発現ベクターをＰｖｕＩ及びＢａｍＨＩ にて消化することにより調製した約６ｋｂｐ の断片内に上記ＤＮＡ 断片を挿入し、再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖発現ベクターＤＨＦＲ− ΔＥ−ＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１ を構築した。
【０１８６】
５−２． ＣＨＯ 細胞への遺伝子導入
再構成ヒト抗ＨＭ１．２４抗体安定産生系を樹立するために、ＰｖｕＩで消化して直鎖状にした前記発現ベクターＤＨＦＲ− ΔＥ−ＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１ 及びＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκをエレクトロポレーション法により前述と同様（前記ＣＯＳ−７ 細胞へのトランスフェクション）の条件下で同時にＣＨＯ 細胞ＤＸＢ−１１に遺伝子導入した。
【０１８７】
５−３． ＭＴＸ による遺伝子増幅
遺伝子導入した ＣＨＯ細胞は５００ μｇ／ｍｌのＧ４１８（ＧＩＢＣＯ−ＢＲＬ 社製）及び１０％のウシ胎児血清を添加したヌクレオシド不含α−ＭＥＭ培養液中（ＧＩＢＣＯ−ＢＲＬ 社製）ではＬ鎖及びＨ鎖発現ベクターが共に導入されたＣＨＯ 細胞のみが増殖でき、それらを選別した。次に、上記培養液中に１０ ｎＭ のＭＴＸ（Ｓｉｇｍａ 社製）を加え、増殖したクローンのうち再構成ヒト抗ＨＭ１．２４抗体の産生量が高いものを選択した結果、約３μｇ／ｍｌの再構成ヒト抗ＨＭ１．２４抗体産生率を示すクローン＃１を得、再構成ヒト抗ＨＭ１．２４抗体産生細胞株とした。
【０１８８】
５−４． 再構成ヒト抗ＨＭ１．２４抗体の作製
再構成ヒト抗ＨＭ１．２４抗体の作製は以下の方法で行った。上記再構成ヒト抗ＨＭ１．２４抗体産生ＣＨＯ 細胞を、培地として１０％のγ−グロブリンフリーウシ胎児血清（ＧＩＢＣＯ−ＢＲＬ 社製）を含有する５００ μｇ／ｍｌのＧ４１８（ＧＩＢＣＯ−ＢＲＬ 社製）を添加したヌクレオシド不含α−ＭＥＭ培養液（ＧＩＢＣＯ−ＢＲＬ 社製）を用い、３７℃、５％ＣＯ_２ の条件下で１０日の培養をＣＯ_２ インキュベーターＢＮＡ１２０Ｄ （ＴＡＢＡＩ 社製）を用いて行った。培養開始後８、１０日目に培養液を回収し、ＴＳ−９ローターを装着した遠心機ＲＬ−５００ＳＰ（トミー精工社製）を用いて２０００ｒｐｍ 、１０分間の遠心分離を行い培養液中の細胞破片を除去した後、０．４５μｍ 径のメンブレンをもつボトルトップフィルター（ＦＡＬＣＯＮ社製）により濾過滅菌した。
【０１８９】
この再構成ヒト抗ＨＭ１．２４抗体産生ＣＨＯ 細胞培養液に等量のＰＢＳ（−）を加えた後、高速抗体精製装置ＣｏｎＳｅｐ ＬＣ１００（ＭＩＬＬＩＰＯＲＥ 社製）およびＨｙｐｅｒ Ｄ Ｐｒｏｔｅｉｎ Ａ カラム（日本ガイシ社製）を用い、付属の説明書に基づき吸着緩衝液としてＰＢＳ（−）、溶出緩衝液として０．１Ｍ クエン酸ナトリウム緩衝液（ｐＨ３ ）を用いて再構成ヒト抗ＨＭ１．２４抗体をアフィニティー精製した。溶出画分は直ちに１Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０ ）を添加してｐＨ７．４ 付近に調整した後、遠心限外濃縮器Ｃｅｎｔｒｉｐｒｅｐ １０ （ＭＩＬＬＩＰＯＲＥ 社製）を用いて濃縮およびＰＢＳ（−）への緩衝液置換を行い、孔径０．２２μｍのメンブレンフィルターＭＩＬＬＥＸ−ＧＶ （ＭＩＬＬＩＰＯＲＥ 社製）を用いて濾過滅菌し精製再構成ヒト抗ＨＭ１．２４抗体を得た。精製抗体の濃度は、２８０ｎｍ の吸光度を測定し、１ｍｇ／ｍｌ を１．３５０Ｄとして算出した。
【０１９０】
実施例１１ ． 再構成ヒト抗 ＨＭ１．２４ 抗体の活性測定
再構成ヒト抗ＨＭ１．２４抗体は下記の抗原結合活性および結合阻害活性にて評価を行った。
１．抗原結合活性および結合阻害活性の測定法
１−１． 抗原結合活性の測定
抗原結合活性の測定は、ＷＩＳＨ細胞を用いたＣｅｌｌ−ＥＬＩＳＡで行った。Ｃｅｌｌ−ＥＬＩＳＡ プレートは前記実施例７．１−２で記載の通り作製した。
【０１９１】
ブロッキングの後、ＣＯＳ−７ 細胞の培養上清を濃縮して得られた、またはＣＨＯ 細胞の培養上清より精製された再構成ヒト抗ＨＭ１．２４抗体を段階希釈して各穴に１００ μｌ 加え、室温にて２時間インキュベーションおよび洗浄の後、ペルオキシダーゼ標識ウサギ抗ヒトＩｇＧ 抗体（ＤＡＫＯ社製）を加えた。室温にて１時間インキュベーションおよび洗浄の後、基質溶液を加えインキュベーションの後、６Ｎ 硫酸５０μｌ で反応を停止させ、ＭＩＣＲＯＰＬＡＴＥ ＲＥＡＤＥＲ Ｍｏｄｅｌ ３５５０（Ｂｉｏ−Ｒａｄ 社製）を用いて４９０ｎｍ での吸光度を測定した。
【０１９２】
１−２． 結合阻害活性の測定
ビオチン標識マウス抗ＨＭ１．２４抗体による結合阻害活性は、ＷＩＳＨ細胞を用いたＣｅｌｌ−ＥＬＩＳＡで行った。Ｃｅｌｌ−ＥＬＩＳＡ プレートは前述の通り作製した。ブロッキングの後、ＣＯＳ−７ 細胞の培養上清を濃縮して得られた、またはＣＨＯ 細胞の培養上清より精製された再構成ヒト抗ＨＭ１．２４抗体を段階希釈して各穴に５０μｌ 加え、同時に２ μｇ／ｍｌのビオチン標識マウス抗ＨＭ１．２４抗体５０μｌ を添加し、室温にて２時間インキュベーションおよび洗浄の後、ペルオキシダーゼ標識ストレプトアビジン（ＤＡＫＯ社製）を加えた。室温にて１時間インキュベーションした後洗浄し、基質溶液を加えインキュベーションの後、６Ｎ 硫酸５０μｌ で反応を停止させ、ＭＩＣＲＯＰＬＡＴＥ ＲＥＡＤＥＲ Ｍｏｄｅｌ ３５５０（Ｂｉｏ−Ｒａｄ 社製）を用いて４９０ｎｍ での吸光度を測定した。
【０１９３】
２． 再構成ヒト抗ＨＭ１．２４抗体の評価
２−１． Ｌ鎖
再構成ヒト抗ＨＭ１．２４抗体のＬ鎖バージョンａの評価は、前記の抗原結合活性の測定により行った。図８に示す通り、Ｌ鎖バージョンａはキメラＨ鎖と組合わせて発現させると、キメラ抗ＨＭ１．２４抗体と同程度の抗原結合活性を示した。しかし、さらなる活性の上昇またはＨ 鎖との相性を考慮し、新たにＬ鎖バージョンｂを作製した。そして、Ｈ 鎖のバージョンａ、ｂ、ｆ又はｈと組み合わせたときの抗原結合活性および結合阻害活性の測定を行いＬ 鎖バージョンａ、ｂを共に評価した。図９、１０、１１及び１２に示すとおり、Ｈ 鎖ａ、ｂ、ｆ及びｈの全てのバージョンで、Ｌ鎖バージョンａがバージョンｂに比べて両活性とも強かった。従って、再構成ヒト抗ＨＭ１．２４抗体のＬ鎖バージョンａを以下の実験に用いた。
【０１９４】
２−２． Ｈ鎖バージョンａ− ｅ
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖バージョンａ− ｅの評価はＬ鎖バージョンａとの組合せで、前記の抗原結合活性および結合阻害活性の測定により行った。その結果、図１１、１３、１４及び１５に示す通り、全てのバージョンにおいてキメラ抗ＨＭ１．２４抗体と比較して両活性とも弱く、さらなるアミノ酸の変換が必要であると考えられた。
【０１９５】
２−３． Ｈ鎖ハイブリッド抗体
Ｈ鎖ハイブリッド抗体の評価は前記の抗原結合活性の測定により行った。その結果、図１６に示す通り、抗原結合活性はヒト− マウスハイブリッド抗ＨＭ１．２４抗体ではキメラ抗ＨＭ１．２４抗体と同等の活性を有している一方、マウス・ヒトハイブリッド抗ＨＭ１．２４抗体はキメラ抗ＨＭ１．２４抗体と比較してその活性が弱かった。従って、マウス抗ＨＭ１．２４抗体、あるいはキメラ抗ＨＭ１．２４抗体と同等の抗原結合活性を有する再構成ヒトＨＭ１．２４抗体を作成するためには、Ｈ 鎖Ｖ 領域のうち、ＦＲ３ あるいはＦＲ４ に含まれるアミノ酸を変換する必要があることが示された。
【０１９６】
２−４． Ｈ鎖バージョン ｆ− ｒ
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖バージョンｆの評価は前記の抗原結合活性測定により行った。その結果、図１７に示す通り、抗原結合活性はキメラ抗ＨＭ１．２４抗体と比較すると劣るが、上記バージョンａ− ｃと比較して活性が向上したことから、本バージョンで新たに変換した６７、６９、７５及び７８番目の４つのアミノ酸のうちいずれかが再構成ヒト抗体の活性に関与していることが示唆された。
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖バージョンｇの評価は前記の抗原結合活性、および結合阻害活性の測定により行った。その結果、図１８及び１９に示す通り、本バージョンは上記バージョンａと同程度の活性しか示さなかったことから、上記Ｈ鎖ヒト・マウスハイブリッド抗体の評価で示した通り、本バージョンで変換した４０番目のアミノ酸は再構成ヒト抗体の活性の向上には寄与していないことが示された。
【０１９７】
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖バージョンｈ− ｊの評価は前記の抗原結合活性、および結合阻害活性の測定により行った。その結果、図２０、２１、２２、２３に示す通り、全てのｖｅｒｓｉｏｎ で両活性ともキメラ抗ＨＭ１．２４抗体と比較すると弱く、上記バージョンｆと同程度であることから、バージョンｆで新たに変換した４アミノ酸のうち、６７及び６９番目のアミノ酸は再構成ヒト抗体の活性の向上に寄与していないことが示唆された。
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖バージョンｋ− ｐの評価は前記の抗原結合活性、および結合阻害活性の測定により行った。その結果、図２４、２５、２６及び２７に示す通り、全てのバージョンで両活性ともキメラ抗ＨＭ１．２４抗体と比較すると弱く、上記バージョンｈと同程度であることから、これら６つのバージョンで新たに変換した８０番目以降のアミノ酸は再構成ヒト抗体の活性の向上に寄与していないことが示唆された。
【０１９８】
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖バージョンｑの評価は前記の抗原結合活性、および結合阻害活性の測定により行った。その結果、図２５及び２７に示す通り、本バージョンは両活性とも上記バージョンｈあるいはバージョンｐと比較すると弱く、上記バージョンａと同程度の活性しか持たなかったことから、７８番目のアミノ酸の置換が再構成ヒト抗体の活性の向上に必須であることが示唆された。
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖バージョンｒの評価は前記の測定により行った。その結果、図１５及び２８に示す通り、バージョンｒはキメラ抗ＨＭ１．２４抗体と同程度の抗原結合活性および結合阻害活性を有することが示された。
【０１９９】
以上の結果より、再構成ヒト抗ＨＭ１．２４抗体がマウス抗ＨＭ１．２４抗体あるいはキメラ抗ＨＭ１．２４抗体と同程度の抗原結合能を持つための、Ｈ 鎖における必要で最小の変換は３０、７１及び７８番目、さらには７３番目のアミノ酸であることが示された。
なお、再構成ヒト抗ＨＭ１．２４抗体Ｈ鎖バージョンａ− ｒについて、その抗原結合活性、および結合阻害活性を表６にまとめた。
【０２００】
【表６】

【０２０１】
２．５． Ｈ鎖バージョンｓ
再構成ヒト抗ＨＭ１．２４抗体のＨ鎖バージョンｓの評価は、Ｌ鎖バージョンａとの組合せで前記の抗原結合活性、および結合阻害活性の測定により行った。その結果、図２９、３０に示すようにバージョンｓはバージョンｒと同程度の抗原結合活性および結合阻害活性を有することが示された。
また、上記のごとく、本発明の再構成ヒト抗ＨＭ１．２４抗体はＦＲ中の１個又は複数個のアミノ酸残基を他のアミノ酸に置換してもなお、抗原に結合する能力を維持している。したがって、本発明は、その本来の性質を維持している限り、Ｈ鎖又はＬ鎖のＶ領域において、１個又は複数個のアミノ酸残基が他のアミノ酸残基に置換されている再構成ヒト抗ＨＭ１．２４抗体をも包含する。
【０２０２】
３． 精製再構成ヒト抗ＨＭ１．２４抗体の評価
前記精製再構成ヒト抗ＨＭ１．２４抗体は前記の抗原結合活性および結合阻害活性にて評価を行った。その結果、図３１及び３２に示す通り再構成ヒト抗ＨＭ１．２４抗体は、キメラ抗ＨＭ１．２４抗体と同程度の抗原結合活性および結合阻害活性を有することが示された。このことより、再構成ヒト抗ＨＭ１．２４抗体はマウス抗ＨＭ１．２４抗体と同じ抗原結合能を持つことが示された。
【０２０３】
実施例１２ ． キメラ抗 ＨＭ１．２４ 抗体のヒト骨髄腫マウスモデルに対する抗腫瘍効果
１． 投与抗体の調製
１−１． キメラ抗ＨＭ１．２４抗体の調製
前記実施例６で得られた精製キメラ抗ＨＭ１．２４抗体を、遠心限外濃縮器Ｃｅｎｔｒｉｐｒｅｐ １０ （ＭＩＬＬＩＰＯＲＥ 社製）で濃縮およびＰＢＳ（−）への緩衝液置換を行い、孔径０．２２μｍのメンブレンフィルターＭＩＬＬＥＸ−ＧＶ （ＭＩＬＬＩＰＯＲＥ 社製）を用いて濾過滅菌した。これを濾過滅菌したＰＢＳ（−）を用いて２００ μｇ／ｍｌに調製し、以下の実験に用いた。抗体濃度は、２８０ｎｍ の吸光度を測定し、１ｍｇ／ｍｌ を１．３５０Ｄとして算出した。
【０２０４】
１−２． コントロールヒトＩｇＧ１の精製
キメラ抗ＨＭ１．２４抗体のコントロールとして用いるヒトＩｇＧ１は以下のように精製した。Ｈｕ ＩｇＧ１ Ｋａｐｐａ Ｐｕｒｉｆｉｅｄ（ＢＩＮＤＩＮＧ ＳＩＴＥ社製）に等量のＰＢＳ（−）を加えた後、高速抗体精製装置ＣｏｎＳｅｐ ＬＣ１００（ＭＩＬＬＩＰＯＲＥ 社製）およびＨｙｐｅｒ Ｄ Ｐｒｏｔｅｉｎ Ａ カラム（日本ガイシ社製）を用い、付属の説明書に基づき吸着緩衝液としてＰＢＳ（−）、溶出緩衝液として０．１Ｍ クエン酸ナトリウム緩衝液（ｐＨ３ ）を用いてアフィニティー精製した。溶出画分は直ちに１Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０ ）を添加してｐＨ７．４ 付近に調整した後、遠心限外濃縮器Ｃｅｎｔｒｉｐｒｅｐ １０ （ＭＩＬＬＩＰＯＲＥ 社製）を用いて濃縮およびＰＢＳ（−）への緩衝液置換を行い、孔径０．２２μｍのメンブレンフィルターＭＩＬＬＥＸ−ＧＶ （ＭＩＬＬＩＰＯＲＥ 社製）を用いて濾過滅菌した。これを濾過滅菌したＰＢＳ（−）を用いて２００ μｇ／ｍｌに調製し、以下の実験に用いた。抗体の濃度は、２８０ｎｍ の吸光度を測定し、１ｍｇ／ｍｌ を１．３５０Ｄとして算出した。
【０２０５】
２． マウス血清ヒトＩｇＧ 定量法
マウスの血清中に含まれるヒトＩｇＧ の定量は以下のＥＬＩＳＡ で行った。０．１Ｍ重炭酸緩衝液（ｐＨ９．６ ）で１ μｇ／ｍｌに希釈したヤギ抗ヒトＩｇＧ （ＴＡＧＯ社製）１００ μｌ を９６穴プレート（Ｎｕｎｃ社製）に加え、４℃で一晩インキュベーションし、抗体を固相化した。ブロッキングの後、段階希釈したマウス血清あるいは標品としてヒトＩｇＧ （ＣＡＰＰＥＬ社製）１００ μｌ を添加し、室温にて１時間インキュベーションした。洗浄後、２０００倍希釈したアルカリフォスファターゼ標識抗ヒトＩｇＧ （ＣＡＰＰＥＬ社製）１００ μｌ を加え、室温にて１時間インキュベートした。洗浄後、基質溶液を加えインキュベーションの後、ＭＩＣＲＯＰＬＡＴＥ ＲＥＡＤＥＲ Ｍｏｄｅｌ ３５５０（Ｂｉｏ−Ｒａｄ 社製）を用いて４０５ｎｍ での吸光度を測定した。
【０２０６】
３． キメラ抗ＨＭ１．２４抗体のヒト骨髄腫移植マウスに対する抗腫瘍効果
３−１． ヒト骨髄腫移植マウスの作製
ヒト骨髄腫移植マウスは以下のように作製した。ＳＣＩＤマウス（日本クレア）を用いてｉｎ ｖｉｖｏ 継代したＫＰＭＭ２ 細胞を、１０％ウシ胎児血清（ＧＩＢＣＯ−ＢＲＬ 社製）を含むＲＰＭＩ１６４０培地（ＧＩＢＣＯ−ＢＲＬ 社製）で３ ｘ １０^７ 個 ／ ｍｌ になるように調製した。あらかじめ前日抗アシアロＧＭ１ （和光純薬社製）１００ μｌ を腹腔内投与したＳＣＩＤマウス（オス、８週令）（日本クレア）に上記ＫＰＭＭ２ 細胞懸濁液 ２００μｌ を尾静脈より注入した。
【０２０７】
３−２． 抗体投与
上記ヒト骨髄腫移植マウスよりＫＰＭＭ２ 細胞移植後１２日目に血清を採取し、上記２ のＥＬＩＳＡ を用いて、血清中のヒトＩｇＧ を定量した。血清中のヒトＩｇＧ の上昇によりＫＰＭＭ２ 細胞の骨髄生着を確認した。これらマウスに対し、ＫＰＭＭ２ 細胞移植後１４、２１、２８日目に上記１で調製した抗体をそれぞれ１００ μｌ 腹腔内投与した。
３−３． キメラ抗ＨＭ１．２４抗体のヒト骨髄腫移植マウスに対する抗腫瘍効果の評価
キメラ抗ＨＭ１．２４抗体の抗腫瘍効果については、マウスの生存期間で評価した。図３３に示す通り、キメラ抗ＨＭ１．２４抗体を投与したマウスではコントロールヒトＩｇＧ１を投与したマウスと比較して、生存期間の延長が認められた。従って、キメラ抗ＨＭ１．２４抗体がヒト骨髄腫移植マウスに対して抗腫瘍効果を有することが示された。
【０２０８】
実施例 １３ ． 再構成ヒト抗 ＨＭ１．２４ 抗体の ＡＤＣＣ 活性の測定
ＡＤＣＣ （Ａｎｔｉｂｏｄｙ−ｄｅｐｅｎｄｅｎｔ Ｃｅｌｌｕｌａｒ Ｃｙｔｏｔｏｘｉｃｉｔｙ） 活性の測定はＣｕｒｒｅｎｔ ｐｒｏｔｏｃｏｌｓ ｉｎ Ｉｍｍｕｎｏｌｏｇｙ， Ｃｈａｐｔｅｒ ７． Ｉｍｍｕｎｏｌｏｇｉｃ ｓｔｕｄｉｅｓ ｉｎ ｈｕｍａｎｓ， Ｅｄｉｔｏｒ， Ｊｏｈｎ Ｅ， Ｃｏｌｉｇａｎ ｅｔ ａｌ．， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｉｎｃ．， １９９３の方法に従った。
１． エフェクター細胞の調製
健常人の末梢血より比重遠心法で単核球を分離した。すなわち健常人の末梢血に等量のＰＢＳ（−）を加え、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＬＵＳ （Ｐｈａｒｍａｃｉａ社製）に積層し、 ４００ｇで４０分間遠心した。単核球層を分取し、１０％ウシ胎児血清（ＧＩＢＣＯ ＢＲＬ社製）を含むＲＰＭＩ １６４０ （ＧＩＢＣＯ ＢＲＬ社製）で４回洗浄後、同培養液で細胞数が５ ｘ １０^６／ｍｌになるように調製した。
【０２０９】
ＳＣＩＤマウス（日本クレア）の骨髄細胞よりＬＡＫ （Ｌｉｍｐｈｏｋｉｎｅ Ａｃｔｉｖａｔｅｄ Ｋｉｌｌｅｒ Ｃｅｌｌ）を誘導した。すなわちマウスの大腿骨より骨髄細胞を分離し１０％ウシ胎児血清（ＧＩＢＣＯ ＢＲＬ社製）を含むＲＰＭＩ １６４０ （ＧＩＢＣＯ ＢＲＬ社製）で２回洗浄後、同培養液で細胞数が２ ｘ １０^５／ｍｌになるように調製した。５０ｎｇ／ｍｌ のリコンビナントヒトＩＬ−２（Ｒ ＆ Ｄ ＳＹＳＴＥＭＳ社製）および１０ｎｇ／ｍｌ のリコンビナントマウスＧＭ−ＣＳＦ（Ｒ ＆ Ｄ ＳＹＳＴＥＭＳ社製）とともに、炭酸ガス培養器（ＴＡＢＡＩ社製）内で７日間培養した。同培養液で細胞数が２ ｘ １０^６／ｍｌになるように調製した。
【０２１０】
２． 標的細胞の調製
ヒト骨髄腫細胞株ＫＰＭＭ２（特開平７−２３６４７５）あるいは形質細胞腫由来ＡＲＨ−７７ （ＡＴＣＣ ＣＲＬ−１６２１）を０．１ｍＣｉの^５１Ｃｒ−ｓｏｄｉｕｍ ｃｈｒｏｍａｔｅ（ＩＣＮ社製）とともに１０％ウシ胎児血清（ＧＩＢＣＯ ＢＲＬ社製）を含むＲＰＭＩ １６４０ （ＧＩＢＣＯ ＢＲＬ社製）中で３７℃にて６０分インキュベートすることにより放射性標識した。放射性標識の後、細胞を同培養液で３回洗浄し、２ ｘ １０^５／ｍｌに調製した。
【０２１１】
３． ＡＤＣＣアッセイ
９６ウエルＵ底プレート（ベクトンディッキンソン社製）に放射性標識した２ ｘ １０^５／ｍｌの標的細胞を５０μｌ と、再構成ヒト抗ＨＭ１．２４抗体、マウス抗ＨＭ１．２４抗体、コントロールヒトＩｇＧ１（Ｔｈｅ Ｂｉｎｄｉｎｇ Ｓｉｔｅ Ｌｉｍｉｔｅｄ社製）、あるいはコントロールマウスＩｇＧ２ａ（ＵＰＣ１０ 、ＣＡＰＰＥＬ社製）５０μｌ を加え、４℃で１５分間反応させた。
その後、エフェクター細胞１００ μｌ を、炭酸ガス培養器内で４時間培養した。その際、エフェクター細胞（Ｅ）と標的細胞（Ｔ）の比（Ｅ：Ｔ）を ０：１、３．２：１ 、８：１ 、２０：１又は５０：１とした。
【０２１２】
１００ μｌ の上清をとり、ガンマカウンター （ＡＲＣ−３００ 、Ａｌｏｋａ 社製）で培養上清中に遊離された放射活性を測定した。最大遊離放射能測定用には１％ＮＰ−４０ （半井社製）を用いた。細胞障害活性（％）は（Ａ−Ｃ）／（Ｂ−Ｃ）Ｘ １００ で計算した。なおＡは抗体存在下において遊離された放射活性（ｃｐｍ）、ＢはＮＰ−４０ により遊離された放射活性（ｃｐｍ）およびＣは抗体を含まず培養液のみで遊離された放射活性（ｃｐｍ）を示す。
【０２１３】
図３４にエフェクター細胞として健常人末梢血より調製した細胞を用い、標的細胞にＫＰＭＭ２ を用いた場合の結果を示す。図３５にエフェクター細胞として健常人末梢血より調製した細胞を用い、標的細胞にＡＲＨ７７ を用いた場合の結果を示す。コントロールヒトＩｇＧ１と比較して再構成ヒト抗ＨＭ１．２４抗体を添加した場合、抗体濃度の上昇に従い細胞障害活性が上昇したことから、この再構成ヒト抗ＨＭ１．２４抗体がＡＤＣＣ活性を有することが示された。
【０２１４】
また、再構成ヒト抗ＨＭ１．２４抗体を添加した場合、マウス抗ＨＭ１．２４抗体を添加した場合と比べて明らかに細胞障害活性が上昇したことから、この再構成ヒト抗ＨＭ１．２４抗体がマウス抗ＨＭ１．２４抗体よりも高いＡＤＣＣ活性を有することが示された。さらに、標的細胞がＫＰＭＭ２ の場合、 ０．１μｇ／ｍｌ以上の濃度で再構成ヒト抗ＨＭ１．２４抗体を添加した場合、細胞障害活性が変わらないことから、 ０．１μｇ／ｍｌ以上の濃度で十分なＡＤＣＣ活性を有することが示された。標的細胞がＡＲＨ７７ の場合、１μｇ／ｍｌ以上の濃度で再構成ヒト抗ＨＭ１．２４抗体を添加した場合、細胞障害活性が変わらないことから、１μｇ／ｍｌ以上の濃度で十分なＡＤＣＣ活性を有することが示された。
【０２１５】
図３６にエフェクター細胞としてＳＣＩＤマウス骨髄より調製した細胞を用いた場合の結果を示す。コントロールヒトＩｇＧ１と比較して再構成ヒト抗ＨＭ１．２４抗体を添加した場合、抗体濃度の上昇に従い細胞障害活性が上昇したことから、この再構成ヒト抗ＨＭ１．２４抗体がＡＤＣＣ活性を有することが示された。また、 ０．１μｇ／ｍｌ以上の濃度で再構成ヒト抗ＨＭ１．２４抗体を添加した場合、細胞障害活性が変わらないことから、 ０．１μｇ／ｍｌ以上の濃度で十分なＡＤＣＣ活性を有することが示された。
これらの結果から再構成ヒト抗ＨＭ１．２４抗体は、エフェクター細胞としてヒト由来、あるいはマウス由来の細胞を用いた場合でもＡＤＣＣ活性を有することが示された。
【０２１６】
実施例 １４ ． 再構成ヒト抗 ＨＭ１．２４ 抗体のヒト骨髄腫マウスモデルに対する抗腫瘍効果
１． 投与抗体の調製
プラスミドＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκとプラスミドＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１を細胞に導入して得られた再構成ヒト抗ＨＭ１．２４抗体については、濾過滅菌したＰＢＳ（−）を用いて４０、２００ 、１０００μ／ｍｌ に、また、実施例１２．１−２で得たコントロールヒトＩｇＧ１については、濾過滅菌したＰＢＳ（−）を用いて ２００μｇ／ｍｌに調製し、投与抗体とした。
２． 再構成ヒト抗ＨＭ１．２４抗体のヒト骨髄腫移植マウスに対する抗腫瘍効果
２−１． ヒト骨髄腫移植マウスの作製
ヒト骨髄腫移植マウスは、実施例１２．３−１に従い作製した。マウスは、ＳＣＩＤマウス（５週令）（日本クレア）を用いた。
【０２１７】
２−２． 抗体投与
上記２−１ で作製したヒト骨髄腫移植マウスよりＫＰＭＭ２ 細胞移植９日目に血清を採取し、実施例１２．２のＥＬＩＳＡ を用いて、血清中のヒトＩｇＧ を定量した。血清中のヒトＩｇＧ の上昇によりＫＰＭＭ２ 細胞の骨髄生着を確認した。これらマウスに対し、ＫＰＭＭ２ 細胞移植後１０日目に上記１で調製した抗体をそれぞれ １００μｌ 静脈内投与した。
【０２１８】
２−３． 再構成ヒト抗ＨＭ１．２４抗体のヒト骨髄腫移植マウスに対する抗腫瘍効果の評価
再構成ヒト抗ＨＭ１．２４抗体の抗腫瘍効果については、マウスの血清ヒトＩｇＧ 量の変化、および生存期間で評価した。
マウスの血清ヒトＩｇＧ 量の変化については、ＫＰＭＭ２ 細胞移植３５日目に血清を採取し、実施例１２．２のＥＬＩＳＡ を用いてヒトＩｇＧ を定量した。その結果、図３７に示すようにコントロールヒトＩｇＧ１投与群では、ＫＰＭＭ２ 細胞移植３５日目の血清ヒトＩｇＧ 量は移植９日目（抗体投与前日）と比較して約１０００倍程度まで増加しているのに対し、再構成ヒト抗ＨＭ１．２４抗体投与群ではいずれの投与量でも、移植９日目とほぼ同じかあるいはそれ以下であり、再構成ヒト抗ＨＭ１．２４抗体がＫＰＭＭ２ 細胞の増殖を抑制していることが示された。
【０２１９】
一方、生存期間についても図３８に示す通り、再構成ヒト抗ＨＭ１．２４抗体投与群ではコントロールヒトＩｇＧ１投与群と比較して、生存期間の延長が認められた。以上より、再構成ヒト抗ＨＭ１．２４抗体投与がヒト骨髄腫移植マウスに対して抗腫瘍効果を有することが示された。
【０２２０】
実施例 １５ ． ヒト骨髄腫マウスモデルにおける、再構成ヒト抗 ＨＭ１．２４ 抗体と既存薬剤メルファランとの抗腫瘍効果の比較
１． 投与薬剤の調製
１−１． 投与抗体の調製
プラスミドＨＥＦ−ＲＶＬａ−ＡＨＭ−ｇκとプラスミドＨＥＦ−ＲＶＨｒ−ＡＨＭ−ｇγ１を細胞に導入して得られた再構成ヒト抗ＨＭ１．２４抗体については、濾過滅菌したＰＢＳ（−）を用いて４０、２００ μｇ／ｍｌに、また、実施例１２．１−２で得たコントロールヒトＩｇＧ１については、濾過滅菌したＰＢＳ（−）を用いて ２００μｇ／ｍｌに調製し、投与抗体とした。
１−２． メルファランの調製
骨髄腫に対する既存薬剤であるメルファラン（ＳＩＧＭＡ製）は、 ０．２％カルボメチルセルロース（ＣＭＣ）（ダイセル化学工業製）を用いて、０．１ｍｇ／ｍｌになるように調製した。
【０２２１】
２． ヒト骨髄腫移植マウスに対する再構成ヒト抗ＨＭ１．２４抗体およびメルファランの抗腫瘍効果
２−１． ヒト骨髄腫移植マウスの作製
ヒト骨髄腫移植マウスは、実施例１４．２−１に従い作製した。
２−２． 薬剤投与
上記２−１ で作製したヒト骨髄腫移植マウスよりＫＰＭＭ２ 細胞移植９日目に血清を採取し、実施例１２．２のＥＬＩＳＡ を用いて、血清中のヒトＩｇＧ を定量した。血清中のヒトＩｇＧ の上昇によりＫＰＭＭ２ 細胞の骨髄生着を確認した。これらマウスに対し、ＫＰＭＭ２ 細胞移植後１０日目に上記１−１ で調製した抗体をそれぞれ １００μｌ 静脈内投与した。さらに、移植後１０日目から１日１回、５日間 ０．２％ ＣＭＣ溶液 ２００μｌ を経口投与した。一方、メルファラン投与群については、上記１−２ で調製したメルファラン溶液をＫＰＭＭ２ 細胞移植後１０日目から１日１回、５日間体重１０ｇ あたり １００μｌ（メルファランとして１ｍｇ／ｋｇ） を経口投与した。
【０２２２】
２−３． 再構成ヒト抗ＨＭ１．２４抗体のヒト骨髄腫移植マウスに対する抗腫瘍効果の評価
再構成ヒト抗ＨＭ１．２４抗体の抗腫瘍効果については、マウスの血清ヒトＩｇＧ 量の変化、および生存期間で評価した。
マウスの血清ヒトＩｇＧ 量の変化については、ＫＰＭＭ２ 細胞移植３５日目に血清を採取し、実施例１２．２のＥＬＩＳＡ を用いてヒトＩｇＧ を定量した。その結果、図３９に示すようにコントロールヒトＩｇＧ１投与群では、ＫＰＭＭ２ 細胞移植３５日目の血清ヒトＩｇＧ 量は移植９日目（抗体投与前日）と比較して１０００倍程度増加し、マウス中でＫＰＭＭ２ 細胞が増殖しているものと思われた。
【０２２３】
また、既存薬であるメルファランを投与した群でも、コントロールヒトＩｇＧ１投与群ほどではないものの、血清ヒトＩｇＧ 量は薬剤投与前より増加しており、メルファランの投与ではマウス中のＫＰＭＭ２ 細胞の増殖を完全には抑制できないと思われた。一方、再構成ヒト抗ＨＭ１．２４抗体投与群ではいずれの投与量でも、移植９日目より血清ヒトＩｇＧ 量が減少しており、再構成ヒト抗ＨＭ１．２４抗体がＫＰＭＭ２ 細胞の増殖を抑制していることが示された。
【０２２４】
一方、生存期間についても図４０に示す通り、再構成ヒト抗ＨＭ１．２４抗体投与群ではコントロールヒトＩｇＧ１投与群、あるいはメルファラン投与群と比較して、生存期間の延長が認められた。以上より、再構成ヒト抗ＨＭ１．２４抗体投与がヒト骨髄腫移植マウスに対して抗腫瘍効果を有すること、さらに本抗体の抗腫瘍効果は既存薬剤のメルファランよりも強いことが示された。
以上より、ヒト由来のエフェクター細胞を用いた場合、マウス抗ＨＭ１．２４抗体は殆どヒト骨髄腫細胞に対して、細胞障害活性を示さなかったのに比して、再構成ヒト抗ＨＭ１．２４抗体およびキメラ抗ＨＭ１．２４抗体は強い細胞障害活性を示した。この事実は抗体をヒト型化することの重要性を示し、再構成ヒト抗ＨＭ１．２４抗体のヒトでの有用性を期待させる。
【０２２５】
ヒト骨髄腫移植ＳＣＩＤマウスにおいて、再構成ヒト抗ＨＭ１．２４抗体が非常に強い抗腫瘍効果を示したが、ヒトにおいては当然エフェクターはヒト由来であり、リンパ球も正常に存在していることから、再構成ヒト抗ＨＭ１．２４抗体のさらに強い抗腫瘍効果が期待できる。
骨髄腫モデルにおいて、再構成ヒト抗ＨＭ１．２４抗体は既存の骨髄腫治療薬に比べ強い抗腫瘍効果を示したことより、再構成ヒト抗ＨＭ１．２４抗体が画期的な骨髄腫治療薬になることが期待される。
【０２２６】
参考例１． マウス抗 ＨＭ１．２４ モノクローナル抗体産生ハイブリドーマの調製
Ｇｏｔｏ， Ｔ． ｅｔ ａｌ．， Ｂｌｏｏｄ （１９９４） ８４， １９９２−１９３０ に記載の方法にて、マウス抗ＨＭ１．２４モノクローナル抗体産生ハイブリドーマを調製した。
ヒト多発性骨髄腫患者の骨髄に由来するエプスタインーバーウィルスー核抗原（ＥＢＮＡ）ー陰性形質細胞株ＫＰＣ−３２（１ｘ１０^７ 個）（Ｇｏｔｏ， Ｔ． ｅｔ ａｌ．， Ｊｐｎ． Ｊ． Ｃｌｉｎ． Ｈｅｍａｔｏｌ． （１１９９１） ３２， １４００）をＢＡＬＢ／Ｃマウス（チャールスリバー製）の腹腔内に６ 週間おきに２ 回注射した。
【０２２７】
このマウスを屠殺する３ 日前にマウスの抗体産生価をさらに上昇させるために、１．５ ｘ １０^６ 個のＫＰＣ−３２細胞をマウスの脾臓内に注射した（Ｇｏｔｏ， Ｔ． ｅｔ ａｌ．， Ｔｏｋｕｓｈｉｍａ Ｊ． Ｅｘｐ． Ｍｅｄ． （１９９０） ３７， ８９ ）。マウスを屠殺した後に脾臓を摘出し、Ｇｒｏｔｈ， ｄｅ Ｓｔ． ＆ Ｓｃｈｒｅｉｄｅｇｇｅｒの方法（Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ （１９８１） ４１， ３４６５ ）に従い摘出した脾臓細胞とミエローマ細胞ＳＰ２／０ を細胞融合に付した。
【０２２８】
ＫＰＣ−３２細胞をコートしたプレートを使用するＥＬＩＳＡ （Ｐｏｓｎｅｒ， Ｍ． Ｒ． ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ． Ｍｅｔｈｏｄｓ （１９８２） ４８， ２３ ）によりハイブリドーマ培養上清中の抗体のスクリーニングを行った。５ ｘ １０^４ 個のＫＰＣ−３２細胞を５０ ｍｌ のＰＢＳ に懸濁し、９６ウエルプレート（Ｕ 底型、Ｃｏｒｎｉｎｇ 、Ｉｗａｋｉ 製）に分注した。１％ウシ血清アルブミン（ＢＳＡ ）を含むＰＢＳ でブロックした後、ハイブリドーマ培養上清を加え４ ℃にて２ 時間インキュベートした。次いで、４ ℃にて１ 時間ペルオキシダーゼ標識抗マウスＩｇＧ ヤギ抗体（Ｚｙｍｅｄ 製）を反応させ、一度洗浄して、室温にて３０分間ｏ−フェニレンジアミン基質溶液（Ｓｕｍｉｔｏｍｏ Ｂａｋｅｌｉｔｅ 製）を反応させた。
【０２２９】
２Ｎ硫酸で反応を停止させ、ＥＬＩＳＡ ｒｅａｄｅｒ（Ｂｉｏ−Ｒａｄ 製）で４９２ｎｍ における吸光度を測定した。ヒト免疫グロブリンに対する抗体を産生するハイブリドーマを除去するために、陽性ハイブリドーマ培養上清をヒト血清にあらかじめ吸着させ、他の細胞下部に対する反応性をＥＬＩＳＡ にてスクリーニングした。陽性のハイブリドーマを選択し、種々の細胞株およびヒトの標本に対する反応性をフローサイトメトリーで調べた。最後に選択されたハイブリドーマクローンを二度クローン化し、これをプリスタン処理したＢＡＬＢ／Ｃマウスの腹腔に注射して、腹水を取得した。
【０２３０】
モノクローナル抗体は、硫酸アンモニウムによる沈澱とプロテインＡ アフィニティクロマトグラフィーキット（Ａｍｐｕｒｅ ＰＡ 、Ａｍｅｒｓｈａｍ製）によりマウス腹水より精製した。精製抗体は、Ｑｕｉｃｋ Ｔａｇ ＦＩＴＣ結合キット（ベーリンガーマンハイム製）を使用することによりフルオロセイニチオシアネート（ＦＩＴＣ）と結合させた。
その結果、３０のハイブリドーマクローンが産生するモノクローナル抗体がＫＰＣ−３２およびＲＰＭＩ ８２２６ 細胞と反応した。クローニングの後、これらのハイブリドーマの培養上清を他の細胞株と末梢血由来単核球との反応性を調べた。
【０２３１】
このうち、３ つのクローンが形質細胞に特異的に反応するモノクローナル抗体であった。これらの３ つのクローンのうち、最もフローサイトメトリー分析に有用であり、かつＲＰＭＩ ８２２６ 細胞に対する補体依存性細胞障害活性を有するハイブリドーマクローンを選択し、ＨＭ１．２４と名付けた。このハイブリドーマが産生するモノクローナル抗体のサブクラスを、サブクラス特異的抗マウスウサギ抗体（Ｚｙｍｅｄ 製）を用いたＥＬＩＳＡ にて決定した。抗ＨＭ１．２４抗体は、ＩｇＧ２ａ κのサブクラスを有していた。抗ＨＭ１．２４抗体を産生するハイブリドーマＨＭ１．２４は、工業技術院生命工学工業研究所（茨城県つくば市東１丁目１番３号）に、平成７年９月１４日にＦＥＲＭ ＢＰ−５２３３としてブタペスト条約に基づき国際寄託された。
【０２３２】
参考例２． ＨＭ１．２４ 抗原ポリペプチドをコードする ｃＤＮＡ のクローニング
１． ｃＤＮＡライブラリーの作製
１）全ＲＮＡ の調製
マウスモノクローナル抗体ＨＭ１．２４が特異的に認識する抗原ポリペプチドであるＨＭ１．２４抗原をコードするｃＤＮＡを以下のように単離した。
ヒト多発性骨髄腫細胞株ＫＰＭＭ２ から、全ＲＮＡ をＣｈｉｒｇｗｉｎら（Ｂｉｏｃｈｃｍｉｓｔｒｙ， １８， ５２９４ （１９７９））の方法に従って調製した。すなわち、２．２ ｘ １０^８ 個のＫＰＭＭ２ を２０ｍｌの４Ｍグアニジンチオシアネート（ナカライテスク製）中で完全にホモジナイズさせた。
【０２３３】
ホモジネートを遠心管中の５．３Ｍ塩化セシウム溶液層状に重層し、次にこれをＢｅｃｋｍａｎ ＳＷ４０ローター中で３１，０００ｒｐｍ にて２０℃で２４時間遠心分離することによりＲＮＡ を沈殿させた。ＲＮＡ 沈殿物を７０％エタノールにより洗浄し、そして１ｍＭ ＥＤＴＡ及び ０．５％ ＳＤＳを含有する１０ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ ７．４） ３００μｌ 中に溶解し、それにＰｒｏｎａｓｅ（Ｂｏｅｈｒｉｎｇｅｒ製）を０．５ｍｇ／ｍｌとなるように添加した後、３７℃にて３０分間インキュベートした。混合物をフェノール及びクロロホルムで抽出し、ＲＮＡ をエタノールで沈殿させた。次に、ＲＮＡ 沈殿物を１ｍＭ ＥＤＴＡを含有する１０ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ ７．４） ２００μｌ に溶解した。
【０２３４】
２）ｐｏｌｙ（Ａ） ＋ＲＮＡ の調製
前記のようにして調製した全ＲＮＡ の約 ５００μｇ を材料としてＦａｓｔ Ｔｒａｃｋ ２．０ｍ ＲＮＡ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（Ｉｎｖｉｔｒｏｇｅｎ製）を用いてキット添付の処方に従ってｐｏｌｙ（Ａ） ＋ＲＮＡ を精製した。
３）ｃＤＮＡライブラリーの構築
上記ｐｏｌｙ（Ａ） ＋ＲＮＡ １０μｇ を材料としてｃＤＮＡ合成キットＴｉｍｅＳａｖｅｒ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ（Ｐｈａｒｍａｃｉａ製）を用いてキット添付の処方に従って二本鎖ｃＤＮＡを合成し、更にＤｉｒｅｃｔｉｏｎａｌ Ｃｌｏｎｉｎｇ Ｔｏｏｌｂｏｘ （Ｐｈａｒｍａｃｉａ製）を用いてキット付属のＥｃｏＲＩ アダプターをキット添付の処方に従って連結した。ＥｃｏＲＩ アダプターのカイネーション及び制限酵素ＮｏｔＩ処理はキット添付の処方に従って行った。更に、約５００ｂｐ 以上の大きさのアダプター付加二本鎖ｃＤＮＡを １．５％低融点アガロースゲル（Ｓｉｇｍａ製）を用いて分離、精製し、アダプター付加二本鎖ｃＤＮＡ約４０μｌ を得た。
【０２３５】
このようにして作製したアダプター付加二本鎖ｃＤＮＡを、あらかじめ制限酵素ＥｃｏＲＩ 、ＮｏｔＩ及びアルカリフォスファターゼ（宝酒造製）処理したｐＣＯＳ１ ベクター（特願平８−２５５１９６）とＴ４ ＤＮＡリガーゼ（ＧＩＢＣＯ−ＢＲＬ製）を用いて連結し、ｃＤＮＡライブラリーを構築した。構築したｃＤＮＡライブラリーは、大腸菌細胞株ＤＨ５ α（ＧＩＢＣＯ−ＢＲＬ製）に形質導入され、全体のサイズは約２．５ ｘ １０^６ 個の独立したクローンであると推定された。
【０２３６】
２． 直接発現法によるクローニング
１）ＣＯＳ−７ 細胞へのトランスフェクション
上記の形質導入した大腸菌約５ ｘ １０^５ クローンを５０μｇ／ｍｌのアンピシリンを含む２−ＹＴ培地（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｎｕａｌ． Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ （１９８９）） にて培養することによりｃＤＮＡの増幅を行い、アルカリ法（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｎｕａｌ． Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ （１９８９）） により大腸菌からプラスミドＤＮＡ を回収した。得られたプラスミドＤＮＡ はＧｅｎｅ Ｐｕｌｓｅｒ 装置（Ｂｉｏ−Ｒａｄ製）を用いてエレクトロポレーション法によりＣＯＳ−７ 細胞にトランスフェクションした。
【０２３７】
すなわち、精製したプラスミドＤＮＡ １０μｇ を１ ｘ １０^７ 細胞／ｍｌ でＰＢＳ 中に懸濁したＣＯＳ−７ 細胞液０．８ｍｌ に加え、１５００Ｖ 、２５μＦＤの容量にてパルスを与えた。室温にて１０分間の回復期間の後、エレクトロポレーション処理された細胞は、１０％牛胎児血清（ＧＩＢＣＯ−ＢＲＬ製）を含むＤＭＥＭ培養液（ＧＩＢＣＯ−ＢＲＬ製）にて、３７℃、５％ ＣＯ_２の条件下で３日間培養した。
【０２３８】
２）パンニングデイッシュの調製
マウス抗ＨＭ１．２４抗体をコーティングしたパンニングデイッシュを、Ｂ．Ｓｅｅｄら（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ． ＵＳＡ， ８４， ３３６５−３３６９ （１９８７）） の方法に従って調製した。すなわち、マウス抗ＨＭ１．２４抗体を１０μｇ／ｍｌになるように５０ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ ９．５）に加えた。このようにして調製した抗体溶液３ｍｌ を直径６０ｍｍの細胞培養皿に加え、室温にて２時間インキュベートした。０．１５Ｍ ＮａＣｌ溶液にて３回洗浄した後、５％牛胎児血清、１ｍＭ ＥＤＴＡ、０．０２％ ＮａＮ_３ を含むＰＢＳ を加え、ブロッキングした後、下記クローニングに用いた。
【０２３９】
３）ｃＤＮＡライブラリーのクローニング
前述のようにトランスフェクトしたＣＯＳ−７ 細胞は、５ｍＭ ＥＤＴＡを含むＰＢＳ にて剥がし、５％牛胎児血清を含むＰＢＳ で一回洗浄した後、約１ ｘ １０^６ 細胞／ｍｌ となるように５％牛胎児血清及び０．０２％ ＮａＮ_３ を含むＰＢＳ に懸濁し、上記のように調製したパンニングデイシュに加え、室温にて約２時間インキュベートした。５％牛胎児血清及び０．０２％ ＮａＮ_３ を含むＰＢＳ で３度緩やかに洗浄した後、 ０．６％ ＳＤＳ及び１０ｍＭ ＥＤＴＡ を含む溶液を用いてパンニングデイシュに結合した細胞からプラスミドＤＮＡ の回収を行った。
【０２４０】
回収したプラスミドＤＮＡ を再び大腸菌ＤＨ５ αに形質導入し、前述のようにプラスミドＤＮＡ を増幅後、アルカリ法にて回収した。回収したプラスミドＤＮＡ をＣＯＳ−７ 細胞にエレクトロポレーション法によりトランスフェクトして前述と同様に結合した細胞よりプラスミドＤＮＡ の回収を行った。同様の操作を更に１回繰り返し、回収したプラスミドＤＮＡ を制限酵素ＥｃｏＲＩ およびＮｏｔＩで消化した結果、約０．９ｋｂｐのサイズのインサートの濃縮が確認された。さらに、回収したプラスミドＤＮＡ の一部を形質導入した大腸菌を５０μｇ／ｍｌのアンピシリンを含む２−ＹＴアガープレートに接種し、一晩培養後、単一のコロニーよりプラスミドＤＮＡ を回収した。制限酵素ＥｃｏＲＩ およびＮｏｔＩにて消化し、インサートのサイズが約０．９ｋｂｐを示すクローンｐ３．１９ を得た。
【０２４１】
本クローンについては、ＰＲＩＳＭ， Ｄｙｅ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇキット（Ｐｅｒｋｉｎ Ｅｌｍｅｒ製）を用いて、キット添付の処方に従い反応を行い、ＡＢＩ ３７３Ａ ＤＮＡ Ｓｅｑｕｅｎｃｅｒ （Ｐｅｒｋｉｎ Ｅｌｍｅｒ製）にて塩基配列の決定を行った。このアミノ酸配列および塩基配列を配列番号１０３ に示す。
配列番号：１０３ に示すアミノ酸配列を有するポリペプチドをコードするｃＤＮＡはｐＵＣ１９ ベクターのＸｂａＩ切断部位の間に挿入されて、プラスミドｐＲＳ３８−ｐＵＣ１９ として調製されている。このプラスミドｐＲＳ３８−ｐＵＣ１９ を含む大腸菌（Ｅ． ｃｏｌｉ） は平成５年（１９９３年）１０月５日付で工業技術院生命工学工業技術研究所（茨城県つくば市東１丁目１番３号）にＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α （ｐＲＳ３８−ｐＵＣ１９）として、受託番号ＦＥＲＭ ＢＰ−４４３４としてブダペスト条約に基づき国際寄託されている（特開平７−１９６６９４参照）。
【０２４２】
【発明の効果】
キメラ抗ＨＭ１．２４抗体はマウス抗ＨＭ１．２４抗体の可変領域とヒト抗体定常領域からなり、再構成抗ＨＭ１．２４抗体はマウス抗ＨＭ１．２４抗体の相捕性決定領域とヒト抗体フレームワーク領域およびヒト抗体定常領域からなることから、ヒトにおける抗原性が低く、それ故に医薬組成物、特に骨髄腫治療剤として期待される。
【０２４３】
【配列表】

【０２４４】

【０２４５】

【０２４６】

【０２４７】

【０２４８】

【０２４９】

【０２５０】

【０２５１】

【０２５２】

【０２５３】

【０２５４】

【０２５５】

【０２５６】

【０２５７】

【０２５８】

【０２５９】

【０２６０】

【０２６１】

【０２６２】

【０２６３】

【０２６４】

【０２６５】

【０２６６】

【０２６７】

【０２６８】

【０２６９】

【０２７０】

【０２７１】

【０２７２】

【０２７３】

【０２７４】

【０２７５】

【０２７６】

【０２７７】

【０２７８】

【０２７９】

【０２８０】

【０２８１】

【０２８２】

【０２８３】

【０２８４】

【０２８５】

【０２８６】

【０２８７】

【０２８８】

【０２８９】

【０２９０】

【０２９１】

【０２９２】

【０２９３】

【０２９４】

【０２９５】

【０２９６】

【０２９７】

【０２９８】

【０２９９】

【０３００】

【０３０１】

【０３０２】

【０３０３】

【０３０４】

【０３０５】

【０３０６】

【０３０７】

【０３０８】

【０３０９】

【０３１０】

【０３１１】

【０３１２】

【０３１３】

【０３１４】

【０３１５】

【０３１６】

【０３１７】

【０３１８】

【０３１９】

【０３２０】

【０３２１】

【０３２２】

【０３２３】

【０３２４】

【０３２５】

【０３２６】

【０３２７】

【０３２８】

【０３２９】

【０３３０】

【０３３１】

【０３３２】

【０３３３】

【０３３４】

【０３３５】

【０３３６】

【０３３７】

【０３３８】

【０３３９】

【０３４０】

【０３４１】

【０３４２】

【０３４３】

【０３４４】

【０３４５】

【図面の簡単な説明】
【図１】図１は、ヒト骨髄腫細胞株ＫＰＭＭ２ を用いたＦＣＭ 解析において、キメラ抗ＨＭ１．２４抗体の蛍光強度がマウス抗ＨＭ１．２４抗体の蛍光強度と同様に、コントロール抗体に比べシフトしていることを示すグラフである。
【図２】図２は、ＷＩＳＨ細胞を用いたＣｅｌｌ−ＥＬＩＳＡにおいて、キメラ抗ＨＭ１．２４抗体はマウス抗ＨＭ１．２４抗体と同様に、ビオチン化マウス抗ＨＭ１．２４抗体のＷＩＳＨ細胞への結合を濃度依存的阻害していることを示すグラフである。
【図３】図３は、コントロールヒトＩｇＧ１、あるいはマウス抗ＨＭ１．２４抗体は、ＲＰＭＩ８２２６細胞に対する細胞障害活性を持たないのに対し、キメラ抗ＨＭ１．２４抗体はＥ／Ｔ 比の上昇に伴い、ＲＰＭＩ ８２２６ 細胞に対する細胞障害活性が上昇していることを示すグラフである。
【図４】図４は、ＰＣＲ 法によるＣＤＲ グラフティングにより再構成ヒト抗ＨＭ１．２４抗体Ｌ 鎖を、作製する方法を示す模式図である。
【図５】図５は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖の作製において、ＰＣＲ 法によりＲＶＨ１、ＲＶＨ２、ＲＶＨ３及びＲＶＨ４のオリゴヌクレオチドをアセンブリーする方法を示す模式図である。
【図６】図６は、ＰＣＲ 法によりヒト・マウスハイブリッド抗ＨＭ１．２４抗体Ｈ 鎖Ｖ 領域を作製する方法を示す模式図である。
【図７】図７は、ＰＣＲ 法によりマウス・ヒトハイブリッド抗ＨＭ１．２４抗体Ｈ 鎖Ｖ 領域を作製する方法を示す模式図である。
【図８】図８は、再構成ヒト抗ＨＭ１．２４抗体Ｌ 鎖バージョンａはキメラ抗ＨＭ１．２４抗体と同程度の抗原結合活性を有することを示すグラフである。なお、−１， −２はロットの違いを示す。
【図９】図９は、Ｌ 鎖バージョンａとＨ 鎖バージョンａ、ｂ、ｆ又はｈを組み合わせた再構成ヒト抗ＨＭ１．２４抗体およびキメラ抗体ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図１０】図１０は、Ｌ 鎖バージョンｂとＨ 鎖バージョンａ、ｂ、ｆ又はｈを組み合わせた再構成ヒト抗ＨＭ１．２４抗体およびキメラ抗体ＨＭ１．２４抗体の結合活性を示すグラフである。
【図１１】図１１は、Ｌ 鎖バージョンａとＨ 鎖バージョンａ、ｂ、ｆ又はｈを組み合わせた再構成ヒト抗ＨＭ１．２４抗体およびキメラ抗体ＨＭ１．２４抗体の結合阻害活性を示すグラフである。
【図１２】図１２は、Ｌ 鎖バージョンｂとＨ 鎖バージョンａ、ｂ、ｆ又はｈを組み合わせた再構成ヒト抗ＨＭ１．２４抗体およびキメラ抗体ＨＭ１．２４抗体の結合阻害活性を示すグラフである。
【図１３】図１３は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｂ、ｃ、ｄ及びキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図１４】図１４は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｅ及びキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。なお、−１， −２はロットの違いを示す。
【図１５】図１５は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｃ、ｐ、ｒ及びキメラ抗ＨＭ１．２４抗体の結合阻害活性を示すグラフである。
【図１６】図１６は、ヒト・マウスハイブリッド抗ＨＭ１．２４抗体、マウス・ヒトハイブリッド抗ＨＭ１．２４抗体およびキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図１７】図１７は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｂ、ｃ、ｆ及びキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図１８】図１８は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｇ及びキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図１９】図１９は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｇ及びキメラ抗ＨＭ１．２４抗体の結合阻害活性を示すグラフである。
【図２０】図２０は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンｈ、ｉ及びキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図２１】図２１は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンｆ、ｈ、ｊ及びにキメラ抗ＨＭ１．２４抗体の抗原結合活性を示す。
【図２２】図２２は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンｈ、ｉ及びキメラ抗ＨＭ１．２４抗体の結合阻害活性を示すグラフである。
【図２３】図２３は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンｆ、ｈ、ｊ及びキメラ抗ＨＭ１．２４抗体の結合阻害活性を示すグラフである。
【図２４】図２４は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンｈ、ｋ、ｌ、ｍ、ｎ、ｏ及びキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図２５】図２５は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｈ、ｐ、ｑ及びキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図２６】図２６は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンｈ、ｋ、ｌ、ｍ、ｎ、ｏ及びキメラ抗ＨＭ１．２４抗体のＷＩＳＨ細胞への結合阻害活性を示すグラフである。
【図２７】図２７は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｈ、ｐ、ｑ及びキメラ抗ＨＭ１．２４抗体の結合阻害活性を示すグラフである。
【図２８】図２８は、再構成ヒト抗ＨＭ１．２４抗体Ｈ 鎖バージョンａ、ｃ、ｐ、ｒ及びキメラ抗ＨＭ１．２４抗体の抗原結合活性を示すグラフである。
【図２９】図２９は、再構成ヒト抗ＨＭ１．２４抗体バージョンｓが、再構成ヒト抗ＨＭ１．２４抗体バージョンｒと同程度の抗原結合活性を有することを示すグラフである。
【図３０】図３０は、再構成ヒト抗ＨＭ１．２４抗体バージョンｓが、再構成ヒト抗ＨＭ１．２４抗体バージョンｒと同程度の結合阻害活性を有することを示すグラフである。
【図３１】図３１は、精製再構成ヒト抗ＨＭ１．２４抗体は、キメラ抗ＨＭ１．２４抗体と同程度の抗原結合活性を有することを示すグラフである。
【図３２】図３２は、精製再構成ヒト抗ＨＭ１．２４抗体は、キメラ抗ＨＭ１．２４抗体と同程度の結合阻害活性を有することを示すグラフである。
【図３３】図３３は、ヒト骨髄腫移植マウスにおいて、キメラ抗ＨＭ１．２４抗体の投与により、コントロールヒトＩｇＧ１投与に比べて、生存期間が延長していることを示すグラフである。
【図３４】図３４は、エフェクター細胞としてヒト健常人末梢血由来細胞を用いた場合、コントロールヒトＩｇＧ１はＫＰＭＭ２ 細胞に対して細胞障害活性を示さず、また、マウス抗ＨＭ１．２４抗体も細胞障害活性が弱いのに対し、再構成ヒト抗ＨＭ１．２４抗体は、ＫＰＭＭ２ 細胞に対して強い細胞障害活性を示していることを示す表すグラフである。
【図３５】図３５は、エフェクター細胞としてヒト健常人末梢血由来細胞を用いた場合、コントロールヒトＩｇＧ１はＡＲＨ−７７細胞に対して細胞障害活性を示さず、また、マウス抗ＨＭ１．２４抗体も細胞障害活性が弱いのに対し、再構成ヒト抗ＨＭ１．２４抗体は、ＡＲＨ−７７細胞に対して強い細胞障害活性を示していることを示す表すグラフである。
【図３６】図３６は、エフェクター細胞としてＳＣＩＤマウス骨髄由来細胞を用いた場合、コントロールヒトＩｇＧ１はＫＰＭＭ２ 細胞に対する細胞障害活性を持たないのに対し、再構成ヒト抗ＨＭ１．２４抗体は抗体温度の上昇に伴い、ＫＰＭＭ２ 細胞に対する細胞障害活性が上昇していることを表すグラフである。
【図３７】図３７は、ヒト骨髄腫移植マウスにおいて、コントロールヒトＩｇＧ１では投与前に比べ投与後も血清ヒトＩｇＧ 量が増加しているのに対し、再構成ヒト抗ＨＭ１．２４抗体では、抗体投与により、血清ヒトＩｇＧ 量の増加を抑制していることを示すグラフである。
【図３８】図３８は、ヒト骨髄腫移植マウスにおいて、再構成ヒト抗ＨＭ１．２４抗体の投与により、コントロールヒトＩｇＧ１投与に比べ、生存期間が延長していることを示すグラフである。
【図３９】図３９は、ヒト骨髄腫移植マウスにおいて、メルファラン、およびコントロールヒトＩｇＧ１では投与前に比べ投与後も血清ヒトＩｇＧ 量が増加しているのに対し、再構成ヒト抗ＨＭ１．２４抗体では、抗体投与により、血清ヒトＩｇＧ 量の増加を抑制していることを示すグラフである。
【図４０】図４０は、ヒト骨髄腫移植マウスにおいて、再構成ヒト抗ＨＭ１．２４抗体の投与により、メルファラン、あるいはコントロールヒトＩｇＧ１投与に比べ、生存期間が延長していることを示すグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a reshaped human anti-HM1.24 antibody and a chimeric anti-HM1.24 antibody, a gene encoding them, a method for producing the antibody, and use of the antibody. The reshaped human antibody and chimeric antibody of the present invention are particularly useful as therapeutic agents for myeloma and the like.
[0002]
[Prior art]
Human B cells eventually mature into antibody-producing plasma cells through several stages that are classified by the surface antigens that are expressed. In the terminal differentiation stage of B cells, B cell-related antigens such as cell surface immunoglobulin, HLA-DR, CD20, Fc receptor and complement C3 receptor are lost while acquiring the ability to produce cytoplasmic immunoglobulin (Ling, N). R. et al., Leucocyte Typing III (1986) p320, Oxford, UK, Oxford).
[0003]
Heretofore, anti-PCA-1 (Anderson, KC et al., J. Immunol. (1983) 130, 1132), which recognizes an antigen on the plasma membrane of plasma cells, and anti-PC-1 (Anderson, KC) Et al., J. Immunol. (1983) 132, 3172), and monoclonal antibodies such as anti-MM4 (Tong, AW et al., Blood (1987) 69, 238) have been reported. Anti-CD38 monoclonal antibodies are still used for the detection of cells and myeloma cells (Epstein, J. et al., N. Engl. J. Med. (1990) 322, 664, Terstappen, L.W. M. M. et al., Blood (1. 90) 76, 1739, Leo, R. et al., Ann. Hematol. (1992) 64, 132, Shimakizaki, C. et al., Am. J. Hematol. (1992) 39, 159, Hata, H., et al. et al., Blood (1993) 81, 3357, Harada, H. et al., Blood (1993) 81, 2658, Billadeau, D. et al., J. Exp. Med. (1993) 178, 1023).
[0004]
However, the anti-CD38 monoclonal antibody is an antigen associated with T cell activation rather than an antigen associated with B cell differentiation, and is expressed on various cells other than B cells. Furthermore, CD38 is strongly expressed on hematopoietic progenitor cells, although it is not expressed on some lymphoplasmacytoids. For these reasons, anti-CD38 monoclonal antibodies are not considered suitable for studies on human B cell differentiation and maturation and for the treatment of plasma cell disorders.
[0005]
Goto, T .; Have reported a mouse monoclonal antibody, HM1.24, obtained by immunizing human plasma cells and specifically recognizing an antigen with a molecular weight of 29-33 kDa, which is specifically expressed in the B cell lineage (Blood (1994) 84, 1922-1930). The antigen recognized by the monoclonal antibody HM1.24 is considered to be an antigen associated with terminal differentiation of B cells (Goto, T. et al., Jpn. J. Clin. Immun. (1992) 16, 688-). 691), and that when the monoclonal antibody HM1.24 was administered to mice transplanted with plasmacytoma, this antibody specifically accumulated in the tumor (Shuji Ozaki et al., Program of the 19th Annual Meeting of the Myeloma Society of Japan, General Abstracts) From 3), it has been suggested that the monoclonal antibody HM1.24 can be applied to diagnosis of tumor localization by labeling with a radioisotope, and to missile therapy such as radioimmunotherapy.
[0006]
The above-mentioned Blood also states that the monoclonal antibody HM1.24 has complement-dependent cytotoxicity against the human myeloma cell line RPMI8226 in vitro.
Myeloma is a neoplastic disease characterized by accumulation of monoclonal plasma cells (myeloma cells) in the bone marrow. Myeloma is a disease in which terminally differentiated B cells that produce and secrete immunoglobulins, that is, plasma cells mainly increase in the bone marrow in a monoclonal manner. Monoclonal immunoglobulins or their constituent light chains such as light chains and heavy chains are detected in serum. (Masaaki Kosaka et al., Japanese Clinical Laboratory (1995) 53, 91-99).
Chemotherapeutic agents have been used to treat myeloma, but no effective therapeutic agent has been found to lead to remission of myeloma and prolong the survival of myeloma patients. The emergence of drugs that have therapeutic effects on tumors has been awaited.
[0007]
Mouse monoclonal antibodies are highly immunogenic (sometimes referred to as "antigenic") in humans, which limits the therapeutic value of mouse monoclonal antibodies in humans. For example, when a mouse antibody is administered to humans, it can be metabolized as a foreign substance, so that the half-life of mouse antibodies in humans is relatively short, and the expected effects cannot be sufficiently exerted. In addition, human anti-mouse antibodies (HAMA) raised against administered mouse antibodies elicit inconvenient and dangerous immune responses to patients, such as serum sickness or other allergic reactions. Therefore, the Mau monoclonal antibody cannot be administered to humans frequently.
[0008]
In order to solve these problems, methods have been developed to reduce the immunogenicity of non-human-derived antibodies, such as mouse-derived monoclonal antibodies. One method is to prepare a chimeric antibody in which the variable region (V region) of the antibody is derived from the original mouse monoclonal antibody and the constant region (C region) is derived from an appropriate human antibody.
Since the resulting chimeric antibody contains the complete variable region of the original mouse antibody, it can be expected to bind to the antigen with the same specificity as the original mouse antibody. Furthermore, the ratio of amino acid sequences derived from non-human is substantially reduced in the chimeric antibody, and therefore, it is expected that the immunogenicity is lower than that of the original mouse antibody. Chimeric antibodies bind antigen similarly to the original mouse monoclonal antibody and are less immunogenic, but may still produce an immune response to the mouse variable regions (LoBuglio, AF et al., Proc. Natl. Acad.Sci.USA, 86, 4220-4224, 1989.).
[0009]
The second method for reducing the immunogenicity of a mouse antibody is more complex, but one that further reduces the potential immunogenicity of the mouse antibody. In this method, only a complementarity determining region (CDR) is transplanted from a mouse antibody variable region into a human antibody variable region to prepare a "reshaped" human antibody variable region.
[0010]
However, if necessary, in order to make the CDR structure of the reshaped human antibody variable region more similar to that of the original mouse antibody, the amino acid sequence of a part of the framework region (FR) supporting the CDR may be replaced with a mouse. In some cases, a variable region of an antibody is transplanted to a human antibody variable region. Next, these humanized reshaped human antibody variable regions are ligated to human antibody constant regions. The portion of the finally reconstructed humanized antibody derived from the non-human amino acid sequence is only CDRs and only a part of FRs. CDRs are composed of hypervariable amino acid sequences, which do not show species-specific sequences. Thus, a humanized antibody carrying a mouse CDR should no longer have stronger immunogenicity than a native human antibody containing a human antibody CDR.
[0011]
Humanized antibodies are further described in Riechmann, L .; Et al., Nature, 332, 323-327, l988; Verhoey, M .; Et al., Science, 239, l534-l536, l988; Kettleborough, C. et al. A. Et al., Protein Engng. Maeda, H., 4,773-783, 199l; Gorman, S. et al., Human Antibodies and Hybridoma, 2, 124-134, 199l; D. Proc. Natl. Acad. Sci. USA, 88, 418- 185, 199l; Tempest, P .; R. Et al., Bio / Technology, 9, 266-271, 199l; S. Proc. Natl. Acad. Sci. USA, 88, 2869-2873, 199l; Carter, P .; Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992; S. J. et al. Immunol. Sato, K., 148, 114-114, 1992; Et al., Cancer Res. , 53, 851-856, 1993.
[0012]
Queen et al (International Patent Application Publication No. WO 90-07861) describes a method for preparing a humanized anti-IL-2 receptor antibody Anti-Tac antibody. However, it is difficult to completely humanize all antibodies even according to the method for preparing humanized antibodies described in WO90-07861. That is, WO90-07861 does not describe a general method for humanizing an antibody, but merely humanizes an anti-Tac antibody which is a specific antibody which is one of anti-IL-2 receptor antibodies. Only the method is described. Also, even according to the method of WO90-07861, it is difficult to prepare a humanized antibody having the same activity as the original mouse antibody completely.
[0013]
In general, the CDR / FR amino acid sequences of individual antibodies are different. Therefore, the determination of the amino acid residue to be substituted and the selection of the amino acid residue to be substituted for the amino acid residue required for constructing a humanized antibody differ depending on each antibody. Therefore, the method for producing a humanized antibody described in WO90-07861 cannot be applied to humanization of all antibodies.
Queen et al. , Proc. Natl. Acad. Sci. USA (1989) 86, 10029-1000033 describes the same contents as WO90-07861. This document describes that only about 1/3 of the activity of the original humanized mouse antibody was obtained by the method of WO90-07861. In other words, it shows that the method of WO90-07861 itself cannot produce a fully humanized antibody having the same activity as the original mouse antibody.
[0014]
Co et al. , Cancer Research (1996) 56, 1118-1125, was issued by the group of Queen et al., Supra. This document describes that a humanized antibody having the same activity as that of the original mouse antibody could not be constructed even according to the method for preparing a humanized antibody of WO90-07861. That is, the method of WO90-07861 itself cannot produce a fully humanized antibody having the same activity as that of the original mouse antibody, and the method of producing the humanized antibody of WO90-07861 requires all antibodies. Is not applicable to humanization of
[0015]
See Ohtomo et al. , Molecular Immunology (1995) 32, 407-416 describes the humanization of the mouse ONS-M21 antibody. This document shows that the amino acid residues suggested in the humanization of the Anti-Tac antibody described in WO90-07861 are not related to any activity, and that the method described in WO90-07861 cannot be applied.
Kettleborough et al. , Protein Eng. (1991) 4, 773-783 describes that some humanized antibodies were prepared from mouse antibodies by substituting amino acid residues. However, substitution of amino acid residues beyond those suggested by the humanization method of Anti-Tac antibody described in WO90-07861 was required.
These documents indicate that the method for preparing a humanized antibody described in WO90-07861 is a technique applicable only to humanization of the Anti-Tac antibody described therein, and that the technique Cannot achieve the same level of activity as the original mouse antibody.
[0016]
The original mouse antibody described in these documents has an amino acid sequence different from that of the Anti-Tac antibody described in WO90-07861. Therefore, the humanized antibody preparation method applicable to the Anti-Tac antibody could not be applied to other antibodies. Similarly, since the mouse anti-HM1.24 antibody of the present invention has an amino acid sequence different from that of the Anti-Tac antibody, the method for preparing a humanized Anti-Tac antibody cannot be applied. Furthermore, the humanized antibody of the present invention, which has been successfully constructed, has an amino acid sequence different from that of the humanized Anti-Tac antibody described in WO90-07861, and this is also an antibody having a different CDR-FR sequence. Shows that the exact same technique is not applicable for humanizing
[0017]
Therefore, even if a mouse antibody that is the source of humanization is known, it is only after trial and error experiments that the humanized antibody having any CDR / FR sequence exhibits the activity. WO90-07861 describes not only the CDR sequence, but also the FR sequence combined with the humanized antibody constructed according to the present invention and that a humanized antibody having an activity can be obtained by combination with the FR. It has not been. As noted above, humanized antibodies are expected to be useful for therapeutic purposes, but humanized anti-HM1.24 antibodies are not known or suggested. In addition, there is no uniform method that can be universally applied to any antibody in the method for producing a humanized antibody, and it shows sufficient binding activity, binding inhibitory activity and neutralizing activity for a specific antigen Various devices are required to prepare a humanized antibody (for example, Sato, K., et al., Cancer Res., 53, 851-856, 1993).
[0018]
[Problems to be solved by the invention]
The present invention provides a reshaped human antibody of the anti-HM1.24 antibody. The present invention also provides a human / mouse chimeric antibody useful in the process of producing the reshaped human antibody. The present invention further provides fragments of the reshaped human antibody. In addition, the present invention provides an expression system for producing chimeric antibodies, reshaped human antibodies and fragments thereof. The present invention further provides a method for producing a chimeric antibody of an anti-HM1.24 antibody and a fragment thereof, and a method for producing a reshaped human antibody of an anti-HM1.24 antibody and a fragment thereof.
[0019]
[Means for Solving the Problems]
More specifically, the present invention provides chimeric antibodies and reshaped human antibodies that specifically recognize a polypeptide having the amino acid sequence shown in SEQ ID NO: 103. The cDNA encoding the polypeptide has been inserted into the pUC19 vector between the XbaI sites and prepared as plasmid pRS38-pUC19. Escherichia coli (E. coli) containing this plasmid pRS38-pUC19 was sent to Escherichia coli on October 5, 1993 at the Institute of Biotechnology and Industrial Technology (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture) on October 5, 1993. It has been deposited internationally under the Budapest Treaty under the accession number FERM BP-4434 as DH5α (pRS38-pUC19) (see JP-A-7-196694).
[0020]
One embodiment of such a chimeric antibody or a reshaped human antibody includes a chimeric anti-HM 1.24 antibody or a reshaped human anti-HM 1.24 antibody. Hereinafter, the chimeric anti-HM1.24 antibody and the reshaped human anti-HM1.24 antibody will be described in detail as specific examples.
That is, the present invention also provides
A chimeric L chain comprising a human light (L) chain constant region (C region), and an L chain variable (V) region of an anti-HM 1.24 antibody, and a human heavy (H) chain C region, and anti-HM 1.24 A chimeric H chain comprising the V region of the H chain of the antibody is provided.
The present invention also provides
(1) a light chain comprising a human light chain C region and a light chain V region of an anti-HM1.24 antibody;
(2) an H chain comprising a human H chain C region and an H chain V region of an anti-HM1.24 antibody;
And a chimeric antibody comprising:
[0021]
The present invention further provides
(1) framework region (FR) of human L chain V region, and
(2) a reshaped human L chain V region of the anti-HM 1.24 antibody, comprising a CDR of the L chain V region of the anti-HM 1.24 antibody;
(1) FR of human H chain V region, and
(2) a reshaped human H chain V region of the anti-HM 1.24 antibody, comprising a CDR of the H chain V region of the anti-HM 1.24 antibody;
I will provide a.
[0022]
The present invention further provides
(1) human L chain C region, and
(2) a reshaped human L chain of an anti-HM 1.24 antibody comprising a human L chain FR and an L chain V region comprising the L chain CDR of the anti-HM 1.24 antibody;
(1) human H chain C region, and
(2) a reshaped human H chain of an anti-HM 1.24 antibody comprising a human H chain FR and an H chain V region comprising the H chain CDR of the anti-HM 1.24 antibody;
I will provide a.
[0023]
The present invention also provides
(A) (1) human L chain C region, and
(2) an L chain comprising a human L chain FR and an L chain V region comprising an L chain CDR of an anti-HM1.24 antibody; and
(B) (1) human H chain C region, and
(2) an H chain comprising a human H chain FR and an H chain V region comprising an H chain CDR of an anti-HM 1.24 antibody;
A reshaped human antibody of an anti-HM1.24 antibody, comprising:
I will provide a.
[0024]
The present invention also provides a DNA encoding the L chain V region of the anti-HM 1.24 antibody and a DNA encoding the H chain V region of the anti-HM 1.24 antibody.
The present invention further provides
(1) a human L chain C region;
(2) a DNA encoding a chimeric L chain, comprising a V region of an L chain of an anti-HM1.24 antibody;
(1) human H chain C region; and
(2) a DNA encoding the chimeric H chain, comprising the V region of the H chain of the anti-HM 1.24 antibody;
I will provide a.
[0025]
(1) FR of human L chain V region, and
(2) a DNA encoding the reshaped human L chain V region of the anti-HM 1.24 antibody, comprising a CDR of the L chain V region of the anti-HM 1.24 antibody;
(1) FR of human H chain V region, and
(2) a DNA encoding the reshaped human H chain V region of the anti-HM 1.24 antibody, comprising a CDR of the H chain V region of the anti-HM 1.24 antibody;
I will provide a.
[0026]
The present invention further provides
(1) human L chain C region;
(2) a DNA encoding a reshaped human L chain of an anti-HM 1.24 antibody comprising: a human L chain FR; and an L chain V region comprising the L chain CDR of the anti-HM 1.24 antibody;
(1) human H chain C region;
(2) a DNA encoding a reshaped human H chain of an anti-HM 1.24 antibody, comprising: a human H chain FR; and an H chain V region comprising the H chain CDR of the anti-HM 1.24 antibody;
I will provide a.
The present invention further provides a vector comprising any of the various DNAs described above.
The present invention further provides a host cell transformed with the above vector.
[0027]
The present invention also relates to a method for producing a chimeric antibody of an anti-HM1.24 antibody, wherein the expression vector comprises a DNA encoding the chimeric L chain and an expression vector comprising the DNA encoding the H chain. Culturing a co-transformed host cell and recovering the antibody of interest.
The present invention further relates to a method for producing a reshaped human antibody of an anti-HM1.24 antibody, comprising an expression vector comprising the DNA encoding the reshaped human L chain and a DNA encoding the structured human H chain. And culturing a host cell co-transformed with an expression vector comprising: recovering an antibody of interest.
[0028]
The present invention further provides a pharmaceutical composition, particularly a therapeutic agent for myeloma, comprising the above-mentioned chimeric antibody or reshaped human antibody.
The present invention further provides a pharmaceutical composition containing, as an active ingredient, a chimeric antibody that specifically recognizes a polypeptide having the amino acid sequence shown in SEQ ID NO: 103, and a polypeptide having the amino acid sequence shown in SEQ ID NO: 103. Provided is a pharmaceutical composition comprising, as an active ingredient, a reshaped human antibody that specifically recognizes. As the pharmaceutical composition, in particular, a therapeutic agent for myeloma is provided.
[0029]
BEST MODE FOR CARRYING OUT THE INVENTION
1. Construction of chimeric antibody
(1) Cloning of DNA encoding V region of mouse anti-HM1.24 monoclonal antibody
mRNA Preparation of
To perform cloning of the DNA encoding the V region of the mouse anti-HM1.24 monoclonal antibody, a method known from the recovered hybridomas, for example, guanidine-ultracentrifugation (Chirgwin, JM et al., Biochemistry, (1979), 18, 5294-5299), the total RNA is prepared by the AGPC method (Chomczynski, P et al. (1987), 162, 156-159) and the like, and Oligo (dT) -cellulose spun column attached to the mRNA Purification Kit (Pharmacia). The mRNA is prepared as described above. In addition, by using QuickPrep mRNA Purification Kit (manufactured by Pharmacia), mRNA can be prepared without performing an extraction operation of total RNA.
[0030]
Preparation and amplification of cDNA
the abovemRNA Preparation ofCDNAs in the V regions of the L chain and the H chain are synthesized from the mRNA obtained in the above using reverse transcriptase. The synthesis of the cDNA for the L chain V region is performed using the AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit. Amplification of the synthesized cDNA is carried out by using an appropriate primer that hybridizes with the leader sequence and C region of the antibody gene (for example, an MKV primer having the nucleotide sequence represented by SEQ ID NOS: 29 to 39 and an MKC having the nucleotide sequence represented by SEQ ID NO: 40) Primer).
[0031]
The synthesis and amplification of the cDNA of the H chain V region is carried out by the 5′-RACE method using 5′-Ampli FINDER RACE kit (CLONTECH) (Frohman, MA, et al., Proc. Natl. Acad. USA 85, 8998-). 9002, 1988, Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-2932, 1989) by PCR (polymerase chain reaction). An Ampli FINDER Anchor was ligated to the 5 'end of the cDNA synthesized above, and specifically hybridized to an Anchor primer (SEQ ID NO: 77) and a mouse H chain constant region (Cγ region) as primers for amplifying the H chain V region. A soybean primer (for example, an MHC2a primer having the base sequence represented by SEQ ID NO: 42) can be used.
[0032]
DNA purification and nucleotide sequence determination
The PCR product is subjected to agarose gel electrophoresis according to a known method to cut out a target DNA fragment, and then the DNA is recovered and purified, and ligated to a vector DNA.
The DNA is purified using a commercially available kit (for example, GENECLEAN II; BIO101). Known vector DNAs (eg, pUC19, Bluescript, etc.) can be used as the vector DNA for retaining the DNA fragments.
[0033]
The DNA and the vector DNA are ligated using a known ligation kit (Takara Shuzo) to obtain a recombinant vector. Next, the resulting recombinant vector is introduced into Escherichia coli JM109 or the like, and ampicillin-resistant colonies are selected, and vector DNA is prepared according to a known method (J. Sambrook, et al., "Molecular Cloning", Cold Spring Harbor). Laboratory Press, 1989). The base sequence of the target DNA is determined by a known method (for example, the dideoxy method) after digesting the vector DNA with a restriction enzyme (J. Sambrook, et al., “Molecular Cloning”, Cold Spring Harbor Laboratory Press, J. Am. 1989). In the present invention, an automatic base sequencer (DNA Sequencer 373A; ABI) can be used.
[0034]
Complementarity determining region
The H chain V region and the L chain V region form an antigen binding site, and their overall structures are similar to each other. That is, each of the four framework regions (FR) is connected by three hypervariable regions, namely, complementarity determining regions (CDRs). The amino acid sequence of the FR is relatively well conserved, while the amino acid sequence of the CDR region has extremely high variability (Kabat, EA, et al., "Sequence of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983).
[0035]
Most of the four FRs have a β-sheet structure, so that the three CDRs form a loop. CDRs may in some cases form part of a β-sheet structure. The three CDRs are held sterically very close to each other by the FR and form an antigen binding site with the three CDRs of the paired region.
Based on these facts, the amino acid sequence of the variable region of the mouse anti-HM1.24 monoclonal antibody was converted into a database of the amino acid sequence of the antibody prepared by Kabat et al. (“Sequence of Proteins of Immunological Interest” US Dept. Health and Human Services, 1983). The CDR regions can be found by examining the homology by applying the above method.
[0036]
(2) Preparation of expression vector for chimeric antibody
Once the DNA fragments encoding the mouse L and H chain V regions of the mouse monoclonal antibody have been cloned, these mouse V regions are ligated with the DNA encoding the human antibody constant region and expressed to produce chimeric anti-HM 1.24. An antibody is obtained.
The basic method for producing a chimeric antibody is to ligate the mouse leader sequence and V region sequence present in the cloned cDNA to a sequence encoding a human antibody C region already present in a mammalian cell expression vector. Comprising. Alternatively, the method comprises ligating a mouse leader sequence and a V region sequence present in the cloned cDNA to a sequence encoding a human antibody C region, and then ligating the sequence to a mammalian cell expression vector.
[0037]
The human antibody C region can be any human H chain C region and human L chain C region, and examples include human H chain Cγ1, Cγ2, Cγ3 and Cγ4, and L chain Cλ and Cκ.
For the production of chimeric antibodies, two different expression vectors are used, namely an expression comprising a DNA encoding a mouse L chain V region and a human L chain C region under the control of an expression control region such as an enhancer / promoter system. A vector and an expression vector comprising DNA encoding a mouse H chain V region and a human H chain C region under an expression control region such as an enhancer / promoter system are prepared. Next, host cells, such as mammalian cells, are co-transformed with these expression vectors, and the transformed cells are cultured in vitro or in vivo to produce chimeric antibodies (eg, WO 91-16928).
[0038]
Alternatively, the DNA encoding the mouse leader sequence and the L chain V region and the human L chain C region and the DNA encoding the mouse leader sequence and the H chain V region and the human H chain C region present in the cloned cDNA are combined into a single An expression vector is introduced (see International Patent Application Publication No. WO94-11523), and a host cell is transformed with the vector, and the transformed host is cultured in vivo or in vitro to obtain the desired chimera. Produce antibodies.
[0039]
1) Construction of chimeric antibody H chain
An H chain expression vector for a chimeric antibody can be obtained by introducing a cDNA encoding the mouse H chain V region into an appropriate expression vector containing genomic DNA or cDNA encoding the H chain C region of a human antibody. Examples of the H chain C region include Cγ1, Cγ2, Cγ3 and Cγ4.
[0040]
Construction of chimeric H chain expression vector containing Cγ1 genomic DNA
Examples of expression vectors having Cγ1 genomic DNA as the H chain C region include HEF-PMh-gγ1 (see International Patent Application Publication No. WO92 / 19759) or DHFR-ΔE-RVh-PM1f (International Patent Application Publication). No. WO92 / 19759) can be used.
[0041]
In order to insert the cDNA encoding the mouse H chain V region into these expression vectors, an appropriate nucleotide sequence can be introduced by PCR. For example, a PCR primer designed to have an appropriate restriction enzyme recognition sequence at the 5'-end, a Kozak consensus sequence immediately before the initiation codon to improve translation efficiency, and an appropriate restriction enzyme at the 3'-end These appropriate nucleotide sequences are introduced by performing PCR using a PCR primer designed to have a splice donor site for correctly recognizing the recognition sequence and the primary transcript of genomic DNA into mRNA.
The cDNA encoding the mouse H chain V region thus constructed is treated with an appropriate restriction enzyme and inserted into the above expression vector to construct a chimeric H chain expression vector containing Cγ1 genomic DNA.
[0042]
Construction of cDNA chimeric H chain expression vector
An expression vector having Cγ1 cDNA as the H chain C region can be constructed as follows. That is, an expression vector DHFR-ΔE-RVh encoding the genomic DNA of the humanized PM1 antibody H chain V region and human antibody H chain C region Cγ1 (N. Takahashi, et al., Cell 29, 671-679 1982). -PM1f (see International Patent Application Publication No. WO92 / 19759) and an expression vector RV1-PM1a encoding genomic DNA of a humanized PM1 antibody L chain V region and human antibody L chain κ chain C region (International Patent Application Publication No. MRNA was prepared from the CHO cells into which the humanized PM1 antibody H chain V region and human antibody H chain C region Cγ1 were cloned by RT-PCR, and used for appropriate animal cell expression. The vector can be linked and constructed by using an appropriate restriction enzyme site.
[0043]
In order to directly link the cDNA encoding the mouse H chain V region to the cDNA containing the human antibody H chain C region Cγ1, an appropriate nucleotide sequence can be introduced by PCR. For example, a PCR primer designed to have an appropriate restriction enzyme recognition sequence at the 5′-end, a Kozak consensus sequence immediately before the start codon to improve translation efficiency, and an H chain C region at the 3′-end These appropriate nucleotide sequences are introduced by performing PCR using PCR primers designed to have a recognition sequence for an appropriate restriction enzyme for direct ligation with Cγ1.
[0044]
The thus constructed cDNA encoding the mouse H chain V region is treated with an appropriate restriction enzyme, ligated to the cDNA containing the H chain C region Cγ1, and inserted into an expression vector such as pCOS1 or pCHO1, thereby obtaining a cDNA. An expression vector containing a chimeric H chain can be constructed.
2) Construction of chimeric antibody L chain
An L-chain expression vector for a chimeric antibody can be obtained by ligating a cDNA encoding the mouse L-chain V region and a genomic DNA or cDNA encoding the L-chain C region of a human antibody, and introducing the resultant into an appropriate expression vector. Can be done. Examples of the L chain C region include a κ chain and a λ chain.
[0045]
Construction of cDNA chimeric L chain κ chain expression vector
In order to construct an expression vector containing a cDNA encoding the mouse L chain V region, an appropriate nucleotide sequence can be introduced by PCR. For example, an appropriate restriction enzyme recognition sequence at the 5′-end, a PCR primer designed to have a Kozak consensus sequence for improving translation efficiency, and an appropriate restriction enzyme recognition sequence at the 3′-end. The appropriate base sequence is introduced by performing PCR using a PCR primer designed to have such a base sequence.
[0046]
The human L chain κ chain C region to be linked to the mouse L chain V region can be constructed, for example, from HEF-PM1k-gk containing genomic DNA (see International Patent Application Publication No. WO92 / 19759). The mouse L chain V region and the L region constructed as described above were constructed by introducing appropriate restriction enzyme recognition sequences into the 5'-end and 3'-end of the DNA encoding the L chain κ chain C region by PCR. By linking the chain k chain C regions and inserting it into an expression vector such as pCOS1 or pCHO1, an expression vector for the cDNA chimeric antibody L chain k chain can be constructed.
[0047]
2. Production of reshaped human antibody
(1) Design of reshaped human anti-HM1.24 antibody V region
In order to produce a reshaped human antibody in which the CDRs of a mouse monoclonal antibody have been grafted onto a human antibody, it is desirable that there be high homology between the FR of the mouse monoclonal antibody and the FR of the human antibody. Therefore, the V regions of the L chain and the H chain of the mouse anti-HM1.24 antibody are compared with the V regions of all known antibodies whose structures have been elucidated using Protein Data Bank.
[0048]
The L chain V region of the mouse anti-HM 1.24 antibody is most similar to the consensus sequence of human antibody L chain V region subgroup IV (HSGIV), with 66.4% homology. On the other hand, they show 56.9%, 55.8% and 61.5% homology with HSGI, HSGII and HSGIII, respectively.
The L-chain V region of the mouse anti-HM1.24 antibody was compared to the L-chain V region of a known human antibody by adding 67. It shows 0% homology. Therefore, FR of REI was used as a starting material for production of the reshaped human anti-HM1.24 antibody L chain V region.
[0049]
Version a of the reshaped human anti-HM1.24 antibody L chain V region was designed. In this version, the human antibody FR is based on the REI-based FR present in the reshaped human CAMPATH-1H antibody (see Riechmann, L. et al., Nature 322, 21-25, (1988); International Patent Application Publication No. WO 92 And the mouse CDR is the same as the CDR in the L chain V region of the mouse anti-HM1.24 antibody. And
[0050]
The H chain V region of the mouse anti-HM 1.24 antibody is most similar to the HSGI consensus sequence of the H chain V region of the human antibody, with 54.7% homology. On the other hand, HSGII and HSGIII show 34.6% and 48.1% homology, respectively. In comparison with the known human antibody H chain V region, the H chain V region of the mouse anti-HM 1.24 antibody shows that from FR1 to FR3, the human antibody HG3, which is one of the subgroup I of the human antibody H chain V region, It is very similar to the H chain V region (Rechavi, G. et al., Proc. Nat. Acad. Sci. USA 80, 855-859) and shows 67.3% homology.
[0051]
For this reason, the FR of the human antibody HG3 was used as a starting material for the production of the V region of the H chain of the reshaped human anti-HM1.24 antibody.
However, since the amino acid sequence of FR4 of the human antibody HG3 is not described, the human antibody JH6 (Ravetch, JV, et al.) Showing the highest homology with FR4 of the H chain of the mouse anti-HM1.24 antibody is not described. , Cell, 27, 583-591). FR4 of JH6 has the same amino acid sequence as FR4 of the H chain of mouse anti-HM1.24 antibody except for one amino acid.
[0052]
In the first version a of the H chain V region of the reshaped human anti-HM 1.24 antibody, except that the amino acid at position 30 in human FR1 and the amino acid at position 71 in human FR3 were the same as the amino acid of the mouse anti-HM 1.24 antibody. , FR1 to FR3 were the same as FR1 to FR3 of the human antibody HG3, and the CDR was the same as the CDR in the H chain V region of the mouse anti-HM1.24 antibody.
[0053]
(2) Preparation of V region of reshaped human anti-HM1.24 antibody L chain
A reshaped human anti-HM1.24 antibody L chain is prepared by CDR grafting by PCR. This method is schematically shown in FIG. Eight PCR primers are used to generate a reshaped human anti-HM1.24 antibody (version a) with FR from human antibody REI. The external primers A (SEQ ID NO: 47) and H (SEQ ID NO: 48) are designed to hybridize with the DNA sequence of the HEF expression vector HEF-VL-gκ.
[0054]
The CDR-grafting primers L1S (SEQ ID NO: 49), L2S (SEQ ID NO: 50) and L3S (SEQ ID NO: 51) have a sense DNA sequence. The CDR-grafting primers L1A (SEQ ID NO: 52), L2A (SEQ ID NO: 53) and L3A (SEQ ID NO: 54) have an anti-sense DNA sequence, and the 5'-primers L1S, L2S and L3S, respectively. It has a complementary DNA sequence (20-23 bp) to the terminal DNA sequence.
[0055]
In the first PCR stage, four reactions A-L1A, L1S-L2A, L2S-L3A, and L3S-H are performed, and each PCR product is purified. The four PCR products from the first PCR are assembled by their own complementarity (see WO 92-19759). Next, external primers A and H are added to amplify the full-length DNA encoding the reshaped human anti-HM1.24 antibody L chain V region (second PCR). In the PCR, the plasmid HEF-RVL-M21a encoding the reshaped human ONS-M21 antibody L chain V region version a based on FR from the human antibody REI (see International Patent Application Publication No. WO95-14401) was used as a template. Can be used.
[0056]
In the first PCR step, a template DNA and each primer are used.
The PCR products A-L1A (215 bp), L1S-L2A (98 bp), L2S-L3A (140 bp) and L3S-H (151 bp) were purified using a 1.5% low melting point agarose gel and assembled in a second PCR. I do. In the second PCR, the products of each first PCR and each external primer (A and H) are used.
The 516 bp DNA fragment generated by the second PCR is purified on a 1.5% low melting point agarose gel, digested with BamHI and HindIII, and the obtained DNA fragment is cloned into the HEF expression vector HEF-VL-gκ. After DNA sequencing, the plasmid containing the DNA fragment encoding the correct amino acid sequence of the reshaped human anti-HM1.24 antibody L chain V region was named HEF-RVLa-AHM-gκ. The amino acid sequence and the base sequence of the L chain V region contained in this plasmid HEF-RVLa-AHM-gκ are shown in SEQ ID NO: 9.
[0057]
Version b of the reshaped human anti-HM1.24 antibody L chain V region can be prepared by a mutagenesis method using PCR. The mutagenic primers FTY-1 (SEQ ID NO: 55) and FTY-2 (SEQ ID NO: 56) are designed so that phenylalanine at position 71 is mutated to tyrosine.
After amplification using the plasmid HEF-RVLa-AHM-gκ as a template and the above primers, the final product was purified, digested with BamHI and HindIII, and the obtained DNA fragment was inserted into an HEF expression vector HEF-VL-gκ. Cloning yields plasmid HEF-RVLb-AHM-gκ. The amino acid sequence and the base sequence of the L chain V region contained in this plasmid HEF-RVLb-AHM-gκ are shown in SEQ ID NO: 10.
[0058]
(3) Production of reshaped human anti-HM1.24 antibody H chain V region
3-1. Production of reshaped human anti-HM1.24 antibody H chain V region version ae
DNA encoding the reshaped human anti-HM1.24 antibody H chain V region can be designed as follows. A reshaped human anti-HM1.24 antibody was obtained by connecting a DNA sequence encoding FR1 to FR3 of human antibody HG3 and FR4 of human antibody JH6 to a DNA sequence encoding the CDR of the H chain V region of mouse anti-HM1.24 antibody. A full-length DNA encoding the H chain V region is designed.
Next, a HindIII recognition site / KOZAK consensus sequence and a BamHI recognition site / splice donor sequence are added to the 5'-side and 3'-side of this DNA sequence, respectively, so that they can be inserted into the HEF expression vector.
[0059]
The DNA sequence thus designed is divided into four oligonucleotides, and then the computer analyzes for secondary structures in the oligonucleotides that may interfere with the assembly of these oligonucleotides.
The four oligonucleotide sequences RVH1-RVH4 are shown in SEQ ID NOs: 57-60. These oligonucleotides are 119-144 bases in length and have an overlap region of 25-26 bp. Of the oligonucleotides, RVH2 (SEQ ID NO: 58), RVH4 (SEQ ID NO: 60) have a sense DNA sequence, and the other RVH1 (SEQ ID NO: 57), RVH3 (SEQ ID NO: 59) are antisense DNA Has an array. The method of assembling these four oligonucleotides by PCR is shown in the figure (see FIG. 5).
[0060]
PCR is performed using four oligonucleotides and RHP1 (SEQ ID NO: 60) and RHP2 (SEQ ID NO: 62) as external primers.
The amplified 438 bp DNA fragment is purified, digested with HindIII and BamHI, and then cloned into the HEF expression vector HEF-VH-gγ1. After DNA sequencing, the plasmid containing the DNA fragment encoding the amino acid sequence of the correct H chain V region was named HEF-RVHa-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHa-AHM-gγ1 are shown in SEQ ID NO: 11.
[0061]
Each version b, c, d, and e of the reshaped human anti-HM1.24 antibody H chain V region is prepared as follows. In preparing each version of the reshaped human anti-HM1.24 antibody H chain V region after version b, a mouse anti-HM1.24 antibody was used to infer the position of the amino acid residue to be substituted in the antibody molecule. A three-dimensional structure model of the V region can be constructed.
Version b uses BS (SEQ ID NO: 63) and BA (SEQ ID NO: 64) designed such that arginine at position 66 is mutated to lysine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA. To obtain the plasmid HEF-RVHb-AHM-gγ1. The amino acid sequence and the base sequence of the H chain V region contained in this plasmid HEF-RVHb-AHM-gγ1 are shown in SEQ ID NO: 12.
[0062]
Version c uses CS (SEQ ID NO: 65) and CA (SEQ ID NO: 66) designed such that threonine at position 73 mutates to lysine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA. To obtain the plasmid HEF-RVHc-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHc-AHM-gγ1 are shown in SEQ ID NO: 13.
Version d uses DS (SEQ ID NO: 67) and DA (SEQ ID NO: 68) designed to mutate arginine at position 66 to lysine and threonine at position 73 to lysine as mutagenic primers. Plasmid HEF-RVHd-AHM-gγ1 is obtained using RVHa-AHM-gγ1 as a template DNA. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHd-AHM-gγ1 are shown in SEQ ID NO: 14.
[0063]
Version e was used as a mutagenic primer using ES (SEQ ID NO: 69) and EA (SEQ ID NO: 70) designed to mutate valine at position 67 to alanine and methionine at position 69 to leucine, and used plasmid HEF- Amplification is performed using RVHa-AHM-gγ1 as a template DNA to obtain plasmid HEF-RVHe-AHM-gγ1. The amino acid sequence and the base sequence contained in the V region of the H chain contained in this plasmid HEF-RVHe-AHM-gγ1 are shown in SEQ ID NO: 15.
[0064]
3-2. Construction of H chain hybrid V region
By constructing the H chain hybrid V region, it is possible to examine which FR of the humanized antibody V region contributes to the binding activity and the binding inhibitory activity of the humanized antibody. Of the two types constructed, one is that the amino acid sequences of FR1 and FR2 are derived from mouse anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 are version a of the H chain V region of the reshaped human anti-HM1.24 antibody. The other (mouse / human hybrid anti-HM1.24 antibody), and the other is that the amino acid sequences of FR1 and FR2 are derived from version a of the H chain V region of reshaped human anti-HM1.24, and that of FR3 and FR4. The amino acid sequence is derived from a mouse anti-HM 1.24 antibody (human / mouse hybrid anti-HM 1.24 antibody). All amino acid sequences of CDR regions are derived from mouse anti-HM1.24 antibody.
[0065]
Two types of H chain hybrid V regions are prepared by the PCR method. This method is schematically illustrated in FIGS. Four primers can be used to create two H chain hybrid V regions. External primers a (SEQ ID NO: 71) and h (SEQ ID NO: 72) are designed to hybridize with the DNA sequence of the HEF expression vector HEF-VH-gγ1. The H-chain hybrid generation primer HYS (SEQ ID NO: 73) has a sense DNA sequence, and the H-chain hybrid primer HYA (SEQ ID NO: 74) has an antisense DNA sequence and thus has a complementary DNA sequence. Designed.
[0066]
Preparation of H chain hybrid V region in which amino acid sequences of FR1 and FR2 are derived from mouse anti-HM 1.24 antibody, and amino acid sequences of FR3 and FR4 are derived from version a of H chain V region of reshaped human anti-HM 1.24 antibody In the first PCR step, PCR using plasmid HEF-1.24H-gγ1 as a template and external primer a and H-chain hybrid primer HYA, and H-chain hybrid primer using plasmid HEF-RVHa-AHM-gγ1 as a template Perform PCR using HYS and external primer h and purify each PCR product.
[0067]
The two PCR purified products from the first PCR are assembled by their own complementarity (see International Patent Application Publication No. WO 92-19759). Next, by adding external primers a and h, the amino acid sequences of FR1 and FR2 are derived from the mouse anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 are derived from the H chain V region of the reshaped human anti-HM1.24 antibody. The full-length DNA encoding the heavy chain hybrid V region from version a is amplified in a second PCR step.
[0068]
Preparation of H-chain hybrid V region in which amino acid sequences of FR1 and FR2 are derived from version a of H-chain V region of reshaped human anti-HM1.24 antibody, and amino acid sequences of FR3 and FR4 are derived from mouse anti-HM1.24 antibody Therefore, in the first PCR step, PCR using the plasmid HEF-RVHa-AHM-gγ1 as a template and the external primer a and the H-chain hybrid primer HYA, and the H-chain hybrid primer using the plasmid HEF-1.24H-gγ1 as a template Perform PCR using HYS and external primer h and purify each PCR product.
[0069]
The two PCR purified products from the first PCR are assembled by their own complementarity (see International Patent Application Publication No. WO 92-19759). Next, by adding external primers a and h, the amino acid sequences of FR1 and FR2 are derived from version a of the V region of the H chain of the reshaped human anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 are derived from mouse anti-HM1. The full-length DNA encoding the H chain hybrid V region derived from antibody 24 is amplified in the second PCR step.
[0070]
The method of the first PCR, purification of the PCR product, assembly, second PCR, and cloning into the HEF expression vector HEF-VH-gγ1 are described in Example 9. The method can be performed according to the method described in Preparation of L chain V region of reshaped human HM1.24 antibody. After DNA sequencing, the amino acid sequences of FR1 and FR2 were derived from mouse anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 were derived from version a of the H chain V region of reshaped human anti-HM1.24 antibody. A plasmid containing a DNA fragment encoding the correct amino acid sequence of the chain hybrid V region was designated as HEF-MH-RVH-AHM-gγ1.
[0071]
The amino acid sequence and the base sequence of the H chain V region contained in this plasmid HEF-MH-RVH-AHM-gγ1 are shown in SEQ ID NO: 75. Further, the amino acid sequences of FR1 and FR2 are derived from the reshaped human anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 are derived from version a of the H chain V region of the mouse anti-HM1.24 antibody. The plasmid containing the DNA fragment coding for the correct amino acid sequence was named HEF-HM-RVH-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-HM-RVH-AHM-gγ1 are shown in SEQ ID NO: 76.
[0072]
3-3. Preparation of reshaped human anti-HM1.24 antibody H chain V region version fs
Each version f, g, h, i, j, k, l, m, n, o, p, q, r, and s of the reshaped human anti-HM1.24 antibody H chain V region is prepared as follows. . When preparing each version of the human anti-HM1.24 antibody H chain V region to be reconstructed after version f, in order to infer the position of the amino acid residue to be substituted in the antibody molecule, as described above, the mouse antibody A three-dimensional structural model of the HM1.24 antibody V region can be constructed.
[0073]
Version f uses FS (SEQ ID NO: 78) and FA (SEQ ID NO: 79) designed to mutate threonine at position 75 to serine and valine at position 78 to alanine as mutagenic primers. Using RVHe-AHM-gγ1 as a template DNA, amplification is performed by the PCR method to obtain plasmid HEF-RVHf-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHf-AHM-gγ1 are shown in SEQ ID NO: 16.
[0074]
Version g uses GS (SEQ ID NO: 80) and GA (SEQ ID NO: 81) designed such that alanine at position 40 is mutated to arginine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA To obtain plasmid HEF-RVHg-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHg-AHM-gγ1 are shown in SEQ ID NO: 17.
[0075]
Version h uses FS and FA as mutagenic primers and amplifies plasmid HEF-RVHb-AHM-gγ1 as template DNA to obtain plasmid HEF-RVHh-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHh-AHM-gγ1 are shown in SEQ ID NO: 18.
Version i was designed using mutagenic primers such as IS (SEQ ID NO: 82) and IA (SEQ ID NO: 83) designed to mutate arginine at position 83 to alanine and serine at position 84 to phenylalanine, and used plasmid HEF- Amplification is performed using RVHh-AHM-gγ1 as a template DNA to obtain plasmid HEF-RVHi-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHi-AHM-gγ1 are shown in SEQ ID NO: 19.
[0076]
Version j uses JS (SEQ ID NO: 84) and JA (SEQ ID NO: 85) designed such that arginine at position 66 mutates to lysine as a mutagenic primer, and uses plasmid HEF-RVHf-AHM-gγ1 as a template DNA. To obtain plasmid HEF-RVHj-AHM-gγ1. The amino acid sequence and the base sequence of the H chain V region contained in this plasmid HEF-RVHj-AHM-gγ1 are shown in SEQ ID NO: 20.
[0077]
Version k uses KS (SEQ ID NO: 86) and KA (SEQ ID NO: 87) designed such that glutamic acid at position 81 mutates to glutamine as a mutagenic primer, and uses plasmid HEF-RVHh-AHM-gγ1 as a template DNA To obtain plasmid HEF-RVHk-AHM-gγ1. The amino acid sequence and the base sequence of the H chain V region contained in this plasmid HEF-RVHk-AHM-gγ1 are shown in SEQ ID NO: 21.
[0078]
Version 1 used LS (SEQ ID NO: 88) and LA (SEQ ID NO: 89) designed to mutate glutamic acid at position 81 to glutamine and serine at position 82B to isoleucine as mutagenic primers, and used plasmid HEF- Amplification is performed using RVHh-AHM-gγ1 as a template DNA to obtain plasmid HEF-RVH1-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVH1-AHM-gγ1 are shown in SEQ ID NO: 22.
[0079]
Version m was designed to mutate glutamic acid at position 81 to glutamine, serine at 82b to isoleucine, and threonine at position 87 to serine as mutagenic primers (MS: SEQ ID NO: 90) and MA (SEQ ID NO: 91). ) Is amplified using plasmid HEF-RVHh-AHM-gγ1 as a template DNA to obtain plasmid HEF-RVHm-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHm-AHM-gγ1 are shown in SEQ ID NO: 23.
[0080]
Version n uses NS (SEQ ID NO: 92) and NA (SEQ ID NO: 93) designed so that serine at position 82B mutates to isoleucine as a mutagenic primer, and uses plasmid HEF-RVHh-AHM-gγ1 as a template DNA. To obtain the plasmid HEF-RVHn-AHM-gγ1. The amino acid sequence and the nucleotide sequence of the H chain V region contained in this plasmid HEF-RVHn-AHM-gγ1 are shown in SEQ ID NO: 24.
[0081]
Version o uses OS (SEQ ID NO: 94) and OA (SEQ ID NO: 95) designed to mutate threonine at position 87 to serine as a mutagenic primer, and uses plasmid HEF-RVHh-AHM-gγ1 as a template DNA. Obtain the plasmid HEF-RVHo-AHM-gγ1. The amino acid sequence and the base sequence of the H chain V region contained in this plasmid HEF-RVHo-AHM-gγ1 are shown in SEQ ID NO: 25.
[0082]
Version p uses PS (SEQ ID NO: 96) and PA (SEQ ID NO: 97) designed so that valine at position 78 mutates to alanine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA. To obtain the plasmid HEF-RVHp-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHp-AHM-gγ1 are shown in SEQ ID NO: 26.
[0083]
Version q uses QS (SEQ ID NO: 98) and QA (SEQ ID NO: 99) designed to mutate threonine at position 75 to serine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA. To obtain plasmid HEF-RVHq-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHq-AHM-gγ1 are shown in SEQ ID NO: 27.
[0084]
Version r was amplified by the PCR method using CS (SEQ ID NO: 65) and CA (SEQ ID NO: 66) as mutagenic primers, plasmid HEF-RVHp-AHM-gγ1 as a template DNA, and plasmid HEF-RVHr- AHM-gγ1 is obtained. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHr-AHM-gγ1 are shown in SEQ ID NO: 28.
[0085]
Version s uses a mutagen primer SS (SEQ ID NO: 100) and a mutagen primer SA (SEQ ID NO: 101) designed to mutate methionine at position 69 to isoleucine as a mutagen primer, and uses plasmid HEF-RVHr- Plasmid HEF-RVHs-AHM-gγ1 is obtained using AHM-gγ1 as a template DNA. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHs-AHM-gγ1 are shown in SEQ ID NO: 102.
The amino acid sequence of the prepared L chain V region is shown in Table 1, and the amino acid sequences of the H chain V region are shown in Tables 2 to 4.
[0086]
[Table 1]

[0087]
[Table 2]

[0088]
[Table 3]

[0089]
[Table 4]

[0090]
3. Production of chimeric and reshaped human antibodies
For the production of a chimeric antibody or a reshaped human antibody, the mouse H chain V region and the human H chain are controlled under the control of expression control regions such as an enhancer / promoter system. An expression vector comprising a DNA encoding a C region, an expression vector comprising a DNA encoding a mouse L chain V region and a human L chain C region under an expression control region such as an enhancer / promoter system, or An expression vector comprising a DNA encoding a humanized H chain V region and a human H chain C region under the control of an expression control region such as an enhancer / promoter system, and an expression vector comprising an expression control region such as an enhancer / promoter system. The invention comprises DNA encoding the humanized L chain V region and the human L chain C region. To produce a vector.
[0091]
Next, a host cell such as a mammalian cell is co-transformed with these expression vectors, and the transformed cell is cultured in vitro or in vivo to produce a chimeric antibody or a reshaped human antibody (for example, published in International Patent Application Publication). No. WO91-16928). In addition, a transgenic animal can be prepared by introducing an antibody gene into a mammal such as a goat, and a chimeric antibody or a reshaped human antibody can be obtained from the milk or the like.
[0092]
Further, the H chain V region and H chain C region and the L chain V region and L chain C region are ligated to a single vector, and an appropriate host cell can be transformed to produce an antibody. That is, for expression of the chimeric antibody, DNA encoding the mouse leader sequence, the H chain V region and the human H chain C region, and the mouse leader sequence, the L chain V region and the human L chain C region existing in the cloned cDNA are used. The encoding DNA is introduced into a single expression vector (see International Patent Application Publication No. WO 94-11523).
[0093]
For expression of the reshaped human antibody, a DNA encoding a humanized H chain V region and a human H chain C region and a DNA encoding a humanized L chain V region and a human L chain C region are expressed in a single expression vector. (See International Patent Application Publication No. WO 94-11523). Then, a host cell is transformed with the vector, and then the transformed host is cultured in vivo or in vitro to produce a desired chimeric antibody or reshaped human antibody.
The transformant transformed with the gene encoding the chimeric antibody or reshaped human antibody of interest as described above is cultured, and the produced chimeric antibody or reshaped human antibody is separated from the inside or outside of the cell and homogenized. Can be purified.
[0094]
The chimeric antibody or the reshaped human antibody which is the target protein of the present invention can be separated and purified using an affinity column. For example, as a column using Protein A, HyperD, POROS, Sepharose F. F. And the like. In addition, other separation / purification methods used for ordinary proteins may be used, and there is no particular limitation. For example, a chimeric antibody or a reshaped human antibody can be separated and purified by appropriately selecting and combining various types of chromatography, ultrafiltration, salt filtration, dialysis and the like.
[0095]
For the production of the chimeric anti-HM1.24 antibody or the reshaped human anti-HM1.24 antibody of the present invention, any expression system, such as a eukaryotic cell, such as an animal cell, such as an established mammalian cell line, an insect cell line, a true Filamentous fungal and yeast cells, as well as prokaryotic cells, such as bacterial cells, such as E. coli cells, can be used. Preferably, the chimeric or reshaped human antibodies of the invention are expressed in mammalian cells, such as COS cells, CHO cells, Hela cells, Vero cells, myeloma cells or BHK cells.
[0096]
In these cases, conventional promoters useful for expression in mammalian cells can be used. For example, it is preferable to use the human cytomegalovirus immediate early (HCMV) promoter. Examples of expression vectors containing the HCMV promoter include HCMV-VH-HCγ1, HCMV-VL-HCκ and the like derived from pSV2neo (International Patent Application Publication No. WO92-19759).
[0097]
In addition, other promoters for gene expression in mammalian cells that can be used for the present invention include viral promoters such as retrovirus, polyoma virus, adenovirus, simian virus 40 (SV40), human polypeptides and the like. A promoter derived from a mammalian cell such as chain elongation factor 1α (HEF-1α) may be used. For example, when using the SV40 promoter, the method of Mulligan et al. (Nature 277 108 (1979)), and when using the HEF-1α promoter, see Mizushima, S. et al. According to these methods (Nucleic Acids Research, 18, 5322, 1990), it can be easily carried out.
[0098]
As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can be used. Further, for amplification of gene copy number in a host cell system, an expression vector is selected. Markers can include aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (DHFR) gene and the like.
[0099]
4. Binding inhibitory activity of chimeric and humanized antibodies
(1) Measurement of antibody concentration
The concentration of the purified antibody is measured by ELISA or absorbance measurement.
An ELISA plate for antibody concentration measurement is prepared as follows. Each well of a 96-well plate for ELISA (eg, Maxisorp, NUNC) is immobilized with 100 μl of a goat anti-human IgG antibody prepared at a concentration of, for example, 1 μg / ml.
[0100]
100 μl of dilution buffer (for example, 50 mM Tris-HCl, 1 mM MgCl₂, 0.15 M NaCl, 0.05% Tween 20, 0.02% NaN₃After blocking with 1% bovine serum albumin (BSA), pH 8.1), culture supernatant of cells expressing chimeric, hybrid or reconstituted human antibodies, such as culture supernatant of COS cells or CHO cells or Purified chimeric, hybrid or reconstituted human antibodies are serially diluted and added to each well, then 100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody is added and a 1 mg / ml substrate solution (Sigma 104, p-nitrophenyl phosphate) , SIGMA) and then the absorbance at 405 nm is measured with a microplate reader (Bio Rad). As a standard for concentration measurement, human IgG1κ (The Binding Site) can be used. The concentration of the purified antibody is determined by measuring the absorbance at 280 nm and calculating 1 mg / ml as 1.35 OD.
[0101]
(2) Antigen binding activity
The antigen binding activity can be measured by Cell-ELISA using a human amniotic cell line WISH (ATCC CCL25). A Cell-ELISA plate is prepared as follows. WISH cells adjusted to an appropriate concentration with a PRMI1640 medium containing 10% fetal bovine serum were added to a 96-well plate, cultured overnight, washed twice with PBS (-), and then washed with 0.1% glutaraldehyde (Nacalai Tesque, Inc.). Fixed by the company).
[0102]
After blocking, cells expressing chimeric anti-HM1.24 antibody, hybrid anti-HM1.24 antibody or reshaped human anti-HM1.24 antibody, for example, culture supernatant of COS cells or CHO cells, or purified chimeric anti-HM1. 24 antibody, hybrid anti-HM1.24 antibody or reconstituted human anti-HM1.24 antibody was serially diluted, and 100 μl was added to each well. After incubation and washing at room temperature for 2 hours, peroxidase-labeled rabbit anti-human IgG antibody (DAKO) Made).
After 1 hour incubation and washing at room temperature, the substrate solution is added and incubated. Next, the reaction is stopped with 50 μl of 6N sulfuric acid, and the absorbance at 490 nm is measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad).
[0103]
(3) Measurement of binding inhibitory activity
The binding inhibitory activity by the biotin-labeled mouse anti-HM1.24 antibody can be performed by Cell-ELISA using human amniotic cell line WISH (ATCC CCL25). The Cell-ELISA plate can be prepared according to the above (2). WISH cells adjusted to an appropriate concentration in RPMI1640 medium containing 10% fetal bovine serum were added to a 96-well plate, cultured overnight, washed twice with PBS (-), and then washed with 0.1% glutaraldehyde (Nacalai Tesque, Inc.). Fixed by the company).
[0104]
After blocking, cells expressing chimeric anti-HM1.24 antibody, hybrid anti-HM1.24 antibody or reshaped human anti-HM1.24 antibody, for example, culture supernatant of COS cells or CHO cells, or purified chimeric anti-HM1. 24 antibody, hybrid anti-HM1.24 antibody or reconstituted human anti-HM1.24 antibody was serially diluted and added to each well in an amount of 50 µl. After 2 hours of incubation and washing, a peroxidase-labeled rabbit anti-human IgG antibody (DAKO) is added.
After incubation at room temperature for 1 hour, washing is performed, a substrate solution is added, incubation is performed, the reaction is stopped with 50 μl of 6N sulfuric acid, and absorbance at 490 nm is measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad).
[0105]
Measurement of ADCC activity
ADCC activity of the chimeric antibody or reshaped human antibody of the present invention can be measured as follows. First, mononuclear cells are separated from human peripheral blood and bone marrow by specific gravity centrifugation, and prepared as effector cells. In addition, human myeloma cells, for example, RPMI 8226 cells (ATCC CCL 155)⁵¹Labeled with Cr and prepared as target cells. Next, a chimeric antibody or reconstituted human antibody for measuring ADCC activity is added to the labeled target cells and incubated, and then an effector cell at an appropriate ratio to the target cells is added and incubated.
[0106]
After incubation, remove the supernatant and measure the radioactivity with a gamma counter. At that time, 1% of NP-40 can be used for measuring the maximum free radioactivity. The cytotoxic activity (%) can be calculated by (A−C) / (B−C) × 100. A is the radioactivity released in the presence of the antibody (cpm), B is the radioactivity released by NP-40 (cpm), and C is the radioactivity released in the culture medium alone without the antibody (cpm). ).
When ADCC activity or CDC activity is expected in the antibody C region, human Cγ1 and human Cγ3 can be used as the antibody C region. Furthermore, stronger ADCC activity or CDC activity can be induced by partially adding, altering, or modifying amino acids in the antibody C region.
[0107]
For example, an IgG-like polymerization of IgG by amino acid substitution (Smith, RIFF & Morrison, SL, BIO / TECHNOLOGY (1994) 12, 683-688), and an IgG-like polymerization of IgG by amino acid addition ( Smith, RIFF et al., J. Immunol. (1995) 154, 2226-2236), and the expression of a gene encoding an L chain in tandem ligation (Shuford, W. et al., Science (1991)). 252, 724-727), dimerization of IgG by amino acid substitution (Caron, PC et al., J. Exp. Med. (1992) 176, 1191-1195), Shopes, B. J. Immunology. (1992) 148, 2 18-2922), IgG dimerization by chemical modification (Wolff, EA et al., Cancer Res. (1993) 53, 2560-2565) and introduction of effector functions by amino acid modification of the antibody hinge region. (Norderhaug, L. et al., Eur. J. Immunol. (1991) 21, 2379-2384). These can be achieved by using an oligomer site-directed mutagenesis method using a pramer, adding a base sequence using a restriction enzyme cleavage site, and using a chemical modifier that causes a covalent bond.
[0108]
Myeloma internal diagnostics
The chimeric anti-HM1.24 antibody or the reshaped human anti-HM1.24 antibody of the present invention can be used as a diagnostic agent in myeloma by binding to a labeled compound such as a radioisotope.
Furthermore, chimeric anti-HM1.24 antibodies or fragments of reshaped human anti-HM1.24 antibodies, such as Fab, F (ab ')₂, Fv or a combination of a single chain Fv (scFv) obtained by linking an Fv of an H chain and an L chain with an appropriate linker and a labeling compound such as a radioisotope can also be used as a diagnostic agent in myeloma. .
[0109]
Specifically, fragments of these antibodies can be prepared by constructing a gene encoding these antibody fragments and introducing them into an expression vector and then expressing them in a suitable host cell, or by using a chimeric anti-HM1.24 antibody or It is obtained by digesting the constituent human anti-HM1.24 antibody using an appropriate enzyme.
The above diagnostic agent for myeloma can be systemically administered parenterally.
Pharmaceutical composition and therapeutic agent for myeloma
In order to confirm the therapeutic effect of the chimeric antibody HM1.24 antibody or the humanized anti-HM1.24 antibody of the present invention, the antibody is administered to an animal into which myeloma cells have been transplanted, and the antitumor effect is evaluated. Done.
[0110]
As the myeloma cells to be transplanted into an animal, human myeloma cells are preferable. H. et al., Eur. J. Immunol. (1992) 22, 1989-1993). The animal to be transplanted is preferably an animal having reduced or deleted immune function, and includes nude mice, SCID mice, beige mice, and nude rats.
The antitumor effect to be evaluated can be confirmed according to changes in the amount of human immunoglobulin in serum, measurement of tumor volume and weight, changes in the amount of human benzjones protein in urine, or the life span of animals.
[0111]
The pharmaceutical composition and the therapeutic agent for myeloma containing the chimeric anti-HM1.24 antibody or reshaped human anti-HM1.24 antibody of the present invention as an active ingredient can be administered parenterally systemically or locally. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, or subcutaneous injection can be selected, and the administration method can be appropriately selected depending on the age and symptoms of the patient.
The effective dose is selected in the range of 0.01 mg / kg to 1000 mg / kg of body weight at a time. Alternatively, a dose of 5 mg / body, preferably 50-100 mg / body per patient can be chosen.
The pharmaceutical composition and the therapeutic agent for myeloma containing the chimeric anti-HM1.24 antibody or the reshaped human anti-HM1.24 antibody of the present invention as an active ingredient contain pharmaceutically acceptable carriers and additives depending on the administration route. It may be something.
[0112]
Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, Sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), Mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like. The additives to be used are appropriately or in combination selected from the above according to the dosage form of the present invention, but are not limited thereto.
[0113]
【Example】
Next, the present invention will be described more specifically with reference to examples.
Example 1 .  Mouse anti HM1.24 Encodes an antibody variable region cDNA Cloning
1. Isolation of messenger RNA (mRNA)
2 x 10 producing mouse anti-HM1.24 antibody⁸From the hybridoma cells (FERM BP-5233), mRNA was isolated using Fast Track mRNA Isolation Kit Version 3.2 (manufactured by Invitrogen) according to the instructions attached to the kit.
[0114]
2. Amplification of the gene encoding the antibody variable region by PCR
PCR was performed using a Thermal Cycler (manufactured by Perkin Elmer Cetus).
2-1. Amplification and fragmentation of gene encoding mouse L chain V region
Single-stranded cDNA was synthesized from the isolated mRNA using AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (manufactured by Life Science) and used for PCR. The primers used in the PCR method are MKV (Mouse Kappa Variable) primers (Jones, ST, et al., Bio / Technology, 9) shown in SEQ ID NOs: 29 to 39 that hybridize with the mouse kappa-type L chain leader sequence. , 88-89, (1991)).
[0115]
100 μl of the PCR solution is composed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.1 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl₂5 units of DNA polymerase Ampli Taq (manufactured by Perkin Elmer Cetus), 0.25 mM of MKV primer shown in SEQ ID NOs: 29 to 39, and 3 mM of MKC primer shown in SEQ ID NO: 40 and 100 ng of single-stranded cDNA After covering with 50 μl of mineral oil for 3 minutes at an initial temperature of 94 ° C. and then 1 minute at 94 ° C., 1 minute at 55 ° C. and 1 minute at 72 ° C., in this order. Heated. After repeating this temperature cycle 30 times, the reaction mixture was further incubated at 72 ° C. for 10 minutes. The amplified DNA fragment was purified with low melting point agarose (manufactured by Sigma) and digested at 37 ° C. with Xmal (manufactured by New England Biolabs) and SalI (manufactured by Takara Shuzo).
[0116]
2-2. Amplification and fragmentation of cDNA encoding mouse H chain V region
The gene encoding the mouse H chain V region is obtained by the 5'-RACE method (Rapid Amplification of cDNA ends; Frohman, MA, et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002, (1988)). The amplification was performed by Edwards, JBDM, et al., Nucleic Acids Res., 19, 5227-5232, (1991). After synthesizing cDNA using primer P1 (SEQ ID NO: 41) that specifically hybridizes to the mouse IgG2a constant region, the mouse H chain V region is encoded using 5′-AmpliFINDER RACE KIT (manufactured by CLONETECH). Amplification of the cDNA was performed using a primer MHC2a (SEQ ID NO: 42) that specifically hybridizes to the mouse IgG2a constant region and an anchor primer (SEQ ID NO: 77) attached to the kit. The amplified DNA fragment was purified with low melting point agarose (manufactured by Sigma) and digested with EcoRI (manufactured by Takara Shuzo) and XmaI (manufactured by New England Biolabs) at 37 ° C.
[0117]
3. Ligation and transformation
A pUC19 vector prepared by digesting the DNA fragment containing the gene encoding the mouse kappa-type L chain V region prepared as described above with SalI and XmaI, and 50 mM Tris-HCl (pH 7.6) , 10 mM MgCl₂2.5 hours at 16 ° C. in a reaction mixture containing 10 mM dithiothreitol, 1 mM ATP, 50 mg / ml polyethylene glycol (8000) and 1 unit T4 DNA ligase (GIBCO-BRL). The reaction was performed and ligated. Similarly, a DNA fragment comprising the gene encoding the mouse H chain V region was ligated with a pUC19 vector prepared by digesting with EcoRI and XmaI at 16 ° C. for 3 hours.
[0118]
Next, 10 μl of the ligation mixture was added to 50 μl of competent E. coli DH5α cells, and the cells were placed on ice for 30 minutes, 42 ° C. for 1 minute and again on ice for 1 minute. Then, 400 μl of 2 × YT medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)) was added, and the mixture was incubated at 37 ° C. for 1 hour, and then 50 μg / T The Escherichia coli was spread on a medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)) and incubated at 37 ° C. overnight to obtain an E. coli transformant.
[0119]
The transformants were cultured overnight at 37 ° C. in 10 ml of 2 × YT medium containing 50 μg / ml of ampicillin, and from the culture, an alkaline method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor) was used. The plasmid DNA was prepared according to Laboratory Press, (1989).
The thus obtained plasmid containing the gene encoding the mouse kappa-type L chain V region derived from the hybridoma producing the anti-HM1.24 antibody was named pUCHMVL9. The plasmid containing the gene encoding the mouse H chain V region derived from the hybridoma producing the anti-HM1.24 antibody, obtained according to the above method, was named pUCHMMVHR16.
[0120]
Example 2 .  DNA Of the nucleotide sequence of
The nucleotide sequence of the cDNA coding region in the above plasmid was determined using an automatic DNA sequencer (manufactured by Applied Biosystem Inc.) and a Taq Dye Deoxy Terminator Cycle Sequencing Kit (manufactured by Applied Biosystem Inc. under the protocol specified by Applied Biosystem Inc.) Were determined.
SEQ ID NO: 1 shows the nucleotide sequence of the gene encoding the L chain V region of the mouse anti-HM1.24 antibody contained in the plasmid pUCHMVL9. In addition, SEQ ID NO: 2 shows a nucleotide sequence of a gene encoding a mouse anti-HM1.24 antibody H chain V region contained in plasmid pUCHMMVHR16.
[0121]
Example 3 .  CDR Decision
The overall structure of the V regions of the light and heavy chains is similar to each other, with each of the four framework portions being linked by three hypervariable regions, the complementarity determining regions (CDRs). The amino acid sequence of the framework is relatively well conserved, while the amino acid sequence of the CDR regions is extremely variable (Kabat, EA, et al., "Sequences of Proteins of Immunological Interest", US Dept. Health). and Human Services, 1983).
Based on this fact, the CDR region was determined as shown in Table 5 by applying the amino acid sequence of the variable region of the anti-HM1.24 antibody to a database of the amino acid sequence of the antibody prepared by Kabat et al. And examining homology. did.
[0122]
[Table 5]

[0123]
Example 4 .  Cloned cDNA Confirmation of the expression of HM1.24 Preparation of antibody)
1. Preparation of expression vector
In order to prepare a vector expressing the chimeric anti-HM1.24 antibody, cDNA clones pUCHMVL9 and pUCHMMVHR16 encoding the respective L and H chain V regions of the mouse anti-HM1.24 antibody were modified by PCR. Then, it was introduced into an HEF expression vector (see International Patent Application Publication No. WO92-19759).
[0124]
The rear primer ONS-L722S (SEQ ID NO: 43) for the L chain V region and the rear primer VHR16S (SEQ ID NO: 44) for the H chain V region are DNAs encoding the beginning of the leader sequence of each V region. And a Kozak consensus sequence (Kozak, M, et al., J. Mol. Biol., 196, 947-950, (1987)) and a HindIII restriction enzyme recognition site. The forward primer VL9A for the L chain V region (SEQ ID NO: 45) and the forward primer VHR16A for the H chain V region (SEQ ID NO: 46) hybridize to the DNA sequence encoding the end of the J region and splice. It was designed to have a donor sequence and a BamHI restriction enzyme recognition site.
[0125]
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.1 mM dNTPs, 1.5 mM MgCl₂, 100 pmole of each primer, 100 ng of template DNA (pUCHMVL9 or pUCHMMVHR16), and 5 units of Ampli Taq enzyme. Cover 100 μl of PCR reaction mixture with 50 μl of mineral oil and Thereafter, a cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute was performed 30 times, and finally, incubation was performed at 72 ° C. for 10 minutes.
The PCR product was purified using a 1.5% low melting point agarose gel, digested with HindIII and BamHI, and HEF-VL-gκ for the light chain V region and HEF-VH-gγ1 for the heavy chain V region. Respectively. After DNA sequencing, plasmids containing DNA fragments having the correct DNA sequence were named HEF-1.24L-gκ and HEF-1.24H-gγ1, respectively.
[0126]
The regions encoding the respective variable regions from the plasmids HEF-1.24L-gκ and HEF-1.24H-gγ1 were converted into restriction fragments with the restriction enzymes HindIII and BamHI, and these were used as HindIII and BamHI sites in the plasmid vector pUC19. And were named pUC19-1.24L-gκ and pUC19-1.24H-gγ1, respectively.
In addition, Escherichia coli containing each of the plasmids pUC19-1.24L-gκ or pUC19-1.24H-gγ1 was Escherichia coli DH5α (pUC19-1.24L-gκ) and Escherichia coli DH5α (pUC19-1.24H, respectively). -Gγ1), respectively, as FERM BP-5646 and FERM BP-5644, respectively, at the Institute of Biotechnology and Industrial Technology (1-3 1-3, Higashi, Tsukuba, Ibaraki Prefecture) as a Budapest Treaty on August 29, 1996. Was deposited internationally.
[0127]
2. Transfection into COS-7 cells
The expression vector was tested in COS-7 (ATCC CRL-1651) cells to observe the transient expression of the chimeric anti-HM1.24 antibody. HEF-1.24L-gκ and HEF-1.24H-gγ1 were co-transformed into COS-7 cells by electroporation using a Gene Pulser device (BioRad). Each DNA (10 μg) was added to 1 × 10⁷A 0.8 ml aliquot of cells / ml was added and pulsed at 1500 V, 25 μF volume.
After a 10 minute recovery period at room temperature, the electroporated cells were added to 30 ml of DMEM culture medium (GIBCO) containing 10% γ-globulin-free fetal bovine serum. CO₂After 72 hours of incubation in the incubator BNA120D (TABAI), the culture supernatant was collected and cell debris was removed by centrifugation, which was used in the following experiments.
[0128]
3. FCM analysis
The antigen binding activity of the chimeric anti-HM1.24 antibody was determined by FCM (flow cytometry) analysis using KPMM2 cells. 4.7 x 10⁵After washing the KPMM2 cells (Patent Application Publication No. JP-A-7-236475) with PBS (-), 50 μl of the chimeric anti-HM1.24 antibody-producing COS-7 cell culture and FACS buffer (2% fetal bovine serum) , PBS (-) containing 0.1% sodium azide), 5 μl of 500 μg / ml purified mouse anti-HM1.24 antibody and 95 μl of FACS buffer, and incubated at ice temperature for 1 hour.
[0129]
As a control, 50 μl of 2 μg / ml chimeric SK2 (International Patent Application Publication No. WO94-28159) and 50 μl of FACS buffer instead of the chimeric anti-HM 1.24 antibody-producing COS cell culture solution, or instead of the purified mouse anti-HM 1.24 antibody 5 μl of 500 μg / ml purified mouse IgG2aκ (UPC10) (manufactured by Cappel) and 95 μl of FACS buffer were added thereto, and incubated in the same manner. After washing with FACS buffer, 100 μl of 25 μg / ml FITC-labeled goat anti-human antibody (GAH) (manufactured by Cappel) or 10 μg / ml of FITC-labeled goat anti-mouse antibody (GAM) (manufactured by Becton Dickinson) was used. In addition, the mixture was incubated at an ice temperature for 30 minutes. After washing with a FACS buffer, the cells were suspended in 1 ml of a FACS buffer, and the fluorescence intensity of each cell was measured with a FACScan (manufactured by Becton Dickinson).
[0130]
As shown in FIG. 1, in the cells to which the chimeric anti-HM1.24 antibody was added, the peak of the fluorescence intensity was shifted to the right as compared with the control similarly to the case where the mouse anti-HM1.24 antibody was added. .24 antibody bound to KPMM2 cells. This confirmed that the cloned cDNA encoded the V region of the mouse anti-HM1.24 antibody.
[0131]
Example 5 .  Chimera anti HM1.24 Antibody stable production CHO Establishment of cell line
1. Construction of chimeric H chain expression vector
The plasmid HEF-1.24H-gγ1 was digested with the restriction enzymes PvuI and BamHI, and a fragment of about 2.8 kbp containing the EF1 promoter and DNA encoding the mouse anti-HM1.24 antibody H chain V region was 1.5%. Purification was performed using a low melting point agarose gel. Next, the expression vector used in the human H chain expression vector DHFR-ΔE-RVh-PM1f containing the DHFR gene and the gene encoding the human H chain constant region (see International Patent Application Publication No. WO92 / 19759) was PvuI. And the above DNA fragment was inserted into a fragment of about 6 kbp prepared by digestion with BamHI to construct a chimeric anti-HM1.24 antibody H chain expression vector DHFR-ΔE-HEF-1.24H-gγ1.
[0132]
2. Gene transfer into CHO cells
In order to establish a chimeric anti-HM1.24 antibody stable production system, the expression vectors HEF-1.24L-gκ and DHFR-ΔE-HEF-1.24H-gγ1 which had been linearized by digestion with PvuI were electroporated. The gene was simultaneously transfected into CHO cells DXB11 (provided by Medical Research Council Laboratories Center) by the poration method under the same conditions as described above (transfection into the COS-7 cells).
[0133]
3. Gene amplification by MTX
The transfected CHO cells were cultured in a nucleoside-free α-MEM culture solution (GIBCO-BRL) supplemented with 500 μg / ml G418 (GIBCO-BRL) and 10% fetal bovine serum, and the light chain and H chain were added. Only CHO cells into which the chain expression vector had been introduced were able to survive and were selected. Next, 10 nM MTX (manufactured by Sigma) was added to the above culture solution, and a clone having a high production amount of the chimeric anti-HM1.24 antibody was selected from the grown clones. As a result, about 20 μg / ml of the chimeric antibody was obtained. Clone # 8-13 showing the production efficiency was obtained and used as a chimeric anti-HM1.24 antibody-producing cell line.
[0134]
Example 6 .  Chimera anti HM1.24 Production of antibodies
Preparation of the chimeric anti-HM1.24 antibody was performed by the following method. Using the above-mentioned chimeric anti-HM1.24 antibody-producing CHO cells as a medium, Iscover's Modified Dulbecco's Medium (GIBCO-BRL) containing 5% γ-globulin-free neonatal calf serum (GIBCO-BRL) was used. The cells were continuously cultured in a high-density cell culture apparatus Verax system 20 (manufactured by CELLEX BIOSCIENCE Inc.) for 30 days.
[0135]
At 13, 20, 23, 26, and 30 days after the start of the culture, the culture solution was collected and filtered using a pressurized filtration filter unit SARTOBRAN (manufactured by Sartorius), and then a large-scale antibody fractionation system Afi-Prep System (Japan) GS) and Super Protein A column (bed volume: 100 ml, NGK) based on the attached instructions, using PBS (-) as an adsorption / washing buffer and 0.1M quenching as an elution buffer. The chimeric anti-HM1.24 antibody was affinity-purified using a sodium acid buffer (pH 3). The eluted fraction was immediately adjusted to around pH 7.4 by adding 1 M Tris-HCl (pH 8.0). The antibody concentration was determined by measuring the absorbance at 280 nm and calculating 1 mg / ml as 1.35 OD.
[0136]
Example 7 .  Chimera anti HM1.24 Antibody activity measurement
The chimeric anti-HM1.24 antibody was evaluated based on the following binding inhibitory activities.
1. Measurement of binding inhibitory activity
1-1. Preparation of biotin-labeled anti-HM1.24 antibody
After diluting the mouse anti-HM1.24 antibody to 4 mg / ml with 0.1 M bicarbonate buffer, 4 μl of 50 mg / ml Biotin-N-hydroxysuccinimide (manufactured by EY LABS Inc.) was added. The reaction was performed at room temperature for 3 hours. Thereafter, 1.5 ml of a 0.2 M glycine solution was added, and the mixture was incubated at room temperature for 30 minutes to stop the reaction, and a biotinylated IgG fraction was collected using a PD-10 column (manufactured by Pharmacia Biotech).
[0137]
1-2. Measurement of binding inhibitory activity
The binding inhibition activity by the biotin-labeled mouse anti-HM1.24 antibody was performed by Cell-ELISA using human amniotic cell line WISH cells (ATCC CCL 25). Cell-ELISA plates were prepared as follows. 4 × 10 6 in a 96-well plate with RPMI 1640 medium containing 10% fetal bovine serum.⁵100 μl of a WISH cell suspension prepared at cells / ml was added, cultured overnight, washed twice with PBS (−), and fixed with 0.1% glutaraldehyde (manufactured by Nacalai Tesque).
[0138]
After blocking, the chimeric anti-HM1.24 antibody or mouse anti-HM1.24 antibody obtained by affinity purification was serially diluted and added to each well in an amount of 50 μl. At the same time, 50 μl of 2 μg / ml biotin-labeled mouse anti-HM1.24 antibody was added. After addition, incubation and washing at room temperature for 2 hours, peroxidase-labeled streptavidin (manufactured by DAKO) was added. After incubation at room temperature for 1 hour, washing was performed, a substrate solution was added, and after incubation, the reaction was stopped with 50 μl of 6N sulfuric acid, and the absorbance at 490 nm was measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad). .
As a result, as shown in FIG. 2, the chimeric anti-HM1.24 antibody exhibited the same binding inhibitory activity as the mouse anti-HM1.24 antibody against the biotin-labeled mouse anti-HM1.24 antibody. This indicated that the chimeric antibody had the same V region as the mouse anti-HM1.24 antibody.
[0139]
Example 8 .  Chimera anti HM1.24 Antibody ADCC Activity measurement
ADCC (Antibody-dependent Cellular Cytotoxicity) activity is measured in Current Protocols in Immunology, Chapter 7. See Immunological studies in humans, Editor, John E, Coligan et al. , John Wiley & Sons, Inc. , 1993.
1. Preparation of effector cells
Mononuclear cells were isolated from peripheral blood and bone marrow of healthy individuals and patients with multiple myeloma by specific gravity centrifugation. That is, an equal amount of PBS (-) was added to peripheral blood and bone marrow of a healthy person and a patient with multiple myeloma, and laminated on Ficoll (Pharmacia) -Conley (Daiichi Pharmaceutical) (specific gravity 1.077), Centrifuged at 400 g for 30 minutes. The mononuclear cell layer was separated and washed twice with RPMI1640 (Sigma) containing 10% fetal bovine serum (Witaker), and the cell number was 5 × 10 5 in the same culture solution.⁶/ Ml.
[0140]
2. Preparation of target cells
The human myeloma cell line RPMI 8226 (ATCC CCL 155) was⁵¹Radiolabeling was performed by incubating with Cr-sodium chromate in RPMI1640 (Sigma) containing 10% fetal calf serum (Witaker) at 37 ° C for 60 minutes. After radiolabeling, the cells were washed three times with Hanks balanced salt solution (HBSS) and 2 x 10⁵/ Ml.
[0141]
3. ADCC assay
Radioactively labeled 2 × 10 6 in a 96-well U-bottom plate (Corning)⁵Of 50 μl / ml target cells and 50 μl of 1 μg / ml chimeric anti-HM1.24 antibody, mouse anti-HM1.24 antibody or control human IgG1 (manufactured by Serotec) obtained by affinity purification. Allowed to react for minutes.
Then 5 x 10⁶/ Ml of the effector cells was added to 100 μl, and cultured in a carbon dioxide incubator for 4 hours. At that time, the ratio (E: T) of the effector cell (E) and the target cell (T) was set to 0: 1,: 5: 1, 20: 1 or 50: 1.
[0142]
100 μl of the supernatant was taken, and the radioactivity released into the culture supernatant was measured with a gamma counter (ARC361, manufactured by Aloka). For the measurement of the maximum free radioactivity, 1% NP-40 (manufactured by BRL) was used. The cytotoxic activity (%) was calculated as (A−C) / (B−C) × 100. A is the radioactivity released in the presence of the antibody (cpm), B is the radioactivity released by NP-40 (cpm), and C is the radioactivity released in the culture medium alone without the antibody (cpm). Show.
As shown in FIG. 3, when the chimeric anti-HM 1.24 antibody was added as compared with the human control IgG1, the cytotoxic activity increased with an increase in the E: T ratio. It was shown to have Furthermore, since no cytotoxic activity is observed even when mouse anti-HM1.24 antibody is added, when the effector cells are human-derived cells, the Fc portion of the human antibody is required to obtain ADCC activity. It was shown.
[0143]
Example 9 .  Reconstituted human anti-HM 1.24 Production of antibodies
1. Design of reshaped human anti-HM1.24 antibody V region
In order to produce a reshaped human antibody in which the CDRs of a mouse monoclonal antibody have been grafted onto a human antibody, it is desirable that there be high homology between the FR of the mouse monoclonal antibody and the FR of the human antibody. Therefore, the V regions of the L and H chains of the mouse anti-HM1.24 antibody were compared with the V regions of all known antibodies whose structures were elucidated using Protein Data Bank.
[0144]
The L chain V region of the mouse anti-HM 1.24 antibody is most similar to the consensus sequence of human L chain V region subgroup IV (HSGIV), with 66.4% homology. On the other hand, it showed 56.9%, 55.8% and 61.5% homology with HSGI, HSGII and HSG III, respectively.
Compared with the known human antibody L chain V region, the L chain V region of the mouse anti-HM 1.24 antibody has 67.0% of the human L chain V region REI, which is one of the subgroup I of the human L chain V region. Showed homology. Therefore, FR of REI was used as a starting material for the production of the reshaped human anti-HM1.24 antibody L chain V region.
[0145]
Version a of the reshaped human anti-HM1.24 antibody L chain V region was designed. In this version, the human FRs are based on the REI present in the reshaped human CAMPATH-1H antibody (see Riechmann, L. et al., Nature 322, 21-25, (1988); International Patent Application Publication No. WO92- FR contained in version a of the L chain V region of reshaped human PM-1 described in 19759, and the mouse CDR was the same as the CDR in the L chain V region of the mouse anti-HM1.24 antibody. .
[0146]
The H chain V region of the mouse anti-HM 1.24 antibody is most similar to the HSGI consensus sequence of the human H chain V region, with 54.7% homology. On the other hand, HSGII and HSGIII showed 34.6% and 48.1% homology, respectively. In comparison with the known human antibody H chain V region, the H chain V region of the mouse anti-HM 1.24 antibody shows that H1 of the human antibody HG3, which is one of the subgroup I of the human H chain V region, It is very similar to the chain V region (Rechavi, G. et al., Proc. Nat. Acad. Sci. USA 80, 855-859), with a homology of 67.3%.
[0147]
For this reason, the FR of the human antibody HG3 was used as a starting material for producing the H chain V region of the reshaped human anti-HM1.24 antibody. However, since the amino acid sequence of FR4 of the human antibody HG3 is not described, the human antibody JH6 (Ravetch, J. V. et al.) Showing the highest homology with FR4 of the H chain of the mouse anti-HM1.24 antibody was described. Et al., Cell, 27, 583-591). FR4 of JH6 has the same amino acid sequence as FR4 of the H chain of mouse anti-HM1.24 antibody except for one amino acid.
In the first version a of the V region of the H chain of the reshaped human anti-HM1.24 antibody, the amino acids at position 30 in human FR1 and position 71 in human FR3 were the same as those in the mouse anti-HM1.24 antibody. , FR1 to FR3 were the same as FR1 to FR3 of human HG3, and the CDR was the same as the CDR in the H chain V region of the mouse anti-HM1.24 antibody.
[0148]
2. Preparation of reshaped human anti-HM1.24 antibody L chain V region
A reshaped human anti-HM1.24 antibody L chain was prepared by CDR grafting by PCR. This method is schematically shown in FIG. Eight PCR primers were used for generation of a reshaped human anti-HM1.24 antibody (version a) with FR from human antibody REI. External primers A (SEQ ID NO: 47) and H (SEQ ID NO: 48) were designed to hybridize with the DNA sequence of the HEF expression vector HEF-VL-gκ.
CDR-grafting primers L1S (SEQ ID NO: 49), L2S (SEQ ID NO: 50) and L3S (SEQ ID NO: 51) have a sense DNA sequence and CDR-grafting primer L1A (SEQ ID NO: 52), L2A (SEQ ID NO: 53) and L3A (SEQ ID NO: 54) have an anti-sense DNA sequence and the complementary DNA sequence (20-23 bp) to the DNA sequence at the 5'-end of primers L1S, L2S and L3S, respectively. Having.
[0149]
In the first PCR stage, four reactions A-L1A, L1S-L2A, L2S-L3A, and L3S-H were performed and each PCR product was purified. The four PCR products from the first PCR were assembled by their own complementarity (see International Patent Application Publication No. WO 92-19759). Next, external primers A and H were added to amplify the full-length DNA encoding the reshaped human anti-HM1.24 antibody L chain V region (second PCR). In the PCR, the plasmid HEF-RVL-M21a encoding the reshaped human ONS-M21 antibody L chain V region version a based on FR from the human antibody REI (see International Patent Application Publication No. WO95-14401) was used as a template. Using.
[0150]
In the first PCR step, 10 mM Tri-HCl (pH 8.3), 50 mM KCl, 0.1 mM dNTPs, 1.5 mM MgCl₂, 100 ng of template DNA, 100 pmole of each primer and 5 u of Ampli Taq. Each PCR tube was covered with 50 μl of mineral oil. After first denaturation at 94 ° C, a reaction cycle of 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 1 minute was performed, followed by incubation at 72 ° C for 10 minutes.
The PCR products A-L1A (215 bp), L1S-L2A (98 bp), L2S-L3A (140 bp) and L3S-H (151 bp) were purified using a 1.5% low melting point agarose gel and assembled in a second PCR. did. In the second PCR, 1 μg of each first PCR product and 98 μl of the PCR mixture containing 5 u of Ampli Taq were mixed at 94 ° C. for 2 minutes, 55 ° C. for 2 minutes and 72 ° C. for 2 minutes For 2 cycles and then 100 pmole of each external primer (A and H) was added. The PCR tube was covered with 50 μl of mineral oil and 30 cycles of PCR were performed under the same conditions as above.
[0151]
The 516 bp DNA fragment generated by the second PCR was purified on a 1.5% low melting point agarose gel, digested with BamHI and HindIII, and the obtained DNA fragment was cloned into the HEF expression vector HEF-VL-gκ. After DNA sequencing, the plasmid containing the DNA fragment encoding the correct amino acid sequence of the reshaped human anti-HM1.24 antibody L chain V region was named HEF-RVLa-AHM-gκ. The amino acid sequence and the base sequence of the L chain V region contained in this plasmid HEF-RVLa-AHM-gκ are shown in SEQ ID NO: 9.
Version b of the reshaped human anti-HM1.24 antibody L chain V region was prepared by a mutagenesis method using PCR. Mutagenic primers FTY-1 (SEQ ID NO: 55) and FTY-2 (SEQ ID NO: 56) were designed such that phenylalanine at position 71 was mutated to tyrosine.
[0152]
After amplification using the plasmid HEF-RVLa-AHM-gκ as a template and the above primers, the final product was purified, digested with BamHI and HindIII, and the resulting DNA fragment was inserted into a HEF expression vector HEF-VL-gκ. Cloning resulted in plasmid HEF-RVLb-AHM-gκ. The amino acid sequence and the base sequence of the L chain V region contained in this plasmid HEF-RVLb-AHM-gκ are shown in SEQ ID NO: 10.
[0153]
3. Preparation of H chain V region of reshaped human anti-HM1.24 antibody
3-1. Preparation of reshaped human anti-HM1.24 antibody H chain V region version ae
DNA encoding the reshaped human anti-HM1.24 antibody H chain V region was designed as follows. A reshaped human anti-HM1.24 antibody was obtained by connecting a DNA sequence encoding FR1 to FR3 of the human antibody HG3 and FR4 of the human antibody JH6 to a DNA sequence encoding the CDR of the H chain V region of the mouse anti-HM1.24 antibody. A full-length DNA encoding the H chain V region was designed.
Next, a HindIII recognition site / Kozak consensus sequence and a BamHI recognition site / splice donor sequence were added to the 5'-side and 3'-side of this DNA sequence, respectively, so that the DNA sequence could be inserted into the HEF expression vector.
[0154]
The DNA sequence designed in this way was divided into four oligonucleotides, and then analyzed for secondary structures in the oligonucleotides that could interfere with the assembly of these oligonucleotides. The four oligonucleotide sequences RVH1-RVH4 are shown in SEQ ID NOs: 57-60. These oligonucleotides are 119-144 bases in length and have an overlap region of 25-26 bp. Of the oligonucleotides, RVH2 (SEQ ID NO: 58), RVH4 (SEQ ID NO: 60) have a sense DNA sequence, and the other RVH1 (SEQ ID NO: 57), RVH3 (SEQ ID NO: 59) are antisense DNA Has an array. The method of assembling these four oligonucleotides by PCR is shown in the figure (see FIG. 5).
[0155]
98 μl of the PCR mixture containing 100 ng of each of the four oligonucleotides and 5 u of Ampli Taq was first denatured at 94 ° C. for 2 minutes, followed by 2 minutes at 94 ° C., 2 minutes at 55 ° C. Two cycles of incubation consisting of 2 minutes at 72 ° C. were performed. After adding 100 pmole of RHP1 (SEQ ID NO: 61) and RHP2 (SEQ ID NO: 62) as external primers, cover the PCR tube with 50 μl of mineral oil and after the first denaturation at 94 ° C. for 1 minute, 38 cycles of 1 minute at 94 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C were performed, and then incubated at 72 ° C for 10 minutes.
[0156]
The 438 bp DNA fragment was purified using a 1.5% low melting point agarose gel, digested with HindIII and BamHI, and then cloned into the HEF expression vector HEF-VH-gγ1. After DNA sequencing, the plasmid containing the DNA fragment encoding the amino acid sequence of the correct H chain V region was named HEF-RVHa-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHa-AHM-gγ1 are shown in SEQ ID NO: 11.
Each version b, c, d and e of the reshaped human anti-HM1.24 antibody H chain V region was prepared as follows.
[0157]
Version b uses BS (SEQ ID NO: 63) and BA (SEQ ID NO: 64) designed such that arginine at position 66 is mutated to lysine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA. Was amplified by a PCR method to obtain a plasmid HEF-RVHb-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHb-AHM-gγ1 are shown in SEQ ID NO: 12.
Version c uses CS (SEQ ID NO: 65) and CA (SEQ ID NO: 66) designed such that threonine at position 73 mutates to lysine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA Was amplified by a PCR method to obtain a plasmid HEF-RVHc-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHc-AHM-gγ1 are shown in SEQ ID NO: 13.
[0158]
Version d uses DS (SEQ ID NO: 67) and DA (SEQ ID NO: 68) designed to mutate arginine at position 66 to lysine and threonine at position 73 to lysine as mutagenic primers. Plasmid HEF-RVHd-AHM-gγ1 was obtained using RVHa-AHM-gγ1 as a template DNA. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHd-AHM-gγ1 are shown in SEQ ID NO: 14.
Version e was used as a mutagenic primer using ES (SEQ ID NO: 69) and EA (SEQ ID NO: 70) designed to mutate valine at position 67 to alanine and methionine at position 69 to leucine, and used plasmid HEF- RVHa-AHM-gγ1 was amplified as a template DNA to obtain plasmid HEF-RVHe-AHM-gγ1. The amino acid sequence and the base sequence contained in the V region of the H chain contained in this plasmid HEF-RVHe-AHM-gγ1 are shown in SEQ ID NO: 15.
[0159]
3-2. Construction of H chain hybrid V region
Two types of heavy chain hybrid V regions were constructed. One is a mouse / human hybrid in which the amino acid sequences of FR1 and FR2 are derived from a mouse anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 are derived from version a of the H chain V region of a reshaped human anti-HM1.24 antibody. An anti-HM1.24 antibody; the other is that the amino acid sequence of FR1 and FR2 is derived from version a of the V region of the H chain of the reshaped human anti-HM1.24 antibody, and the amino acid sequence of FR3 and FR4 is a mouse anti-HM1.24 antibody It is a human / mouse hybrid anti-HM1.24 antibody from which it is derived. All amino acid sequences of the CDR regions are derived from a mouse anti-HM1.24 antibody.
[0160]
Two types of H chain hybrid V regions were prepared by a PCR method. This method is schematically illustrated in FIGS. Four primers were used to generate two H chain hybrid V regions. External primers a (SEQ ID NO: 71) and h (SEQ ID NO: 72) were designed to hybridize with the DNA sequence of the HEF expression vector HEF-VH-gγ1. The H-chain hybrid-producing primer HYS (SEQ ID NO: 73) has a sense DNA sequence, and the H-chain hybrid primer HYA (SEQ ID NO: 74) has an antisense DNA sequence and thus has a complementary DNA sequence. Designed.
[0161]
Preparation of H-chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from mouse anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 are derived from version a of H-chain V region of reshaped human anti-HM1.24 antibody In the first PCR step, PCR using the plasmid HEF-1.24H-gγ1 as a template and the external primer a and the H chain hybrid primer HYA, and the plasmid HEF-RVHa-AHM-gγ1 as a template and the H chain hybrid primer PCR using HYS (SEQ ID NO: 73) and external primer h (SEQ ID NO: 72) was performed, and each PCR product was purified. The two PCR products from the first PCR were assembled by their own complementarity (see International Patent Application Publication No. WO 92-19759).
[0162]
Next, by adding external primers a (SEQ ID NO: 71) and h (SEQ ID NO: 72), the amino acid sequences of FR1 and FR2 are derived from mouse anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 are reconstructed. The full-length DNA encoding the H chain hybrid V region derived from version H of the H chain V region of the human anti-HM1.24 antibody was amplified in the second PCR step.
Preparation of H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from version a of the H chain V region of reshaped human anti-HM 1.24 antibody, and the amino acid sequences of FR3 and FR4 are derived from mouse anti-HM 1.24 antibody In the first PCR step, PCR using the plasmid HEF-RVHa-AHM-gγ1 as a template and the external primer a and the H-chain hybrid primer HYA, and the H-chain hybrid primer using the plasmid HEF-1.24H-gγ1 as a template PCR using HYS and external primer h was performed and each PCR product was purified. The two PCR products from the first PCR were assembled by their own complementarity (see International Patent Application Publication No. WO 92-19759).
[0163]
Next, by adding external primers a and h, the amino acid sequences of FR1 and FR2 are derived from version a of the V region of the H chain of the reshaped human anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 are derived from mouse anti-HM1. The full-length DNA encoding the H chain hybrid V region derived from the 24 antibody was amplified in a second PCR step.
The method for the first PCR, purification of the PCR product, assembly, second PCR, and cloning into the HEF expression vector HEF-VH-gγ1 is described in Example 9. The method was the same as that described for the preparation of the reshaped human HM1.24 antibody L chain V region.
[0164]
After DNA sequencing, the amino acid sequences of FR1 and FR2 were derived from the mouse anti-HM1.24 antibody, and the amino acid sequences of FR3 and FR4 were derived from version a of the H chain V region of the reshaped human anti-HM1.24 antibody. The plasmid containing the DNA fragment encoding the correct amino acid sequence of the chain hybrid V region was designated as HEF-MH-RVH-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-MH-RVH-AHM-gγ1 are shown in SEQ ID NO: 75. The amino acid sequences of FR1 and FR2 are derived from version a of the reshaped human anti-HM1.24 antibody H chain V region, and the amino acid sequences of FR3 and FR4 are derived from the mouse antibody HM1.24 antibody. The plasmid containing the DNA fragment encoding the correct amino acid sequence was designated as HEF-HM-RVH-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-HM-RVH-AHM-gγ1 are shown in SEQ ID NO: 76.
[0165]
3-3. Preparation of reshaped human anti-HM1.24 antibody H chain V region version fs
Each version f, g, h, i, j, k, l, m, n, o, p, q, r and s of the reshaped human anti-HM1.24 antibody H chain V region was prepared as follows. .
Version f uses FS (SEQ ID NO: 78) and FA (SEQ ID NO: 79) designed to mutate threonine at position 75 to serine and valine at position 78 to alanine as mutagenic primers. Using RVHe-AHM-gγ1 as a template DNA, amplification was performed by the PCR method to obtain a plasmid HEF-RVHf-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHf-AHM-gγ1 are shown in SEQ ID NO: 16.
[0166]
Version g uses GS (SEQ ID NO: 80) and GA (SEQ ID NO: 81) designed so that alanine at position 40 is mutated to arginine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA To obtain plasmid HEF-RVHg-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHg-AHM-gγ1 are shown in SEQ ID NO: 17.
Version h uses FS (SEQ ID NO: 78) and FA (SEQ ID NO: 79) as mutagenic primers, amplifies plasmid HEF-RVHb-AHM-gγ1 as template DNA, and converts plasmid HEF-RVHh-AHM-gγ1 into plasmid HEF-RVHh-AHM-gγ1. Obtained. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHh-AHM-gγ1 are shown in SEQ ID NO: 18.
[0167]
Version i was designed using mutagenic primers such as IS (SEQ ID NO: 82) and IA (SEQ ID NO: 83) designed to mutate arginine at position 83 to alanine and serine at position 84 to phenylalanine, and used plasmid HEF- Amplification was performed using RVHh-AHM-gγ1 as a template DNA to obtain plasmid HEF-RVHi-AHM-gγ1. The amino acid sequence and base sequence of the V region of the H chain contained in this plasmid HEF-RVHi-AHM-gγ1 are shown in SEQ ID NO: 19.
Version j uses JS (SEQ ID NO: 84) and JA (SEQ ID NO: 85) designed to mutate arginine at position 66 to lysine as a mutagenic primer, and uses plasmid HEF-RVHf-AHM-gγ1 as a template DNA To obtain plasmid HEF-RVHj-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHj-AHM-gγ1 are shown in SEQ ID NO: 20.
[0168]
Version k uses KS (SEQ ID NO: 86) and KA (SEQ ID NO: 87) designed such that glutamic acid at position 81 mutates to glutamine as a mutagenic primer, and uses plasmid HEF-RVHh-AHM-gγ1 as a template DNA To obtain plasmid HEF-RVHk-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHk-AHM-gγ1 are shown in SEQ ID NO: 21.
Version 1 uses LS (SEQ ID NO: 88) and LA (SEQ ID NO: 89) designed to mutate glutamic acid at position 81 to glutamine and serine at position 82B to isoleucine as mutagenic primers, and to use plasmid HEF- RVHh-AHM-gγ1 was amplified as a template DNA to obtain plasmid HEF-RVH1-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVH1-AHM-gγ1 are shown in SEQ ID NO: 22.
[0169]
Version m was designed to mutate glutamic acid at position 81 to glutamine, serine at position 82b to isoleucine, and threonine at position 87 to serine as mutagenic primers, MS (SEQ ID NO: 90) and MA (SEQ ID NO: 91), plasmid HEF-RVHh-AHM-gγ1 was amplified as a template DNA to obtain plasmid HEF-RVHm-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHm-AHM-gγ1 are shown in SEQ ID NO: 23.
Version n uses NS (SEQ ID NO: 92) and NA (SEQ ID NO: 93) designed so that serine at position 82B mutates to isoleucine as a mutagenic primer, and uses plasmid HEF-RVHh-AHM-gγ1 as a template DNA. As a result, a plasmid HEF-RVHn-AHM-gγ1 was obtained. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHn-AHM-gγ1 are shown in SEQ ID NO: 24.
[0170]
Version o uses OS (SEQ ID NO: 94) and OA (SEQ ID NO: 95) designed such that threonine at position 87 mutates to serine as a mutagenic primer, and uses plasmid HEF-RVHh-AHM-gγ1 as a template DNA As a result, a plasmid HEF-RVHo-AHM-gγ1 was obtained. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHo-AHM-gγ1 are shown in SEQ ID NO: 25.
Version p uses PS (SEQ ID NO: 96) and PA (SEQ ID NO: 97) designed so that valine at position 78 mutates to alanine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA. Was amplified by a PCR method to obtain a plasmid HEF-RVHp-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHp-AHM-gγ1 are shown in SEQ ID NO: 26.
[0171]
Version q uses QS (SEQ ID NO: 98) and QA (SEQ ID NO: 99) designed such that threonine at position 75 mutates to serine as a mutagenic primer, and uses plasmid HEF-RVHa-AHM-gγ1 as a template DNA Was amplified by a PCR method to obtain a plasmid HEF-RVHq-AHM-gγ1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHq-AHM-gγ1 are shown in SEQ ID NO: 27.
Version r was amplified by the PCR method using CS (SEQ ID NO: 65) and CA (SEQ ID NO: 66) as mutagenic primers, plasmid HEF-RVHp-AHM-gγ1 as template DNA, and plasmid HEF-RVHr- AHM-gγ1 was obtained. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHr-AHM-gγ1 are shown in SEQ ID NO: 28.
[0172]
Version s of the reshaped human anti-HM1.24 antibody H chain V region was prepared by a mutagenesis method using PCR. Mutagenic primers SS (SEQ ID NO: 100) and SA (SEQ ID NO: 101) were designed such that methionine at position 69 was mutated to isoleucine.
After amplification using the plasmid HEF-RVHr-AHM-gγ1 as a template and the above primers, the final product was purified, digested with BamHI and HindIII, and the resulting DNA fragment was used as a HEF expression vector HEF-VH-gγ1. By cloning, plasmid HEF-RVHs-AHM-gγ1 was obtained. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHs-AHM-gγ1 are shown in SEQ ID NO: 102.
[0173]
The regions encoding the respective variable regions from the plasmids HEF-RVLa-AHM-gκ and HEF-RVHr-AHM-gγ1 were converted into restriction fragments with the restriction enzymes HindIII and BamHI, and these were inserted into the HindIII and BamHI sites of the plasmid vector pUC19. Inserted. The respective plasmids were named pUC19-RVLa-AHM-gκ and pUC19-RVHr-AHM-gγ1.
In addition, Escherichia coli containing each of the plasmids pUC19-RVLa-AHM-gκ and pUC19-RVHr-AHM-gγ1 was Escherichia coli DH5α (pUC19-RVLa-AHM-gκ) and EscherichiaVH-AHC-HHC-DH19-HHC-DH19-E. -Gγ1) and submitted to the Research Institute of Biotechnology and Industrial Technology (1-3 1-3, Higashi, Tsukuba, Ibaraki Prefecture) on August 29, 1996, as FERM BP-5645 and FERM BP-5643, respectively. Was deposited internationally.
[0174]
The region encoding the variable region from the plasmid HEF-RVHs-AHM-gγ1 was converted into a restriction fragment with restriction enzymes HindIII and BamHI, which were inserted into the BamHI and HindIII sites of the plasmid vector pUC19. The resulting plasmid was designated as pUC19-RVHs-AHM-gγ1.
Escherichia coli containing the plasmid pUC19-RVHs-AHM-gγ1 is referred to as Escherichia coli DH5α (pUC19-RVHs-AHM-gγ1), and the Institute of Biotechnology, Industrial Science and Technology Institute (1-1-3 Higashi, Tsukuba, Ibaraki) Was deposited internationally on September 29, 1997 under the Budapest Treaty as FERM BP-6127.
[0175]
4. Reshaped human anti-HM1.24 antibody, chimeric anti-HM1.24 antibody, and heavy chain hybrid antibody
Making the body
To evaluate each chain of the reshaped human anti-HM 1.24 antibody, a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody as a positive control antibody were expressed. When preparing each version of the reshaped human anti-HM1.24 antibody H chain V region after version b, an H chain hybrid antibody was expressed in order to examine which amino acid residue in which FR should be substituted. For evaluation of the reshaped human anti-HM1.24 antibody light chain version a, it was expressed in combination with a chimeric heavy chain.
[0176]
4-1. Expression of reshaped human anti-HM1.24 antibody (1)
Expression vectors for the reshaped human anti-HM 1.24 antibody heavy chain (HEF-RVHa-AHM-gγ1 to HEF-RVHr-AHM-gγ1) and the expression vector for the reshaped human anti-HM 1.24 antibody light chain (HEF) 10 μg each of -RVLa-AHM-gκ or HEF-RVLb-AHM-gκ) was co-transformed into COS-7 cells by electroporation using a Gene Pulser device (BioRad). Each DNA (10 μg) was added to 1 × 10⁷A 0.8 ml aliquot of cells / ml was added and pulsed at 1500 V, 25 μF volume.
[0177]
After a 10 minute recovery period at room temperature, the electroporated cells were added to 30 ml of DMEM culture (GIBCO) containing 10% γ-globulin-free fetal bovine serum. 37 ° C, 5% CO₂Culture for 72 hours under the conditions of₂After performing the culture using an incubator BNA120D (manufactured by TABAI), the culture supernatant was collected, and centrifuged at 1000 rpm for 5 minutes by a centrifuge 15PR-22 (manufactured by HITACHI) equipped with a centrifugal rotor 03 (manufactured by HITACHI). To remove cell debris, and a microconcentrator (Centricon 100, manufactured by Amicon) using a centrifuge J2-21 (manufactured by BECKMAN) equipped with a centrifugal rotor JA-20-1 (manufactured by BECKMAN) at 2000 rpm. The solution was subjected to ultrafiltration and concentration under the following conditions and used for Cell-ELISA.
[0178]
Expression of reshaped human anti-HM1.24 antibody (2)
An expression vector for the reshaped human anti-HM1.24 antibody heavy chain version s (HEF-RVHs-AHM-gγ1) and an expression vector for the reshaped human anti-HM1.24 antibody light chain (HEF-RVLa-AHM-gκ) ) 10 µg of each was co-transformed into COS cells by electroporation using a Gene Pulser device (BioRad). Each DNA (10 μg) was added to 1 × 10⁷A 0.8 ml aliquot of cells / ml was added and pulsed at 1,500 V, 25 μF volume.
[0179]
After a 10 minute recovery period at room temperature, the electroporated cells were added to 30 ml of DMEM culture (GIBCO) containing 10% γ-globulin-free fetal bovine serum. 37 ° C, 5% CO₂Culture for 72 hours under the conditions of₂After performing using an incubator BNA120D (manufactured by TABAI), the culture supernatant was collected, and centrifuged at 1000 rpm for 5 minutes by a centrifuge 05PR-22 (manufactured by HITACHI) equipped with a centrifugal rotor 03 (manufactured by HITACHI). The cell debris was removed, and a microconcentrator (Centricon 100, manufactured by Amicon) was centrifuged with a centrifuge J2-21 (manufactured by BECKMAN) equipped with a centrifugal rotor JA-20-1 (manufactured by BECKMAN) under the condition of 2000 rpm. Ultrafiltration and concentration were performed, and a filter sterilized using a filter, Millex GV 13 mm (manufactured by Millipore) was used for Cell-ELISA.
[0180]
4-2. Expression of chimeric anti-HM1.24 antibody
Using the expression vector HEF-1.24H-gγ1 for the chimeric anti-HM1.24 antibody H chain and 10 μg each of the expression vector HEF-1.24L-gκ for the chimeric anti-HM1.24 antibody L chain, A chimeric anti-HM1.24 antibody for use in Cell-ELISA was prepared according to the method of expressing an anti-HM1.24 antibody.
[0181]
4-3. Expression of anti-HM1.24 antibody consisting of humanized L chain version a and chimeric H chain
Using an expression vector HEF-1.24H-gγ1 for the chimeric anti-HM1.24 antibody heavy chain and 10 μg each of an expression vector HEF-RVLa-AHM-Gκ for the reshaped human anti-HM1.24 antibody light chain version a According to the method for expressing the reshaped human anti-HM 1.24 antibody, an anti-HM 1.24 antibody comprising a humanized L chain version a and a chimeric H chain for use in Cell-ELISA was prepared.
[0182]
4-4. Expression of H chain hybrid antibody
An expression vector (HEF-MH-RVH-AHM-gγ1 or HEF-HM-RVH-AHM-gγ1) for the H chain hybrid V region and an expression vector HEF-RVLa for the reshaped human anti-HM1.24 antibody L chain Using 10 μg of each of -AHM-gκ, an H-chain hybrid antibody to be used for Cell-ELISA was prepared according to the method of expressing the reshaped human anti-HM1.24 antibody.
[0183]
4-5. Measurement of antibody concentration
The concentration of the obtained antibody was measured by ELISA. A coating buffer (0.1 M NaHCO3) was added to each well of a 96-well plate for ELISA (Maxisorp, manufactured by NUNC).₃, 0.02% NaN₃, PH 9.6) and 100 μl of a goat anti-human IgG antibody (manufactured by BIO SOURCE) adjusted to a concentration of 1 μg / ml, and incubated at room temperature for 1 hour to solidify. 100 μl of dilution buffer (50 mM Tris-HCl, 1 mM MgCl₂, 0.15 M NaCl, 0.05% Tween 20, 0.02% NaN₃, 1% bovine serum albumin (BSA), pH 8.1), followed by ultrafiltration and concentration, followed by reconstituted human anti-HM1.24 antibody, chimeric anti-HM1.24 antibody, and H-chain hybrid antibody. After dilution and addition of 100 μl to each well, incubation and washing at room temperature for 1 hour, 100 μl of alkaline phosphatase-labeled goat anti-human IgG antibody (manufactured by DAKO) was added.
[0184]
After 1 hour incubation and washing at room temperature, the substrate buffer (50 mM NaHCO3)₃, 10 mM MgCl₂(PH 9.8)), 100 μl of a 1 mg / ml substrate solution (Sigma 104, p-nitrophenyl phosphate, manufactured by SIGMA) was added, and the absorbance at 405 nm was measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad). It measured using. A human IgG1κ (manufactured by The Binding Site) was used as a sample for concentration measurement.
[0185]
5. Establishment of CHO cell line stably producing reshaped human anti-HM1.24 antibody
5-1. Preparation of H chain expression vector for reshaped human anti-HM1.24 antibody
The plasmid HEF-RVHr-AHM-gγ1 was digested with restriction enzymes PvuI and BamHI, and a fragment of about 2.8 kbp containing the EF1 promoter and DNA encoding the H chain V region of the reshaped human anti-HM1.24 antibody was obtained. Purification was performed using a 5% low melting point agarose gel. Next, the expression vectors used in the human H chain expression vector DHFR-ΔE-RVh-PM1f (International Patent Application Publication No. WO92-19759) containing the DHFR gene and the gene encoding the human H chain constant region were PvuI and BamHI. The above DNA fragment was inserted into a fragment of about 6 kbp prepared by digestion with the above, to construct a reshaped human anti-HM1.24 antibody H chain expression vector DHFR-ΔE-HEF-RVHr-AHM-gγ1.
[0186]
5-2. Gene transfer into CHO cells
In order to establish a stable production system of a reshaped human anti-HM1.24 antibody, the expression vectors DHFR-ΔE-HEF-RVHr-AHM-gγ1 and HEF-RVLa-AHM-gκ which had been digested with PvuI and made linear were used. The gene was simultaneously introduced into CHO cells DXB-11 by electroporation under the same conditions as described above (transfection into COS-7 cells).
[0187]
5-3. Gene amplification by MTX
The transfected CHO cells were isolated from the light chain and H chain in a nucleoside-free α-MEM culture solution (GIBCO-BRL) supplemented with 500 μg / ml G418 (GIBCO-BRL) and 10% fetal bovine serum. Only CHO cells into which the chain expression vector had been introduced were able to grow and were selected. Next, 10 nM MTX (manufactured by Sigma) was added to the above culture solution, and a clone having a high production of reconstituted human anti-HM1.24 antibody was selected from the grown clones. Clone # 1 showing the production rate of the constituent human anti-HM1.24 antibody was obtained and used as a reconstituted human anti-HM1.24 antibody-producing cell line.
[0188]
5-4. Production of reshaped human anti-HM1.24 antibody
Preparation of the reshaped human anti-HM1.24 antibody was performed by the following method. The reconstituted human anti-HM1.24 antibody-producing CHO cells were transformed with 500 μg / ml G418 (GIBCO-BRL) containing 10% γ-globulin-free fetal bovine serum (GIBCO-BRL) as a medium. Using the added nucleoside-free α-MEM culture solution (manufactured by GIBCO-BRL) at 37 ° C., 5% CO 2₂Culture for 10 days under the conditions of₂This was performed using an incubator BNA120D (manufactured by TABAI). On days 8 and 10 after the start of the culture, the culture solution was collected, and centrifuged at 2,000 rpm for 10 minutes using a centrifuge RL-500SP (manufactured by Tommy Seiko) equipped with a TS-9 rotor to perform cell separation in the culture solution. After removing the debris, the mixture was sterilized by filtration through a bottle top filter (manufactured by FALCON) having a 0.45 μm diameter membrane.
[0189]
After adding an equal amount of PBS (-) to the reconstituted human anti-HM1.24 antibody-producing CHO cell culture solution, a high-speed antibody purifier ConSep LC100 (manufactured by MILLIPORE) and a Hyper D Protein A column (manufactured by NGK Insulators, Ltd.) The reconstituted human anti-HM1.24 antibody was affinity purified using PBS (-) as the adsorption buffer and 0.1 M sodium citrate buffer (pH 3) as the elution buffer based on the attached instructions. Immediately after adding the 1M Tris-HCl (pH 8.0) to adjust the pH to around 7.4, the eluted fraction was concentrated using a centrifugal ultraconcentrator Centriprep 10 (manufactured by MILLIPORE) and concentrated into PBS (-). The solution was replaced with a buffer, and sterilized by filtration using a membrane filter MILLEX-GV (manufactured by MILLIPORE) having a pore size of 0.22 μm to obtain a purified and reconstituted human anti-HM1.24 antibody. The concentration of the purified antibody was calculated by measuring the absorbance at 280 nm and setting 1 mg / ml to 1.350D.
[0190]
Example 11 .  Reconstituted human anti HM1.24 Antibody activity measurement
The reshaped human anti-HM1.24 antibody was evaluated based on the following antigen binding activity and binding inhibition activity.
1. Methods for measuring antigen binding activity and binding inhibitory activity
1-1. Measurement of antigen binding activity
The antigen-binding activity was measured by Cell-ELISA using WISH cells. Cell-ELISA plates were prepared as described in Example 7.1-2 above.
[0191]
After blocking, the reconstituted human anti-HM1.24 antibody obtained by concentrating the culture supernatant of COS-7 cells or purified from the culture supernatant of CHO cells was serially diluted, and 100 μl was added to each well. After incubation and washing at room temperature for 2 hours, a peroxidase-labeled rabbit anti-human IgG antibody (DAKO) was added. After incubation and washing at room temperature for 1 hour, a substrate solution was added, and after incubation, the reaction was stopped with 50 μl of 6N sulfuric acid, and the absorbance at 490 nm was measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad).
[0192]
1-2. Measurement of binding inhibitory activity
The binding inhibition activity by the biotin-labeled mouse anti-HM1.24 antibody was performed by Cell-ELISA using WISH cells. Cell-ELISA plates were prepared as described above. After blocking, the reconstituted human anti-HM1.24 antibody obtained by concentrating the culture supernatant of COS-7 cells or purified from the culture supernatant of CHO cells was serially diluted, and 50 μl was added to each well. At the same time, 50 μl of 2 μg / ml biotin-labeled mouse anti-HM1.24 antibody was added, and after incubation and washing at room temperature for 2 hours, peroxidase-labeled streptavidin (manufactured by DAKO) was added. After incubation at room temperature for 1 hour, washing was performed, a substrate solution was added, and after incubation, the reaction was stopped with 50 μl of 6N sulfuric acid, and the absorbance at 490 nm was measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad).
[0193]
2. Evaluation of reshaped human anti-HM1.24 antibody
2-1. L chain
The evaluation of the light chain version a of the reshaped human anti-HM1.24 antibody was performed by measuring the antigen binding activity described above. As shown in FIG. 8, when the L chain version a was expressed in combination with the chimeric H chain, it exhibited an antigen binding activity comparable to that of the chimeric anti-HM1.24 antibody. However, in consideration of further increase in activity or compatibility with the H chain, a new L chain version b was prepared. Then, the antigen binding activity and the binding inhibitory activity when combined with the H chain versions a, b, f or h were measured to evaluate both the L chain versions a and b. As shown in FIGS. 9, 10, 11 and 12, in all versions of H chains a, b, f and h, L chain version a was stronger in both activities than version b. Therefore, the light chain version a of the reshaped human anti-HM1.24 antibody was used in the following experiments.
[0194]
2-2. H chain version ae
The evaluation of the H chain version ae of the reshaped human anti-HM1.24 antibody was performed by measuring the antigen binding activity and the binding inhibitory activity in combination with the L chain version a. As a result, as shown in FIGS. 11, 13, 14, and 15, both activities were weaker in all versions compared to the chimeric anti-HM1.24 antibody, and it was considered that further amino acid conversion was required.
[0195]
2-3. H chain hybrid antibody
The evaluation of the H-chain hybrid antibody was performed by measuring the antigen-binding activity described above. As a result, as shown in FIG. 16, the antigen-binding activity of the human-mouse hybrid anti-HM1.24 antibody is equivalent to that of the chimeric anti-HM1.24 antibody, while the mouse-human hybrid anti-HM1.24 antibody has Its activity was weaker than that of the chimeric anti-HM1.24 antibody. Therefore, in order to prepare a reshaped human HM1.24 antibody having an antigen-binding activity equivalent to that of a mouse anti-HM1.24 antibody or a chimeric anti-HM1.24 antibody, it is necessary to include FR3 or FR4 in the H chain V region. It has been shown that certain amino acids need to be changed.
[0196]
2-4. H chain version fr
The evaluation of the H chain version f of the reshaped human anti-HM1.24 antibody was performed by the above-mentioned antigen binding activity measurement. As a result, as shown in FIG. 17, the antigen-binding activity was inferior to that of the chimeric anti-HM1.24 antibody, but the activity was improved as compared to the above-mentioned versions ac, and thus the newly converted 67, It was suggested that any of the four amino acids at positions 69, 75 and 78 were involved in the activity of the reshaped human antibody.
The H chain version g of the reshaped human anti-HM1.24 antibody was evaluated by measuring the antigen binding activity and the binding inhibitory activity described above. As a result, as shown in FIGS. 18 and 19, this version showed only the same activity as that of version a, and as shown in the evaluation of the H chain human / mouse hybrid antibody, the version was converted by this version. It was shown that the second amino acid did not contribute to improving the activity of the reshaped human antibody.
[0197]
The H chain version hj of the reshaped human anti-HM1.24 antibody was evaluated by measuring the antigen binding activity and the binding inhibitory activity described above. As a result, as shown in FIGS. 20, 21, 22, and 23, both activities were weak in all versions as compared to the chimeric anti-HM1.24 antibody, and were almost the same as those in version f. It was suggested that out of the four amino acids, amino acids 67 and 69 did not contribute to the improvement of the activity of the reshaped human antibody.
Evaluation of the version k-p of the H chain of the reshaped human anti-HM1.24 antibody was performed by measuring the antigen binding activity and the binding inhibitory activity described above. As a result, as shown in FIGS. 24, 25, 26, and 27, both activities were weaker than those of the chimeric anti-HM1.24 antibody in all versions, and were similar to those of the above-mentioned version h. It has been suggested that the 80th and subsequent amino acids converted to are not contributing to the improvement of the activity of the reshaped human antibody.
[0198]
The evaluation of the H chain version q of the reshaped human anti-HM1.24 antibody was performed by measuring the antigen binding activity and the binding inhibitory activity. As a result, as shown in FIGS. 25 and 27, this version was weaker in both activities as compared to version h or version p, and had only the same activity as version a. It is suggested that it is essential for improving the activity of the reshaped human antibody.
The evaluation of the H chain version r of the reshaped human anti-HM1.24 antibody was performed by the above-described measurement. As a result, as shown in FIGS. 15 and 28, version r was shown to have the same antigen-binding activity and binding-inhibitory activity as the chimeric anti-HM1.24 antibody.
[0199]
From the above results, the minimum necessary conversion in the H chain for the reshaped human anti-HM 1.24 antibody to have the same antigen binding ability as the mouse anti-HM 1.24 antibody or the chimeric anti-HM 1.24 antibody is 30, It was shown to be amino acids 71 and 78, and even amino acid 73.
Table 6 summarizes the antigen-binding activity and the binding-inhibiting activity of the reshaped human anti-HM1.24 antibody H-chain version ar.
[0200]
[Table 6]

[0201]
2.5. H chain version s
Evaluation of the H chain version s of the reshaped human anti-HM1.24 antibody was carried out by measuring the antigen binding activity and the binding inhibitory activity in combination with the L chain version a. As a result, as shown in FIGS. 29 and 30, version s was shown to have the same antigen binding activity and binding inhibitory activity as version r.
Further, as described above, the reshaped human anti-HM1.24 antibody of the present invention retains its ability to bind to an antigen even after substituting one or more amino acid residues in FR with another amino acid. I have. Therefore, the present invention relates to a reshaped human in which one or more amino acid residues are substituted with another amino acid residue in the V region of an H chain or an L chain as long as the original properties are maintained. Also encompasses anti-HM1.24 antibodies.
[0202]
3. Evaluation of purified reshaped human anti-HM1.24 antibody
The purified and reconstituted human anti-HM1.24 antibody was evaluated based on the antigen binding activity and binding inhibition activity described above. As a result, as shown in FIGS. 31 and 32, the reshaped human anti-HM1.24 antibody was shown to have the same antigen binding activity and binding inhibitory activity as the chimeric anti-HM1.24 antibody. This indicated that the reshaped human anti-HM1.24 antibody had the same antigen-binding ability as the mouse anti-HM1.24 antibody.
[0203]
Example 12 .  Chimera anti HM1.24 Antitumor effect of antibody on human myeloma mouse model
1. Preparation of administration antibody
1-1. Preparation of chimeric anti-HM1.24 antibody
The purified chimeric anti-HM 1.24 antibody obtained in Example 6 was concentrated using a centrifugal ultracentrifuge Centriprep 10 (manufactured by MILLIPORE), and the buffer solution was replaced with PBS (-) to give a membrane having a pore size of 0.22 μm. The solution was sterilized by filtration using a filter MILLEX-GV (manufactured by MILLIPORE). This was adjusted to 200 μg / ml using filter-sterilized PBS (−) and used for the following experiments. The antibody concentration was measured by measuring the absorbance at 280 nm and calculating 1 mg / ml as 1.350D.
[0204]
1-2. Purification of control human IgG1
Human IgG1 used as a control for the chimeric anti-HM1.24 antibody was purified as follows. After adding an equal amount of PBS (-) to Hu IgG1 Kappa Purified (manufactured by BINDING SITE), a high-speed antibody purifier ConSep LC100 (manufactured by MILLIPORE) and a Hyper D Protein A column (manufactured by NGK) were used. Affinity purification was carried out using PBS (-) as the adsorption buffer and 0.1 M sodium citrate buffer (pH 3) as the elution buffer based on the instructions in the above. Immediately after adding the 1M Tris-HCl (pH 8.0) to adjust the pH to around 7.4, the eluted fraction was concentrated using a centrifugal ultraconcentrator Centriprep 10 (manufactured by MILLIPORE) and concentrated into PBS (-). The solution was replaced with a buffer, and sterilized by filtration using a membrane filter MILLEX-GV (manufactured by MILLIPORE) having a pore size of 0.22 μm. This was adjusted to 200 μg / ml using filter-sterilized PBS (−) and used for the following experiments. The antibody concentration was measured by measuring the absorbance at 280 nm and calculating 1 mg / ml as 1.350D.
[0205]
2. Mouse serum human IgG assay
Quantification of human IgG contained in mouse serum was performed by the following ELISA. 100 μl of goat anti-human IgG (manufactured by TAGO) diluted to 1 μg / ml with 0.1 M bicarbonate buffer (pH 9.6) was added to a 96-well plate (manufactured by Nunc) and incubated at 4 ° C. overnight. The antibody was immobilized. After blocking, 100 μl of serially diluted mouse serum or human IgG (manufactured by Cappel) as a standard was added, and the mixture was incubated at room temperature for 1 hour. After washing, 100 μl of alkaline phosphatase-labeled anti-human IgG (manufactured by Cappel) diluted 2000-fold was added, and the mixture was incubated at room temperature for 1 hour. After washing, a substrate solution was added, and after incubation, absorbance at 405 nm was measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad).
[0206]
3. Antitumor effect of chimeric anti-HM1.24 antibody on human myeloma transplanted mice
3-1. Preparation of mice transplanted with human myeloma
Mice transplanted with human myeloma were prepared as follows. In vivo passage of KPMM2 cells using SCID mice (CLEA Japan) was performed at 3 × 10 5 in RPMI1640 medium (GIBCO-BRL) containing 10% fetal bovine serum (GIBCO-BRL).⁷/ Ml. 200 μl of the above KPMM2 cell suspension was injected from the tail vein into SCID mice (male, 8 weeks old) (CLEA Japan) to which 100 μl of anti-asialo GM1 (manufactured by Wako Pure Chemical Industries) was intraperitoneally administered the day before.
[0207]
3-2. Antibody administration
Serum was collected from the human myeloma-transplanted mouse on day 12 after KPMM2 cell transplantation, and human IgG in the serum was quantified using the ELISA described in 2 above. Elevation of human IgG in serum confirmed the engraftment of BM in KPMM2 cells. These mice were intraperitoneally administered with 100 μl each of the antibody prepared in 1 above on days 14, 21, and 28 after transplantation of KPMM2 cells.
3-3. Evaluation of antitumor effect of chimeric anti-HM1.24 antibody on human myeloma transplanted mice
The antitumor effect of the chimeric anti-HM1.24 antibody was evaluated by the survival time of the mouse. As shown in FIG. 33, the mice to which the chimeric anti-HM1.24 antibody was administered exhibited a longer survival time than the mice to which control human IgG1 was administered. Therefore, it was shown that the chimeric anti-HM1.24 antibody has an antitumor effect on human myeloma transplanted mice.
[0208]
Example Thirteen .  Reconstituted human anti HM1.24 Antibody ADCC Activity measurement
ADCC (Antibody-dependent Cellular Cytotoxicity) activity was measured in Current Protocols in Immunology, Chapter 7. See Immunological studies in humans, Editor, John E, Coligan et al. , John Wiley & Sons, Inc. , 1993.
1. Preparation of effector cells
Mononuclear cells were separated from peripheral blood of a healthy person by specific gravity centrifugation. That is, an equal amount of PBS (-) was added to peripheral blood of a healthy person, laminated on Ficoll-Paque PLUS (manufactured by Pharmacia), and centrifuged at 400 g for 40 minutes. The mononuclear cell layer was separated, washed four times with RPMI 1640 (GIBCO BRL) containing 10% fetal bovine serum (GIBCO BRL), and the cell number was 5 × 10 5 in the same culture solution.⁶/ Ml.
[0209]
LAK (Limphokine Activated Killer Cell) was induced from bone marrow cells of SCID mice (CLEA Japan). That is, bone marrow cells were separated from the femur of a mouse, washed twice with RPMI 1640 (GIBCO BRL) containing 10% fetal bovine serum (GIBCO BRL), and the cell number was 2 × 10⁵/ Ml. Together with 50 ng / ml recombinant human IL-2 (R & D SYSTEMS) and 10 ng / ml recombinant mouse GM-CSF (R & D SYSTEMS) in a carbon dioxide incubator (TABAI) for 7 days Cultured. 2 x 10 cells in the same culture⁶/ Ml.
[0210]
2. Preparation of target cells
Human myeloma cell line KPMM2 (Japanese Unexamined Patent Publication No. Hei 7-236475) or plasma cell tumor-derived ARH-77 (ATCC CRL-1621) was used at 0.1 mCi.⁵¹Radioactive labeling was carried out by incubating with Cr-sodium chromate (ICN) in RPMI 1640 (GIBCO BRL) containing 10% fetal bovine serum (GIBCO BRL) at 37 ° C for 60 minutes. After radiolabeling, the cells were washed three times with the same medium and 2 x 10⁵/ Ml.
[0211]
3. ADCC assay
Radiolabeled 2 × 10 on a 96-well U-bottom plate (manufactured by Becton Dickinson)⁵/ Ml target cells and 50 μl of reconstituted human anti-HM1.24 antibody, mouse anti-HM1.24 antibody, control human IgG1 (The Binding Site Limited) or control mouse IgG2a (UPC10, Cappel). The reaction was performed at 4 ° C. for 15 minutes.
Thereafter, 100 μl of the effector cells were cultured in a carbon dioxide incubator for 4 hours. At that time, the ratio (E: T) between the effector cells (E) and the target cells (T) was set to 0: 1, 3.2: 1, 8: 1, 20: 1 or 50: 1.
[0212]
100 μl of the supernatant was taken, and the radioactivity released into the culture supernatant was measured with a gamma counter (ARC-300, manufactured by Aloka). For the measurement of the maximum free radioactivity, 1% NP-40 (manufactured by Hanoi) was used. The cytotoxic activity (%) was calculated as (AC) / (BC) X100. A represents the radioactivity released in the presence of the antibody (cpm), B represents the radioactivity released by NP-40 (cpm), and C represents the radioactivity released in the culture medium alone without the antibody (cpm). Show.
[0213]
FIG. 34 shows the results obtained when cells prepared from peripheral blood of a healthy subject were used as effector cells, and KPMM2 was used as target cells. FIG. 35 shows the results obtained when cells prepared from healthy human peripheral blood were used as effector cells and ARH77 was used as target cells. When the reshaped human anti-HM1.24 antibody was added as compared to the control human IgG1, the cytotoxic activity was increased as the antibody concentration was increased. Therefore, it is considered that this reshaped human anti-HM1.24 antibody has ADCC activity. Indicated.
[0214]
In addition, when the reshaped human anti-HM 1.24 antibody was added, the cytotoxic activity was clearly increased as compared with the case where the mouse anti-HM 1.24 antibody was added. It was shown to have higher ADCC activity than anti-HM1.24 antibody. Furthermore, when the target cell is KPMM2, the cytotoxic activity does not change when reconstituted human anti-HM1.24 antibody is added at a concentration of 0.1 μg / ml or more. Therefore, a concentration of 0.1 μg / ml or more is sufficient. It has been shown to have significant ADCC activity. When the target cell is ARH77, the cytotoxic activity does not change when reconstituted human anti-HM1.24 antibody is added at a concentration of 1 μg / ml or more. Therefore, sufficient ADCC activity is required at a concentration of 1 μg / ml or more. It has been shown.
[0215]
FIG. 36 shows the results obtained when cells prepared from the bone marrow of a SCID mouse were used as effector cells. When the reshaped human anti-HM1.24 antibody was added as compared to the control human IgG1, the cytotoxic activity was increased as the antibody concentration was increased. Therefore, it is considered that this reshaped human anti-HM1.24 antibody has ADCC activity. Indicated. When reconstituted human anti-HM1.24 antibody is added at a concentration of 0.1 μg / ml or more, the cytotoxic activity does not change. Indicated.
These results indicated that the reshaped human anti-HM1.24 antibody had ADCC activity even when human- or mouse-derived cells were used as effector cells.
[0216]
Example 14 .  Reconstituted human anti HM1.24 Antitumor effect of antibody on human myeloma mouse model
1. Preparation of administration antibody
The reconstituted human anti-HM1.24 antibody obtained by introducing the plasmid HEF-RVLa-AHM-gκ and the plasmid HEF-RVHr-AHM-gγ1 into cells was subjected to filtration using sterilized PBS (−) to obtain 40, 200 The control human IgG1 obtained in Example 12.1-2 was adjusted to 200 μg / ml using filter-sterilized PBS (−), and used as an administration antibody.
2. Antitumor effect of reshaped human anti-HM1.24 antibody on human myeloma transplanted mice
2-1. Preparation of mice transplanted with human myeloma
Human myeloma-transplanted mice were prepared according to Example 12.3-1. SCID mice (5-week-old) (CLEA Japan) were used as mice.
[0217]
2-2. Antibody administration
From the human myeloma-transplanted mouse prepared in 2-1 above, serum was collected on the 9th day of the KPMM2 cell transplantation, and human IgG in the serum was quantified using the ELISA of Example 12.2. Elevation of human IgG in serum confirmed the engraftment of BM in KPMM2 cells. On day 10 after the transplantation of the KPMM2 cells, 100 μl of the antibody prepared in 1 above was intravenously administered to these mice.
[0218]
2-3. Evaluation of antitumor effect of reshaped human anti-HM1.24 antibody on human myeloma transplanted mice
The antitumor effect of the reshaped human anti-HM1.24 antibody was evaluated based on the change in the amount of mouse serum human IgG and the survival time.
Regarding the change in the amount of mouse serum human IgG, the serum was collected on day 35 of the KPMM2 cell transplantation, and human IgG was quantified using the ELISA of Example 12.2. As a result, as shown in FIG. 37, in the control human IgG1 administration group, the amount of serum human IgG on the 35th day of KPMM2 cell transplantation was increased to about 1000 times as compared with the 9th day of transplantation (the day before the antibody administration). In contrast, in the reconstituted human anti-HM1.24 antibody administration group, the dose was almost the same as or less than that on day 9 after transplantation, and the reconstituted human anti-HM1.24 antibody suppressed the proliferation of KPMM2 cells. It was shown that.
[0219]
On the other hand, as shown in FIG. 38, the survival time of the reconstituted human anti-HM1.24 antibody administration group was longer than that of the control human IgG1 administration group. From the above, it was shown that administration of the reshaped human anti-HM1.24 antibody has an antitumor effect on human myeloma-transplanted mice.
[0220]
Example Fifteen .  Reconstituted human antibodies in a mouse model of human myeloma HM1.24 Comparison of antitumor effect between antibody and existing drug melphalan
1. Preparation of drug for administration
1-1. Preparation of administration antibody
The reconstituted human anti-HM1.24 antibody obtained by introducing the plasmid HEF-RVLa-AHM-gκ and the plasmid HEF-RVHr-AHM-gγ1 into cells was subjected to filtration using sterilized PBS (−) to obtain 40, 200 μg / ml, and the control human IgG1 obtained in Example 12.1-2 was adjusted to 200 μg / ml using filter-sterilized PBS (−), and used as an administration antibody.
1-2. Preparation of melphalan
Melphalan (manufactured by SIGMA), which is an existing drug for myeloma, was prepared to have a concentration of 0.1 mg / ml using 0.2% carbomethylcellulose (CMC) (manufactured by Daicel Chemical Industries, Ltd.).
[0221]
2. Antitumor effect of reshaped human anti-HM1.24 antibody and melphalan on human myeloma transplanted mice
2-1. Preparation of mice transplanted with human myeloma
Human myeloma transplanted mice were prepared according to Example 14.2-1.
2-2. Drug administration
From the human myeloma-transplanted mouse prepared in 2-1 above, serum was collected on the 9th day of the KPMM2 cell transplantation, and human IgG in the serum was quantified using the ELISA of Example 12.2. Elevation of human IgG in serum confirmed the engraftment of BM in KPMM2 cells. On day 10 after KPMM2 cell transplantation, 100 μl of the antibody prepared in 1-1 above was intravenously administered to these mice. Furthermore, 200 μl of a 0.2% CMC solution was orally administered once a day from the 10th day after transplantation for 5 days. On the other hand, for the melphalan-administered group, 100 μl (1 mg / kg as melphalan) of the melphalan solution prepared in the above 1-2 was orally administered once a day from day 10 after the KPMM2 cell transplantation for 5 days for 10 days. did.
[0222]
2-3. Evaluation of antitumor effect of reshaped human anti-HM1.24 antibody on human myeloma transplanted mice
The antitumor effect of the reshaped human anti-HM1.24 antibody was evaluated based on the change in the amount of mouse serum human IgG and the survival time.
Regarding the change in the amount of mouse serum human IgG, the serum was collected on day 35 of the KPMM2 cell transplantation, and human IgG was quantified using the ELISA of Example 12.2. As a result, as shown in FIG. 39, in the control human IgG1-administered group, the amount of serum human IgG on the 35th day of the KPMM2 cell transplantation was increased about 1000-fold compared to the 9th transplantation day (the day before the antibody administration), and KPMM2 cells appeared to be proliferating.
[0223]
In the group to which the existing drug, melphalan, was administered, the serum human IgG amount was higher than that before the drug administration, though not as much as the control human IgG1 administration group, and the proliferation of KPMM2 cells in mice was increased by the administration of melphalan. Did not seem to be completely controlled. On the other hand, in each of the groups to which the reconstituted human anti-HM1.24 antibody was administered, the amount of serum human IgG decreased from day 9 after transplantation, and the reconstituted human anti-HM1.24 antibody suppressed the proliferation of KPMM2 cells. It was shown that.
[0224]
On the other hand, as shown in FIG. 40, the survival period was prolonged in the reshaped human anti-HM1.24 antibody administration group as compared with the control human IgG1 administration group or the melphalan administration group, as shown in FIG. From the above, it was shown that administration of the reshaped human anti-HM1.24 antibody has an antitumor effect on human myeloma-transplanted mice, and that the antitumor effect of this antibody is stronger than that of the existing drug melphalan.
As described above, when human-derived effector cells were used, mouse anti-HM1.24 antibody showed almost no cytotoxic activity against human myeloma cells. And the chimeric anti-HM1.24 antibody showed strong cytotoxic activity. This fact indicates the importance of humanizing the antibody, and expects the usefulness of the reshaped human anti-HM1.24 antibody in humans.
[0225]
In human myeloma-transplanted SCID mice, the reshaped human anti-HM1.24 antibody showed a very strong antitumor effect, but in humans, the effector is naturally derived from humans and lymphocytes are also normally present. Further, a stronger antitumor effect of the reshaped human anti-HM1.24 antibody can be expected.
In a myeloma model, reconstituted human anti-HM1.24 antibody showed a stronger antitumor effect than existing myeloma therapeutic agents, and thus reconstituted human anti-HM1.24 antibody is a breakthrough myeloma therapeutic agent. It is expected to become.
[0226]
Reference Example 1.  Mouse anti HM1.24 Preparation of monoclonal antibody producing hybridoma
Goto, T .; et al. , Blood (1994) 84, 1992-1930, to prepare a mouse anti-HM1.24 monoclonal antibody-producing hybridoma.
Epstein-Barr virus-nuclear antigen (EBNA) derived from bone marrow of a human multiple myeloma patient-negative plasma cell line KPC-32 (1 × 10⁷(Goto, T. et al., Jpn. J. Clin. Hematol. (11991) 32, 1400) were injected twice intraperitoneally into BALB / C mice (Charles River) every 6 weeks.
[0227]
Three days before sacrifice of the mouse, 1.5 × 10 5⁶KPC-32 cells were injected into the spleen of mice (Goto, T. et al., Tokushima J. Exp. Med. (1990) 37, 89). After sacrifice of the mouse, the spleen was removed and Groth, de St. Spleen cells and myeloma cells SP2 / 0 were subjected to cell fusion according to the method of Schreidger (Cancer Research (1981) 41, 3465).
[0228]
Antibodies in the hybridoma culture supernatant were screened by ELISA (Posner, MR et al., J. Immunol. Methods (1982) 48, 23) using a plate coated with KPC-32 cells. 5 x 10⁴KPC-32 cells were suspended in 50 ml of PBS, and dispensed into a 96-well plate (U-bottom type, manufactured by Corning, Iwaki). After blocking with PBS containing 1% bovine serum albumin (BSA), the hybridoma culture supernatant was added and incubated at 4 ° C for 2 hours. Then, a peroxidase-labeled anti-mouse IgG goat antibody (manufactured by Zymed) was reacted at 4 ° C. for 1 hour, washed once, and reacted with an o-phenylenediamine substrate solution (manufactured by Sumitomo Bakerite) at room temperature for 30 minutes.
[0229]
The reaction was stopped with 2N sulfuric acid, and the absorbance at 492 nm was measured using an ELISA reader (manufactured by Bio-Rad). In order to remove hybridomas producing antibodies against human immunoglobulin, the positive hybridoma culture supernatant was adsorbed to human serum in advance, and the reactivity to other lower cells was screened by ELISA. Positive hybridomas were selected and tested for their reactivity to various cell lines and human specimens by flow cytometry. Finally, the selected hybridoma clone was cloned twice and injected into the abdominal cavity of BALB / C mice treated with pristane to obtain ascites.
[0230]
Monoclonal antibodies were purified from mouse ascites by precipitation with ammonium sulfate and Protein A affinity chromatography kit (Ampure PA, manufactured by Amersham). The purified antibody was bound to fluoroseinithiocyanate (FITC) by using a Quick Tag FITC binding kit (Boehringer Mannheim).
As a result, the monoclonal antibodies produced by 30 hybridoma clones reacted with KPC-32 and RPMI 8226 cells. After cloning, the culture supernatants of these hybridomas were examined for reactivity between other cell lines and peripheral blood-derived mononuclear cells.
[0231]
Of these, three clones were monoclonal antibodies that specifically reacted with plasma cells. Among these three clones, a hybridoma clone most useful for flow cytometry analysis and having complement-dependent cytotoxicity against RPMI 8226 cells was selected and named HM1.24. The subclass of the monoclonal antibody produced by this hybridoma was determined by ELISA using a subclass-specific anti-mouse rabbit antibody (Zymed). The anti-HM1.24 antibody had a subclass of IgG2a κ. Hybridoma HM1.24, which produces an anti-HM1.24 antibody, was submitted to Budapest as FERM BP-5233 on September 14, 1995 by the Institute of Biotechnology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan). Deposited internationally under the Convention.
[0232]
Reference example 2.  HM1.24 Encodes an antigenic polypeptide cDNA Cloning
1. Preparation of cDNA library
1) Preparation of total RNA
A cDNA encoding the HM1.24 antigen, which is an antigen polypeptide specifically recognized by the mouse monoclonal antibody HM1.24, was isolated as follows.
Total RNA was prepared from the human multiple myeloma cell line KPMM2 according to the method of Chirgwin et al. (Biochcmistry, 18, 5294 (1979)). That is, 2.2 × 10⁸Pieces of KPMM2 were completely homogenized in 20 ml of 4M guanidine thiocyanate (Nacalai Tesque).
[0233]
The homogenate was overlaid in a 5.3 M cesium chloride solution layer in a centrifuge tube, and the RNA was precipitated by centrifugation at 31,000 rpm in a Beckman SW40 rotor at 20 ° C. for 24 hours. The RNA precipitate is washed with 70% ethanol and dissolved in 300 μl of 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 0.5% SDS, to which Pronase (manufactured by Boehringer) is added at 0.5 mg / ml. After adding to a volume of 0.1 ml, the mixture was incubated at 37 ° C. for 30 minutes. The mixture was extracted with phenol and chloroform, and the RNA was precipitated with ethanol. Next, the RNA precipitate was dissolved in 200 μl of 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA.
[0234]
2) Preparation of poly (A) + RNA
Using about 500 μg of the total RNA prepared as described above as a material, poly (A) + RNA was purified using the Fast Track 2.0 mRNA Isolation Kit (manufactured by Invitrogen) according to the prescription attached to the kit.
3) Construction of cDNA library
Using 10 μg of the above poly (A) + RNA as a material, a double-stranded cDNA was synthesized using a cDNA synthesis kit TimeSaver cDNA Synthesis Kit (manufactured by Pharmacia) according to the instructions attached to the kit, and further using a Directional Cloning Toolbox (manufactured by Pharmacia). EcoRI adapters were ligated according to the instructions included in the kit. EcoRI adaptation of the adapter and NotI restriction enzyme treatment were performed according to the prescription attached to the kit. Further, the adapter-added double-stranded cDNA having a size of about 500 bp or more was separated and purified using 1.5% low melting point agarose gel (manufactured by Sigma) to obtain about 40 μl of the adapter-added double-stranded cDNA.
[0235]
The pCOS1 vector (Japanese Patent Application No. 8-255196) and the T4 DNA ligase (GIBCO-BRL) were prepared by treating the adapter-added double-stranded cDNA thus prepared with the restriction enzymes EcoRI, NotI and alkaline phosphatase (Takara Shuzo) in advance. To construct a cDNA library. The constructed cDNA library was transduced into E. coli cell line DH5α (manufactured by GIBCO-BRL), and the total size was about 2.5 × 10 5⁶Was estimated to be independent clones.
[0236]
2. Cloning by direct expression method
1) Transfection into COS-7 cells
About 5 x 10⁵The cDNA was amplified by culturing the clone by alkaline cloning by culturing the clone by alkaline cloning (Molecular Cloning) by culturing the clone in a 2-YT medium (Molecular Cloning: A Laboratory Manual. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)) containing 50 μg / ml ampicillin (Molecular Cloning). Plasmid DNA was recovered from Escherichia coli by the Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press (1989). The obtained plasmid DNA was transfected into COS-7 cells by an electroporation method using a Gene Pulser apparatus (manufactured by Bio-Rad).
[0237]
That is, 10 μg of the purified plasmid DNA was added to 1 × 10⁷COS-7 cell suspension suspended in PBS at cells / ml was added to 0.8 ml and pulsed at 1500 V, 25 μFD volume. After a recovery period of 10 minutes at room temperature, the electroporated cells were incubated at 37 ° C., 5% CO₂For 3 days.
[0238]
2) Preparation of panning dish
A panning dish coated with a mouse anti-HM1.24 antibody was applied to B.I. Seed et al. (Proc. Natl. Acad. Sci. USA,84, 3365-3369 (1987)). That is, a mouse anti-HM1.24 antibody was added to 50 mM Tris-HCl (pH 9.5) at a concentration of 10 μg / ml. 3 ml of the antibody solution thus prepared was added to a cell culture dish having a diameter of 60 mm, and incubated at room temperature for 2 hours. After washing three times with a 0.15 M NaCl solution, 5% fetal calf serum, 1 mM EDTA, 0.02% NaN₃Was added and PBS was blocked, and then used for the following cloning.
[0239]
3) Cloning of cDNA library
The COS-7 cells transfected as described above were detached with PBS containing 5 mM EDTA, washed once with PBS containing 5% fetal bovine serum, and then washed with about 1 × 10 5⁶5% fetal calf serum and 0.02% NaN to give cells / ml₃Was added to the panning dish prepared as described above, and incubated at room temperature for about 2 hours. 5% fetal calf serum and 0.02% NaN₃After gently washing with PBS containing 3 times, plasmid DNA was recovered from the cells bound to the panning dish using a solution containing 0.6% SDS and 10 mM EDTA.
[0240]
The recovered plasmid DNA was transduced again into Escherichia coli DH5α, and the plasmid DNA was amplified as described above and recovered by the alkali method. The recovered plasmid DNA was transfected into COS-7 cells by electroporation, and the plasmid DNA was recovered from the ligated cells in the same manner as described above. The same operation was repeated once more, and the recovered plasmid DNA was digested with restriction enzymes EcoRI and NotI. As a result, it was confirmed that the insert having a size of about 0.9 kbp was concentrated. Further, Escherichia coli transduced with a part of the recovered plasmid DNA was inoculated on a 2-YT agar plate containing 50 μg / ml of ampicillin, and after overnight culture, plasmid DNA was recovered from a single colony. After digestion with restriction enzymes EcoRI and NotI, clone p3.19 having an insert size of about 0.9 kbp was obtained.
[0241]
This clone was subjected to a reaction using PRISM, Dye Terminator Cycle Sequencing Kit (manufactured by Perkin Elmer) according to the instructions attached to the kit, and the nucleotide sequence was determined using ABI 373A DNA Sequencer (manufactured by Perkin Elmer). The amino acid sequence and the base sequence are shown in SEQ ID NO: 103.
The cDNA encoding the polypeptide having the amino acid sequence shown in SEQ ID NO: 103 is inserted between the XbaI cleavage sites of the pUC19 vector, and prepared as plasmid pRS38-pUC19. Escherichia coli (E. coli) containing this plasmid pRS38-pUC19 was sent to Escherichia coli on October 5, 1993 at the Institute of Biotechnology and Industrial Technology (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture) on October 5, 1993. It has been internationally deposited as DH5α (pRS38-pUC19) under the accession number FERM BP-4434 based on the Budapest Treaty (see Japanese Patent Application Laid-Open No. 7-196694).
[0242]
【The invention's effect】
The chimeric anti-HM 1.24 antibody is composed of the variable region of a mouse anti-HM 1.24 antibody and the constant region of a human antibody. Since it consists of a human antibody constant region and low antigenicity in humans, it is expected as a pharmaceutical composition, particularly as a therapeutic agent for myeloma.
[0243]
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[0345]

[Brief description of the drawings]
FIG. 1 shows that in the FCM analysis using the human myeloma cell line KPMM2, the fluorescence intensity of the chimeric anti-HM1.24 antibody shifted from the control antibody similarly to the fluorescence intensity of the mouse anti-HM1.24 antibody. It is a graph which shows that it is.
FIG. 2 shows that in a Cell-ELISA using WISH cells, a chimeric anti-HM1.24 antibody binds a biotinylated mouse anti-HM1.24 antibody to WISH cells similarly to a mouse anti-HM1.24 antibody. It is a graph which shows that it inhibits concentration-dependently.
FIG. 3 shows that control human IgG1 or mouse anti-HM1.24 antibody has no cytotoxic activity against RPMI8226 cells, whereas chimeric anti-HM1.24 antibody has an increased E / T ratio. It is a graph which shows that the cytotoxic activity with respect to RPMI 8226 cells is increasing.
FIG. 4 is a schematic diagram showing a method for producing a reshaped human anti-HM1.24 antibody L chain by CDR grafting by PCR.
FIG. 5 is a schematic diagram showing a method for assembling oligonucleotides of RVH1, RVH2, RVH3 and RVH4 by PCR in production of a reshaped human anti-HM1.24 antibody H chain.
FIG. 6 is a schematic diagram showing a method for preparing a human / mouse hybrid anti-HM1.24 antibody H chain V region by a PCR method.
FIG. 7 is a schematic diagram showing a method for preparing a mouse / human hybrid anti-HM1.24 antibody H chain V region by a PCR method.
FIG. 8 is a graph showing that reshaped human anti-HM1.24 antibody light chain version a has the same level of antigen binding activity as a chimeric anti-HM1.24 antibody. In addition, -1 and -2 indicate differences between lots.
FIG. 9 is a graph showing the antigen-binding activity of reshaped human anti-HM1.24 antibody and chimeric antibody HM1.24 antibody obtained by combining L-chain version a and H-chain version a, b, f or h. .
FIG. 10 is a graph showing the binding activity of a reshaped human anti-HM1.24 antibody and a chimeric antibody HM1.24 antibody obtained by combining L-chain version b and H-chain version a, b, f or h.
FIG. 11 is a graph showing the binding inhibitory activity of reshaped human anti-HM1.24 antibody and chimeric antibody HM1.24 antibody in which L-chain version a and H-chain version a, b, f or h are combined. .
FIG. 12 is a graph showing the binding inhibitory activity of a reshaped human anti-HM1.24 antibody and a chimeric antibody HM1.24 antibody obtained by combining L-chain version b and H-chain version a, b, f or h. .
FIG. 13 is a graph showing the antigen-binding activity of reshaped human anti-HM1.24 antibody H chain versions a, b, c, d and chimeric anti-HM1.24 antibody.
FIG. 14 is a graph showing the antigen-binding activity of the reshaped human anti-HM1.24 antibody H chain versions a and e and the chimeric anti-HM1.24 antibody. In addition, -1 and -2 indicate differences between lots.
FIG. 15 is a graph showing the binding inhibitory activities of reshaped human anti-HM1.24 antibody H chain versions a, c, p, r and a chimeric anti-HM1.24 antibody.
FIG. 16 is a graph showing the antigen-binding activity of a human-mouse hybrid anti-HM1.24 antibody, a mouse-human hybrid anti-HM1.24 antibody, and a chimeric anti-HM1.24 antibody.
FIG. 17 is a graph showing the antigen-binding activity of reshaped human anti-HM1.24 antibody H chain versions a, b, c, f and chimeric anti-HM1.24 antibody.
FIG. 18 is a graph showing the antigen-binding activity of reshaped human anti-HM1.24 antibody H chain versions a and g and chimeric anti-HM1.24 antibody.
FIG. 19 is a graph showing the binding inhibitory activities of reshaped human anti-HM1.24 antibody H chain versions a and g and a chimeric anti-HM1.24 antibody.
FIG. 20 is a graph showing the antigen-binding activity of the reshaped human anti-HM1.24 antibody H chain versions h and i and the chimeric anti-HM1.24 antibody.
FIG. 21 shows the antigen-binding activity of reshaped human anti-HM1.24 antibody H chain versions f, h, j and chimeric anti-HM1.24 antibody.
FIG. 22 is a graph showing the binding inhibitory activities of reshaped human anti-HM1.24 antibody H chain versions h and i and a chimeric anti-HM1.24 antibody.
FIG. 23 is a graph showing the binding inhibitory activities of reshaped human anti-HM1.24 antibody H chain versions f, h, j and chimeric anti-HM1.24 antibody.
FIG. 24 is a graph showing the antigen-binding activity of the reshaped human anti-HM1.24 antibody H chain version h, k, 1, m, n, o and the chimeric anti-HM1.24 antibody.
FIG. 25 is a graph showing the antigen-binding activity of reshaped human anti-HM1.24 antibody H chain versions a, h, p, q and a chimeric anti-HM1.24 antibody.
FIG. 26 is a graph showing the binding inhibitory activities of reshaped human anti-HM1.24 antibody H chain versions h, k, l, m, n, o and chimeric anti-HM1.24 antibody to WISH cells. .
FIG. 27 is a graph showing the binding inhibitory activities of reshaped human anti-HM1.24 antibody H chain versions a, h, p, q and a chimeric anti-HM1.24 antibody.
FIG. 28 is a graph showing the antigen-binding activity of reshaped human anti-HM1.24 antibody H chain versions a, c, p, r and chimeric anti-HM1.24 antibody.
FIG. 29 is a graph showing that reshaped human anti-HM1.24 antibody version s has the same level of antigen-binding activity as reshaped human anti-HM1.24 antibody version r.
FIG. 30 is a graph showing that reshaped human anti-HM1.24 antibody version s has a binding inhibitory activity comparable to that of reshaped human anti-HM1.24 antibody version r.
FIG. 31 is a graph showing that purified reconstituted human anti-HM1.24 antibody has a similar level of antigen binding activity as chimeric anti-HM1.24 antibody.
FIG. 32 is a graph showing that purified reconstituted human anti-HM1.24 antibody has a similar binding inhibitory activity as a chimeric anti-HM1.24 antibody.
FIG. 33 is a graph showing that administration of a chimeric anti-HM1.24 antibody prolongs survival time in human myeloma-transplanted mice as compared to control human IgG1 administration.
FIG. 34 shows that when human healthy human peripheral blood-derived cells were used as effector cells, control human IgG1 did not show cytotoxic activity against KPMM2 cells, and mouse anti-HM1.24 antibody also showed cytotoxicity. FIG. 4 is a graph showing that the reshaped human anti-HM1.24 antibody shows strong cytotoxic activity against KMPM2 cells, whereas the activity is weak.
FIG. 35 shows that when human healthy human peripheral blood-derived cells were used as effector cells, control human IgG1 did not show cytotoxic activity against ARH-77 cells, and mouse anti-HM1.24 antibody was also used. FIG. 4 is a graph showing that the reshaped human anti-HM1.24 antibody shows strong cytotoxic activity against ARH-77 cells, whereas cytotoxic activity is weak.
FIG. 36 shows that when SCID mouse bone marrow-derived cells were used as effector cells, control human IgG1 had no cytotoxic activity against KPMM2 cells, whereas reshaped human anti-HM1.24 antibody It is a graph showing that the cytotoxic activity with respect to KPMM2 cell is increasing with the rise.
FIG. 37 shows that in human myeloma-transplanted mice, control human IgG1 increased serum human IgG after administration compared to before administration, whereas reconstituted human anti-HM1.24 antibody 4 is a graph showing that administration suppresses an increase in the amount of serum human IgG.
FIG. 38 is a graph showing that administration of a reshaped human anti-HM1.24 antibody prolongs survival time in human myeloma-transplanted mice as compared to control human IgG1 administration.
FIG. 39 shows that in human myeloma-transplanted mice, melphalan and control human IgG1 increased the amount of serum human IgG after administration compared to before administration, whereas reconstituted human anti-HM1.24 3 is a graph showing that administration of an antibody suppresses an increase in the amount of serum human IgG.
FIG. 40 is a graph showing that administration of reconstituted human anti-HM1.24 antibody prolongs survival time in human myeloma-transplanted mice compared to melphalan or control human IgG1 administration. is there.

Claims (9)

A L chain V region of a reshaped human antibody which binds to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 103, wherein the reshaped human L chain V region comprises the amino acid sequence shown in amino acid numbers 1 to 107 of SEQ ID NO: 9. region. A light chain of a reshaped human antibody which binds to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 103, wherein the light chain V region comprises the amino acid sequence shown in amino acid numbers 1 to 107 of SEQ ID NO: 9; A reshaped human light chain comprising a chain C region. The reshaped human L chain according to claim 2, wherein the human L chain C region is CK. An H chain V region of a reshaped human antibody which binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 103, wherein the amino acid sequence is represented by amino acids 1 to 120 of SEQ ID NO: 28 or the amino acids 1 to 120 of SEQ ID NO: 102 3. A reshaped human H chain V region comprising the amino acid sequence shown in FIG. An H chain of a reshaped human antibody which binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 103, which is represented by the amino acid sequence represented by amino acid numbers 1 to 120 of SEQ ID NO: 28 or the amino acid sequence represented by amino acid numbers 1 to 120 of SEQ ID NO: 102 A reshaped human H chain comprising an H chain V region comprising an amino acid sequence and a human H chain C region. The reshaped human H chain according to claim 5, wherein the human H chain C region is Cγ1. A reshaped human antibody which binds to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 103,
(A) a reshaped human L chain comprising an L chain V region comprising the amino acid sequence shown in amino acid Nos. 1 to 107 of SEQ ID NO: 9 and a human L chain C region;
(B) a reshaped human H chain V region comprising the amino acid sequence shown in amino acid numbers 1-120 of SEQ ID NO: 28 or the amino acid sequence shown in amino acid numbers 1-120 of SEQ ID NO: 102, and a human H chain C region A reshaped human heavy chain comprising:
Or a fragment of the reshaped human antibody, wherein the fragment binds to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 103. The reshaped human antibody according to claim 7, wherein the human L chain C region is Cκ and the human H chain C region is Cγ1. The fragment of the reshaped human antibody according to claim 7 or 8, wherein the fragment is Fab, F (ab ') ₂ , Fv or single chain Fv.

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