JP3659405B2 - SH2 domain-containing peptide - Google Patents

Description

【０１４６】
ＤＮＡ調製：
ＤＮＡは培養した細胞系、一次腫瘍、正常ヒト血液から調製した。単離は、全てQuiagenからの、精製キット、バッファーセット及びプロテアーゼを用い、製造者の指示と下記に従って実施した。
細胞培養溶解：
細胞を洗浄し、チップ当たり7.5x10^８の濃度でトリプシン化し、４℃で５分間1000rpmで遠心分離してペレット化し、次いで1/2容量のＰＢＳ再遠心で洗浄した。ペレットを３回洗浄し、懸濁細胞を回収して2xＰＢＳで洗浄した。次いで細胞を10mLのＰＢＳに懸濁させた。バッファーＣ１を４℃で平衡化させた。Quiagenプロテアーゼ#19155を6.25mlの冷ｄｄＨ２Ｏで最終濃度20mg/mlまで希釈して４℃で平衡化させた。10mLのＧ２バッファーを、QuiagenＲＮＡｓｅＡストック（100mg/ml）を200μg/mlの最終濃度まで希釈して調製した。
バッファーＣ１（10mL、４℃）及びｄｄＨ２Ｏ（40mL、４℃）を、次いで10mLの細胞懸濁物に添加し、反転させて混合し、氷上で１０分間インキュベートした。細胞核をBeckmanスイングバケットロータで４℃において2500rpmで１５分間遠心分離することによりペレット化した。上清を捨て、核をボルテックスしながら2mLのバッファーＣ１（４℃）及び6mLのｄｄＨ_２Ｏに懸濁し、１秒後に４℃において2500rpmで１５分間遠心分離した。次いで核を残りのバッファー中にチップ当たり200μlを用いて再懸濁した。Ｇ２バッファー（10ml）を懸濁した核に添加しながら緩いボルテックスを適用した。バッファー添加が完了したら、強いボルテックスを３０秒間適用した。Quiagenプロテアーゼ（200μl上記のように調製）を添加し、５０℃で６０分間インキュベートした。インキュベーション及び遠心分離を、溶解物が透明になるまで繰り返した（例えば、さらに30-60分間インキュベートし、４℃で１０分間3000xgでペレット化する）。
【０１４７】
固体ヒト腫瘍試料の調製及び溶解：
腫瘍試料を秤量し50mlのコニカル管に配して氷上に保持した。加工は調製当たり250mgの組織未満に制限した（１チップ／調製）。プロテアーゼ溶液を6.25mlの冷ｄｄＨ_２Ｏ中に最終濃度20mg/mlまで希釈することにより新たに調製して４℃で貯蔵した。ＤＮＡｓｅＡを最終濃度200mg/mlまで希釈することによりＧ２バッファー（20ml）を調製した（100mg/mlのストックから）。エアロゾルの吸入を避けるために層流ＴＣフード内でポリトロンの大きなチップを用いて、腫瘍組織を19mlのＧ２バッファー中で６０秒間均一化し、室温に保持した。試料間で、各々2LのｄｄＨ_２Ｏで２ｘ３０秒間、次いでＧ２バッファー（50ml）でスピンさせることによりポリトロンを透明化した。組織がジェネレータチップ上に存在する場合は、装置を分解して清浄化した。
Quiagenプロテアーゼ（上記の用に調製、1.0ml）を添加し、次いでボルテックスして５０℃で３時間インキュベートした。インキュベーション及び遠心分離を、溶解物が透明になるまで繰り返した（例えば、さらに30-60分間インキュベートし、４℃で１０分間3000xgでペレット化する）。
【０１４８】
ヒト血液調製及び溶解：
健常なボランティアから標準的な感染薬プロトコールを用いて血液を採りだし、チップ当たり10mlの試料にクエン酸化した。Quiagenプロテアーゼを6.25mlの冷ｄｄＨ_２Ｏ中に最終濃度20mg/mlまで希釈することにより新たに調製して４℃で貯蔵した。ＤＮＡｓｅＡを100mg/mlのストックから最終濃度200μ g/mlまで希釈することによりＧ２バッファーを調製した。血液（10ml）を50mlのコニカル管に配し、10mlのＣ１バッファー及び30mlのｄｄＨ_２Ｏ（ともに４℃で平衡化したもの）を添加し、反転させて混合して氷上に１０分間保持した。Beckmanスイングバケットローターで、４℃において2500rpmで１５分間核をペレット化し、上清を捨てた。ボルテックスしながら、核を2mlのＣ１バッファー（４℃）及び6mlのｄｄＨ２Ｏ（４℃）中に懸濁させた。ボルテックスはペレットが白くなるまで繰り返した。次いで核を残りのバッファー中に200μlチップを用いて懸濁させた。Ｇ２バッファー（10ml）を懸濁核に添加しながら緩くボルテックスし、次いで３０秒間強くボルテックスした。Quiagenプロテアーゼを添加（200μl）し、５０℃で６０分間インキュベートした。インキュベーション及び遠心分離を、溶解物が透明になるまで繰り返した（例えば、さらに30-60分間インキュベートし、４℃で１０分間3000xgでペレット化する）。
【０１４９】
透明化溶解物の精製：
（１）ゲノムＤＮＡの単離：
ゲノムＤＮＡを10mlのＱＢＴバッファーで平衡化した（最大チップ調製当たり１試料）。ＱＦ溶離バッファーを５０℃で平衡化した。試料を３０秒間ボルテックスし、次いで平衡化チップに負荷して重力により排液した。チップを2x15mlのＱＣバッファーで洗浄した。ＤＮＡを、30mlのシラン化したオートクレーブ30mlCortex管に15mlのＱＦバッファー（５０℃）で溶離した。イソプロパノール（10.5ml）を各試料に添加し、管をパラフィンで被覆し、ＤＮＡが沈殿するまで繰り返し反転させて混合した。試料を、SS-34ロータで４℃において15,000rpmで１０分間遠心分離してペレット化した。ペレット位置をマークして上清を捨て、10mlの70%エタノール（４℃）を添加した。試料を、SS-34ロータで４℃において10,000rpmで１０分間遠心分離して再度ペレット化した。ペレット位置をマークして上清を捨てた。次いで管を乾燥棚の各面に置き、３７℃で１０分間乾燥させたが、使用の過剰乾燥には注意した。
乾燥後、ペレットを1.0mlのＴＥ（pH8.5）に溶解し、５０℃に１−２時間置いた。試料を４℃に終夜保持して溶解を続けた。次いでＤＮＡ溶液を、ツベルクリンシリンジ上に２６ゲージの針を具備する1.5ml管に移した。ＤＮＡを剪断するために移行を５ｘ繰り返した。次いで試料を５０℃に１−２時間置いた。
【０１５０】
（２）ゲノムＤＮＡの定量及び遺伝子増幅アッセイのための調製：
各管のＤＮＡレベルを1:20希釈（5μlＤＮＡ＋95μlｄｄＨ_２Ｏ）での標準的なＡ２６０、Ａ２８０スペクトルにより、Beckman DU640分光光度計の0.1ml石英キュベットを用いて定量した。Ａ２６０／Ａ２８０比率は１．８−１．９の範囲であった。次いで各ＤＮＡ試料をＴＥ（pH8.5）中に約200ng/mlまで希釈した。最初の材料が高濃度（約700ng/μl）である場合、材料を５０℃に再懸濁するまで数時間置いた。
次いで、希釈した材料（20-600ng/ml）に対して、製造者の指示を以下のように改変して蛍光ＤＮＡ定量を実施した。これは、Hoeffer DyNA Quant 200蛍光計を約１５分間暖めて実施した。Hoechst染料作業溶液（#H33258、10μl、使用の１２時間以内に調製）を100mlの1xＴＮＥバッファーに希釈した。2mlキュベットを蛍光計溶液で満たし、機械に配し、機械をゼロ調節した。ｐＧＥＭ３Ｚｆ(＋)（２μl、ロット#360851026）を2mlの蛍光計溶液に添加して200単位で校正した。次いで、さらに２μlのｐＧＥＭ３Ｚｆ(＋)ＤＮＡを試験し、400+/-10単位で読みを確認した。次いで各試料を少なくとも３回読んだ。３試料が互いに10%以内であることが見られたとき、それらの平均をとり、この値を定量化値として用いた。
次いで、蛍光測定で決定した濃度を、各試料をｄｄＨ_２Ｏ中に10ng/μlμまで希釈するのに用いた。これは、１回のTaqManプレートアッセイについて全てのテンプレート試料について同時に行い、500-1000アッセイを実施するのに十分な材料で行った。試料は、Taqman(商品名)プライマー及びプローブで３回試験し、Ｂ-アクチン及びＧＡＰＤＨともに正常なヒトＤＮＡを持ちテンプレート対照を持たない単一のプレート上にある。希釈した試料を用いたが、試験ＤＮＡから減算した正常ヒトＤＮＡのＣＴ値は+/-１ＣＴであった。希釈した、ロット定性化したゲノムＤＮＡを、1.0mlアリコートで−８０℃において保存した。続いて遺伝し増幅アッセイに使用するアリコートは、４℃で保存した。各1mlのアリコートは、８−９プレート又は６４試験に十分である。
【０１５１】
遺伝子増幅アッセイ：
本発明のＰＲＯ２０１，ＰＲＯ３０８又はＰＲＯ３０９化合物を以下の一次腫瘍でスクリーニングし、得られたΔＣｔ値を表２に報告する。

【０１５２】
ＤＮＡ３０６７６（配列番号：２）を、フレームワークマッピングでの上記の最初のスクリニングから選択した腫瘍について再試験した。Ｆｉｇ１８及び表３は、この実施例で使用したフレームワークマーカーの染色体マッピングを示す。フレームワークマーカーは約２０メガベース毎に位置し、対照異数性に使用した。
ＤＮＡ３０６７６（配列番号：２）をエピセンターマッピングで再試験した。表４に示したマーカーは、ＤＮＡ３０６７６（配列番号：２）の（ゲノムにおける）直ぐ隣に位置し、対応する分子が局在する染色体１９の直近における相対的増幅の評価に使用する。個々のマーカー間の距離はセンチレイ（ｃＲ）で測定し、これは放射線分解単位であり、２つのマーカー間の分解の１％チャンスとほぼ等しい。１ｃＲは極めて粗くは２０キロベースと等しい。マーカーＳＨＧＣ-３５４４１はＤＮＡ３０６７６がマッピングされる染色体１９の位置の最も近くに見られるマーカーである。
【０１５３】

ＰＲＯ２０１に対する染色体１９に沿った上記のフレームワークマーカーのΔＣｔ値は、表５に選択された腫瘍について示した。
【０１５４】

【０１５５】
表６はＤＮＡ３０６７６（配列番号：２）に対するエピセンターマッピングの結果についてのΔＣｔ値を示し、染色体１９に沿ったＤＮＡ３０６７６（配列番号：２）の実際の位置により近い領域での相対的増幅を示す。

【０１５６】
議論と結論：
種々の肺及び結腸腫瘍におけるＤＮＡ３０６７６（配列番号：２）及びＤＮＡ４０５５６（配列番号：２０）についてのΔＣｔ値を表２に報告する。ΔＣｔ＞１は典型的に増幅評点化の閾値として用い、これは２倍の遺伝子コピーを表す。表２は：一次肺腫瘍ＬＴ１ａ、ＬＴ３、ＬＴ１１、ＬＴ１３、ＬＴ１５、ＬＴ１７、ＬＴ１９；一次結腸腫瘍ＣＴ２、ＣＴ３、ＣＴ８、ＣＴ１０、ＣＴ１２、ＣＴ１４、ＣＴ１５、ＣＴ１６、ＣＴ１７、ＣＴ１、ＣＴ４、ＣＴ５、ＣＴ６、ＣＴ７、ＣＴ９、ＣＴ１１、ＣＴ１８；肺腫瘍細胞系Ｃａｌｕ-１、Ｃａｌｕ-６、Ｈ１５７、Ｈ４４１、ＳＫＭＥＳ１、Ｈ５２２及びＨ８１０；及び結腸腫瘍細胞系Ｃｏｌｏ-３２０及びＣｏｏ-２０５で生じたＤＮＡ３０６７６の有意な増幅を示す。表２は更に：一次肺腫瘍ＬＴ１ａ、ＬＴ８、ＬＴ１１、ＬＴ１５、ＬＴ１７、ＬＴ１９；一次結腸腫瘍ＣＴ１４、ＣＴ５、ＣＴ１１及び肺腫瘍細胞系Ａ５４９におけるＤＮＡ４０５５６の有意な発現を示す。
ＤＮＡ３０６７６（配列番号：２）のΔＣｔ及び平均ΔＣｔ値は、一次肺腫瘍に該当するのは：１．１０、２．０５、１．５２、２．１３。２．５８。１．６３、３．４３；一次結腸腫瘍に該当するのは：３．８１、２．３、１．９７、３．０１、２．７６、３．３４、２．５８、２．４６、２．６４、１．５７、３．３９、２．９５、２．８１、２．７７、２．６３、３．０、２．２３；肺腫瘍細胞系に該当するのは：２．３７、２．６１、２．３９、２．０７，３．０１、２．９１及び２．０７；結腸腫瘍細胞系：１．９５及び１．２６であった。ＤＮＡ４０５５６のΔＣｔ及び平均ΔＣｔ値は、一次肺に該当するのは１．０３、１．１４、１．３３、１．１８、１．００及び１．４２；結腸腫瘍に該当するのは：１．１２、１．００及び１．１３５；肺腫瘍細胞系１．２６であった。
上記の段落で列挙したＤＮＡ３０６７６（配列番号：２）のΔＣｔ及び平均ΔＣｔ値は、
一次肺腫瘍に対する２．１４、４．１４、２．８７、４．３８、５．９８、３．０９、１０．７０：一次結腸腫瘍に対する１４．０３、４．９２、３．９２、８．０６、６．７７、１０．１３、５．９８、５．５０、６．２３、２．９７、１０．４８、７．７３、７．０１、６．８２、６．１９、８．０、４．６９；肺腫瘍細胞系に対する５．１７、６．１０、５．２４、４．２０。８．０６、７．５１、４．２０；結腸腫瘍細胞系に対する３．８６、２．４０という正常組織に対する遺伝子コピーの増加を表している。ＤＮＡ４０５５６については、これらの値は、肺腫瘍に対する２．０４、２．２０、２．５１、２．２７、２．０、２．６８、結腸腫瘍に対する２．１７、２．０、２．２０；２．３９という正常組織に対する遺伝子コピーの増加を表す。
【０１５７】
増幅は、一次肺腫瘍ＬＴ３、ＬＴ１５，ＬＴ１６、ＬＴ１７、ＬＴ１８；及び一次結腸腫瘍ＣＴ２、ＣＴ３、ＣＴ８、ＣＴ９、ＣＴ１０、ＣＴ１２、ＣＴ１４、ＣＴ１５、ＣＴ１６、ＣＴ１７におけるＤＮＡ３０６７６（配列番号：２）のフレームワークマッピングにより確認された。一次肺腫瘍について報告されたΔＣｔ値は、１．０４、１．４３、１．３５、１．５１及び１．２２であり、一次結腸腫瘍については、２．８１、２．０３、１．３９、２．２１、１．９３、２．３７、１．２７、１．７６及び１．６５が報告された。正常組織に対して、これはおよそ肺腫瘍については２．０６、２．６９、２．５５、２．８５及び２．３２倍の増加、結腸腫瘍については７．０１、４．０８、２．６２、４．６３、３．８１、５．１７、２．４１、３．３９、３．１４倍の増加を表す。ＤＮＡ３０６６のエピセンターマッピングは、一次肺腫瘍ＬＴ３、ＬＴ１３、ＬＴ１５、ＬＴ１６、ＬＴ１８；及び結腸腫瘍ＣＴ１、ＣＴ２、ＣＴ３、ＣＴ４、ＣＴ５、ＣＴ６、ＣＴ７、ＣＴ８、ＣＴ９、Ｃｔ１０、ＣＴ１１、ＣＴ１２、ＣＴ１４、ＣＴ１５、ＣＴ１６、ＣＴ１７及びＣＴ１８における有意な増幅を確認した。一次肺腫瘍について報告されたΔＣｔ値は、１．８７、１．４４、１．９９、１．７２及び１．０９であり、一次結腸腫瘍は２．５６、３．５１、２．１９、３．２２、２．８３、２．９３、２．４３、１．９５、２．７２、３．１２、３．２６、２．７７、２．８８、２．６０、２．６１、２．２４及び２．４８のΔＣｔ及び平均ΔＣｔ値を示した。正常組織に対して、これらは、肺腫瘍について３．６６、２．７１、３．９７、３．２９、２．１３倍の遺伝子コピーの増加、及び結腸腫瘍について５．９０、１１．３９、４．５６、９．３２、７．１１、７．６２、５．３９、３．８６、６．５９、８．６９、９．５８、６．８２、７．３６、６．０６、６．１１、４．７２、５．５８倍の遺伝子コピーの増加を表す。
これに対して、最も近いフレームワークマーカー（１つ、即ちＳ５０を除く）（表５）又はエピセンターマーカー（表６）の増幅は、ＤＮＡ３０６７６（配列番号：２）より大きな程度では起こらなかった。このことは、ＤＮＡ３０６７６（配列番号：２）が染色体１９上の特定領域の増幅の原因となる遺伝子であることを強く示唆している。ＤＮＡ３０６７６（配列番号：２）の増幅は種々の肺及び結腸腫瘍及び細胞系（特に結腸）で起こるので、それは腫瘍形成又は成長において有意な役割を果たす可能性が高い。結果として、ＤＮＡ３０６７６（配列番号：２）（即ち、Ｎｓｐ１、配列番号：１）ＤＮＡ４０５７５（配列番号：４）及びＤＮＡ６１６０１（配列番号：６）に対するアンタゴニスト（例えば抗体）は、癌治療における有用性を持つと予測される。
【０１５８】
実施例１５
インサイツハイブリッド形成
インサイツハイブリッド形成は、細胞又は組織調製物内での核酸配列の検出及び局在化のための強力で多用途の技術である。それは、例えば、遺伝子発現部位の同定、転写物の組織分布の分析、ウイルス感染の同定及び局在化、特定ｍＲＮＡ合成及び染色体マッピングにおける追跡に有用である。
インサイツハイブリッド形成は、Lu及びGillett, Cell Vision 1: 169-176 (1994)のプロトコールの最適な変形に従って、ＰＣＲ生成^３３Ｐ-標識リボプローブを用いて実施される。簡単には、ホルマリン固定、パラフィン包埋ヒト組織を切片化し、脱パラフィンし、プロテイナーゼＫ（20g/ml）で１５分間３７℃で脱タンパクし、さらに上掲のLu及びGillettに記載されたようにインサイツハイブリッド形成する。［^３３-Ｐ］ＵＴＰ-標識アンチセンスリボプローブをＰＣＲ産物から生成し、５５℃で終夜ハイブリッド形成する。スライドをKodak NTB2核トラックエマルションに浸漬して４週間露出する。
^３３Ｐ-リボプローブ合成
6.0μl（125mCi）の^３３Ｐ-ＵＴＰ（Amersham BF 1002, SA＜2000 Ci/mmol）をスピード真空乾燥させた。乾燥^３３Ｐ-ＵＴＰを含む管に以下の成分を添加した：
2.0μlの5ｘ転写バッファー
1.0μlのＤＴＴ（100mM）
2.0μlのＮＴＰ混合物（2.5mM: 10μ l; 10mMのGTP, CTP及びATP+10μlのH₂O）
1.0μlのＵＴＰ（50μM）
1.0μlのＲｎａｓｉｎ
1.0μlのＤＮＡテンプレート（1μg）
1.0μlのＨ_２Ｏ
1.0μlのＲＮＡポメラーゼ（ＰＣＲ産物についてT3＝AS, T7=S,通常）
【０１５９】
管を３７℃で１時間インキュベートし、1.0μlのRQ1 ＤＮａｓｅを添加し、次いで３７℃で１５分間インキュベートした。90μlのＴＥ（10mMトリスpH7.6／1mMのＥＤＴＡpH8.0）を添加し、混合物をDE81紙にピペットした。残りの溶液をMicrocon-50限外濾過ユニットに負荷し、プログラム１０を用いてスピンさせた（6分間）。濾過ユニットを第２の管に変換し、プログラム２を用いてスピンさせた（3分間）。最終回収スピンの後、100μlのＴＥを添加した。1μlの最終生成物をDE81紙にピペットし6mlのBiofluor IIで数えた。
プローブをＴＢＥ／尿素ゲル上で走らせた。1-3μlのプローブ又は5μlのＲＮＡ ＭｒｋＩＩＩを3μlの負荷バッファーに添加した。加熱ブロック上で９５℃に３分間加熱した後、ゲルを即座に氷上に置いた。ゲルのウェルをフラッシングし、試料を負荷し、180-250ボルトで45分間走らせた。ゲルをサランラップでラップし、−７０℃冷凍機内で補強スクリーンを持つＸＡＲフィルムに１時間から終夜露出した。
【０１６０】
^３３Ｐ-ハイブリッド形成
凍結切片の前処理
スライドを冷凍機から取り出し、アルミニウムトレイに配置して室温で５分間解凍した。トレイを５５℃のインキュベータに５分間配置して凝結を減らした。スライドを蒸気フード内において4%パラホルムアルデヒド中で５分間固定し、0.5ｘＳＳＣで５分間室温で洗浄した（25ml 20xSSC + 975ml SQ H₂O）。0.5μg/mlのプロテイナーゼＫ中、３７℃で１０分間の脱タンパクの後（250mlの予備加熱ＲＮａｓｅ無しＲＮａｓｅバッファー中の10mg/mlストック12.5μl）、切片を0.5ｘＳＳＣで１０分間室温で洗浄した。切片を、70%、95%、100%エタノール中、各2分間脱水した。
パラフィン包埋切片の前処理
スライドを脱パラフィンし、ＳＱ Ｈ_２Ｏ中に配置し、2ｘＳＳＣで室温において各々５分間２回リンスした。切片を20μg/mlのプロテイナーゼＫ（250ｍｌのＲＮａｓｅ無しＲＮａｓｅバッファー中10mg/mlを500μｌ；３７℃、１５分間）−ヒト胚又は８ｘプロテイナーゼＫ（250mlのＲＮａｓｅバッファー中100μl、３７℃、３０分間）−ホルマリン組織で脱タンパクした。続く0.5ｘＳＳＣでのリンス及び脱水は上記のように実施した。
プレハイブリッド化
スライドをＢｏｘバッファー（4ｘＳＳＣ、50%ホルムアミド）−飽和濾紙で列を作ったプラスチックボックスに並べた。組織を50μlのハイブリッド形成バッファー（3.75gデキストラン硫酸＋6mlＳＱ Ｈ２Ｏ）で被覆し、ボルテックスし、キャップを外して２分間マイクロ波で加熱した。氷上で冷却した後、18.75mlのホルムアミド、3.75ｍｌの20ｘＳＳＣ及び9mlのＳＱ Ｈ２Ｏを添加し、組織を良くボルテックスし、４２℃で1-4時間インキュベートした。
ハイブリッド形成
スライド当たり1.0ｘ10^６cpmのプローブ及び1.0μlのｔＲＮＡ（50mg/mlストック）を９５℃で３分間加熱した。スライドを氷上で冷却し、スライド当たり48μlのハイブリッド形成バッファーを添加した。ボルテックスの後、50μlの^３３Ｐ混合物をスライド上のプレハイブリッド50μlに添加した。スライドを５５℃で終夜インキュベートした。
洗浄
洗浄は、2ｘ10分間、2ｘＳＳＣ、ＥＤＴＡで室温で実施し（400mlの20ｘSSC＋16mlの0.25M EDTA、Ｖｆ＝４Ｌ）、次いでＲＮａｓｅＡ処理を３７℃で３０分間行った（250mlＲＮａｓｅバッファー中10mg/mlを500μｌ＝20μg/ml）。スライドを2ｘ10分間、2x ＳＳＣ、ＥＤＴＡで室温において洗浄した。緊縮性洗浄条件は次の通り：５５℃で２時間、0.1ｘＳＳＣ、ＥＤＴＡ（20mlの20ｘSSC＋16mlのEDTA、Ｖｆ＝４Ｌ）。
【０１６１】
ヒト組織ＩＩＩにおけるＤＮＡ３０６７６ｐａｎｃ．ｓｈｃ(ｎｓｐ-１)の発現 多くの組織でセンス及びアンチセンスプローブで匹敵するバックグラウンドが観察された。発現が特異的であることがわかった部位は、胎児胸腺髄質、胎児脾臓、胎児小腸の上皮及び新たな骨形成の骨細胞領域のみであった。胎児及び正常成人前立腺では特異的なシグナルが観察されなかった。胎児組織は妊娠約１２−１６週であった。使用したオリゴはＢ-１９１Ｄ及びＢ-１９１Ｅであった。
【０１６２】
結腸腫瘍、胎児肝臓、及び形質移入細胞系でのＤＮＡ３０６７６（ＳＨＣｈｌｏｇ／ｎｓｐ１）の発現
この実験の目的は、結腸腺癌におけるＤＮＡ３０６７６の発現を測定することである。このＤＮＡは、結腸癌における増幅が示されている。発現は対照細胞ペレット及び１０の結腸癌において分析した。対照細胞ペレットは、ＳＨＣ形質移入２９３細胞及びＳＷ４８０細胞を含み、これらはＳＨＣを発現する（細胞はＨ９８−７１７として提出されている）。
細胞ペレットの試験は、ＳＨＣ形質移入細胞がセンス及びアンチセンスの両方のプローブにポジティブであり、この実験を問題にしている。ＳＷ４８０細胞は両方のプローブにネガティブであった。結腸癌についてはＡＳプローブのみを実施した。多数の結腸癌が僅かな発現を示し、これは検体９７２７／９８において最も強かった。しかしながら、細胞ペレットのデータから見ると、このシグナルは全部を解釈するのが困難であり、シグナルはポジティブと呼ぶには不十分と感じられる。
【０１６３】
下記のオリゴヌクレオチドを、この実施例に記載したインサイツハイブリッド形成で使用した。
B166A-GGATTCTAATACGACTCACTATAGGGCGCGGAGGCTGCTCTGGGGTAG （配列番号：３０）
B166B-CTATGAAATTAACCCTCACTAAAGGGATGTTGCCCTGGCTGGTCTTGA （配列番号：３１）
B166D-GGATTCTAATACGACTCACTATAGGGCATCTGCCTTGCCCCGAACGAG （配列番号：３２）
B166E-CTATGAAATTAACCCTCACTAAAGGGATCATCCAGAGCCCGCATCAGC （配列番号：３３）
A-322I-GGATTCTAATACGACTCACTATAGGGCAGATGTGGAAGACTGAGGCCT （配列番号：３４）
A-323J-CTATGAAATTAACCCTCACTAAAGGGAATATGTGCCAAATCTGCAGGCT （配列番号：３５）
【０１６４】
材料の寄託
次の細胞系をアメリカン・タイプ・カルチャー・コレクション, １２３０１ パークローンドライブ、ロックビル、メリーランド、米国(12301 Parklawn Drive, Rockville, MD, USA)(ＡＴＣＣ)に寄託した：
材料 ATCC寄託番号 寄託日
DNA30676-1223 209567 1997年12月23日
DNA40575-1223 209565 1997年12月23日
DNA40556-1223 209566 1997年12月23日
DNA40554-1223 209564 1997年12月23日
DNA61601-1223 209713 1998年 3月31日
【０１６５】
この寄託は、特許手続き上の微生物の寄託の国際的承認に関するブダペスト条約及びその規則(ブダペスト条約)の規定に従って行われた。これは、寄託の日付から３０年間、寄託の生存可能な培養が維持されることを保証するものである。寄託物はブダペスト条約の条項に従い、またジェネンテク社とＡＴＣＣとの間の合意に従い、ＡＴＣＣから入手することができ、これは、どれが最初であろうとも、関連した米国特許の発行時又は任意の米国又は外国特許出願の公開時に、寄託培養物の後代を永久かつ非制限的に入手可能とすることを保証し、米国特許法第１２２条及びそれに従う特許庁長官規則(特に参照番号８８６ＯＧ６３８の３７ＣＦＲ第１．１４条を含む)に従って権利を有すると米国特許庁長官が決定した者に子孫を入手可能とすることを保証するものである。
本出願の譲受人は、寄託した培養物が、適切な条件下で培養されていた場合に死亡もしくは損失又は破壊されたならば、材料は通知時に同一の他のものと即座に取り替えることに同意する。寄託物質の入手可能性は、特許法に従いあらゆる政府の権限下で認められた権利に違反して、本発明を実施するライセンスであるとみなされるものではない。
上記の文書による明細書は、当業者に本発明を実施できるようにするために十分であると考えられる。寄託した態様は、本発明のある側面の一つの説明として意図されており、機能的に等価なあらゆる作成物がこの発明の範囲内にあるため、寄託された作成物により、本発明の範囲が限定されるものではない。ここでの物質の寄託は、ここに含まれる文書による説明が、そのベストモードを含む、本発明の任意の側面の実施を可能にするために不十分であることを認めるものではないし、それが表す特定の例証に対して請求の範囲を制限するものと解釈されるものでもない。実際、ここに示し記載したものに加えて、本発明を様々に改変することは、前記の記載から当業者にとっては明らかなものであり、添付の請求の範囲内に入るものである。
【図面の簡単な説明】
【Ｆｉｇ１】 ＤＮＡ３０６７６（配列番号：２）の核酸配列並びに天然配列ＰＲＯ２０１（Ｎｓｐ１）ポリペプチド（配列番号：１）のコード化アミノ酸配列を示す図である。
【Ｆｉｇ２】 ＤＮＡ４０５７５（配列番号：４）の核酸配列並びに天然配列ＰＲＯ３０８（Ｎｓｐ２）ポリペプチド（配列番号：３）のコード化アミノ酸配列を示す図である。
【Ｆｉｇ３】 ＤＮＡ６１６０１（配列番号：６）の核酸配列並びに天然配列ＰＲＯ３０９（Ｎｓｐ３）ポリペプチド（配列番号：５）のコード化アミノ酸配列を示す図である。
【Ｆｉｇ４Ａ−Ｃ】 本発明の全長ＤＮＡ３０６７６（配列番号：２）、ＤＮＡ４０５７５（配列番号：４）及びＤＮＡ６１６０１（配列番号：６）核酸配列の単離に使用した（LIFESEQ(登録商標)データベース、Incyte Pharmaceuticals, Palo Alto, Ca）の、１３２８９３８（配列番号：１３）、１０４１９１（配列番号：１４）及び１６５８１１（配列番号：１５）各々の配列を示す図である。
【Ｆｉｇ５Ａ】 ＤＮＡ３０６７６（配列番号：２）の単離に使用したオリゴヌクレオチド配列（配列番号：７、配列番号：８）を示す図である。
【Ｆｉｇ５Ｂ】 ＤＮＡ４０５７５（配列番号：４）の単離に使用したオリゴヌクレオチド配列（配列番号：９、配列番号：１０）を示す図である。
【Ｆｉｇ５Ｃ】 ＤＮＡ６１６０１（配列番号：６）の単離に使用したオリゴヌクレオチド配列（配列番号：１１、配列番号：１２）を示す図である。
【Ｆｉｇ６Ａ】 Ｎｓｐ１（配列番号：１）、Ｎｓｐ２（配列番号：３）及びＮｓｐ３（配列番号：５）の間の種々のドメインの比較を例示する図である。
【Ｆｉｇ６Ｂ】 前記３つの配列の間の同一性の程度を最大にするために必要ならばギャップを挿入してそれらの実際の配列自体を示す図である。
【Ｆｉｇ７】 Ｎｓｐ１（配列番号：１）の配列を示す図であり、ＳＨ２領域は太字で、Ｐ／Ｓ領域のプロリン及びセリンは各々一本及び二本の下線で示した。
【Ｆｉｇ８】 Ｎｓｐ１（配列番号：１）と、ヒトＳｈｃ（配列番号：１６）、Ｓｃｋ（配列番号：１７）及びＦｅｓ（配列番号：１８）との比較を示す図である。
【Ｆｉｇ９Ａ】 Ｎｓｐ１（配列番号：２）はヒト胎児肝臓で有意に発現されるがＮｓｐ２（配列番号：４）及びＮｓｐ３（配列番号：６）はより広く発現されることを示すノーザンブロットの図である。
【Ｆｉｇ９Ｂ】 造血組織における２つのＮｓｐ３転写物の発現を示す図である。
【Ｆｉｇ１０Ａ−１０Ｃ】 １０Ａ及び１０Ｃはウェスタンブロットであり、抗-ｆｌａｇ免疫沈降が抗-ｆｌａｇ、抗-(Ｐ)Ｔｙｒ又は抗-Ｃａｓ抗体で表示したようにブロットされた。１０Ｂでは、抗-ＥＧＦレセプター（CalBiochem）免疫沈降が抗-ｆｌａｇ又は抗-(Ｐ)Ｔｙｒ抗体で表示したようにブロットされた。
【Ｆｉｇ１１】 抗-Ｆｌａｇ又は抗-ｐ１３０^Ｃａｓでの免疫沈降を示すウェスタンブロット、及び抗-(Ｐ)ＴｙｒＡｂＰＹ-２０又は抗-ｐ１３０^Ｃａｓでのブロットを示す図である。
【Ｆｉｇ１２Ａ】 インシュリン処理時のＮｓｐ１（配列番号：１）の減少したリン酸化を示すウェスタンブロットを示す図である。
【Ｆｉｇ１２Ｂ】 Ｆｉｇ１２Ａのブロットの剥離により精製されたウェスタンブロットを示す図であり、抗-ｐ１３０^Ｃａｓで再プローブして１３０ｋＤタンパク質が実際にｐ１３０^Ｃａｓであることを確認した。
【Ｆｉｇ１３】 種々のＮｓｐ１変異体のウェスタンブロットを示す図であり、それらはＣＯＳ細胞に形質移入され、ＥＧＦで処理され、及び抗-ＦｌａｇＡｂで免疫沈降された細胞溶解物が抗-(Ｐ)Ｔｙｒ、抗-ｐ１３０^Ｃａｓ又は抗-ｆｌａｇＡｂでウェスタンブロットされた。
【Ｆｉｇ１４】 １４Ａはレトロウイルス感染ベクターＭＳＣＶＮＩＨ３Ｔ３細胞の極薄切片の顕微鏡写真であり、１４ＢはＮｓｐ１（配列番号：２）形質移入されたＮＩ３Ｔ３細胞である。１４Ｃは固定され、ブロックされ、ヘマトキシリン及びエオシンで断面染色された４つの弱い腫瘍の極薄切片の顕微鏡写真である。
【Ｆｉｇ１５】 ベクター制御形質移入及びＮｓｐ１．ｓｃ１、．ｓｃ２及び．ｓｃ３細胞の腫瘍サイズを比較する棒グラフである。
【Ｆｉｇ１６】 形質転換サブクローンＮｓｐ．ｓｕｂ１．１、Ｎｓｐ．ｓｕｂ２．１、非形質転換細胞培養（ＮＩＨ３Ｔ３-Ｎｓｐ１．ｎｏｎ-ｔｒａｎｓ）及び対照細胞ＮＩＨ３Ｔ３-ｎｅｏの成長因子飢餓ＮＩＨ３Ｔ３のアポトーシス耐性を示す棒グラフである。
【Ｆｉｇ１７】 ｐＲＫ又はＮｓｐ１で形質移入され、ＥＧＦで処理された又は未処理のＣＯＳ細胞のウェスタンブロットを示す図であり、それはＣｏＩＰバッファーに溶解され、ｐＩ３-キナーゼＮ-末端又はＣ-末端ＳＨ２ドメイン-ＧＳＴビーズ（UBI）とともにインキュベートした。沈殿したＮｓｐ１は抗-ｆｌａｇ抗体で検出した。
【Ｆｉｇ１８】 ヒトゲノムにおけるＤＮＡ３０６７６（配列番号：２）の粗いおよそのマッピングを示す染色体１９の図的表示である。
【Ｆｉｇ１９】 種々の肺及び結腸腫瘍において増幅が見られるＤＮＡ３０６７６（配列番号：２）のスプライシング変異体であるＤＮＡ４０５５６（配列番号：２０）を示す図である。
【Ｆｉｇ２０】 Ｎｓｐ１（配列番号：２）、Ｎｓｐ２（配列番号：４）及びＤＮＡ４０５５６（配列番号：２０）にコードされるタンパク質配列の比較を示す図である。
【配列表】

[0001]
(Field of Invention)
The present invention relates generally to the identification and isolation of novel DNA and the recombinant production of novel polypeptides characterized by the presence of novel SH2-containing proteins (Nsp's).
[0002]
(Background of the Invention)
Interactions between ligands and cognate cell surface receptors are important for a variety of biological processes including maintenance of cell and organism homeostasis, development and tumorigenesis. Many of these ligands can activate multiple independent pathways, and the strength of activating different pathways can be altered by the presence or absence of signals from other receptors. Hotamisligil et al., Proc Natl. Acad. Sci. USA 91: 4854-58 (1994); Kanety et al., J. Biol. Chem. 270: 23780-84 (1995); Luttrell et al., J. Biol. Chem. 272: 4637 -44 (1997). Adapter molecules are important in the integration of multiple signaling cascades and in determining cell type specific responses to extracellular stimuli. These adapter proteins have no apparent catalytic activity. Rather, they contain one or more domains that mediate protein-protein or protein-lipid interactions. The most commonly conserved interaction domains in these adapter molecules are the Src homology (SH2), SH3, phosphotyrosine binding site (PTB) and plectrin homology domains [Pawson and Scott, Science 278: 2075-80 (1997). ).
[0003]
Signals generated by growth factors such as epidermal growth factor (EGF) or insulin growth factor-1 (IGF-1) via growth via receptor tyrosine kinase (RTK) or by extracellular matrix components acting via integrin receptors Can induce changes in the cytoskeleton, Leventhal et al., J. Biol. Chem. 272: 5214-18 (1997); Ojaniemi and Vuori, J. Biol. Chem. 272: 2443-47 (1996). RTKs can also modulate integrin signals, or vice versa, Doerr and Jones, J. Biol. Chem. 271: 2443-47 (1996); Jones et al., Proc. Natl. Acad. Sci. USA 93: 2482-87 (1996); Knight et al., J. Biol. Chem. 270: 10199-203 (1995); Matsumoto et al., Cancer Mates. Rev. 14: 205-17 (1995). However, the details of how RTKs signal to cytoskeletal components are not yet fully understood. In addition, some adapter proteins have a restricted expression pattern [Liu and Roth, Proc. Natl. Acad. Sci. USA 92: 10287-91 (1995); Nakamura et al., Oncogene 13: 1111-21 (1996) )], Many are expressed uniformly [Araki et al., Diabetes 42: 1041-54 (1993); Frantz et al., J. Biol. Chem. 272: 2659-67 (1997)]. Thus, it is unclear how biologically relevant outputs are modulated when cells differentiate.
[0004]
Cancer is derived from normal tissue and forms an abnormal or tumorigenic cell that forms a tumor entity, invasion of adjacent tissue by these tumorigenic tumor cells, and ultimately blood and lymphatic system. It is characterized by the generation of malignant cells that diffuse (metastasize) to local lymph nodes and distant sites. In a cancerous state, cells proliferate under conditions in which normal cells do not grow. Cancer itself manifests in a wide variety of forms characterized by different degrees of invasiveness and aggressiveness.
Altered gene expression is strongly associated with uncontrolled cell growth and dedifferentiation and is a common feature of all cancers. Certain well-studied genomes, usually called tumor suppressor genes, normally prevent malignant cell growth or overexpression of certain dominant genes, such as oncogenes that act to promote malignant growth It has been found to exhibit a phenomenon of recessive gene expression that acts like These genetic changes have been shown to be responsible for the transfer of some of the traits that aggregate and display sufficient oncogenic phenotype (Hunter, Cell 64: 1129 [1991]; Bishop, Cell 64 : 235-248 [1991]).
[0005]
A well-known gene (eg, oncogene) overexpression mechanism in cancer cells is gene amplification. This is the process by which multiple copies of a particular gene are generated in the ancestral cell chromosome. This process involves unplanned replication of the chromosomal region containing the gene, followed by recombination of the replicated segment back to the chromosome (Alitalo et al., Adv. Cancer Res. 47: 235-281 [1986]). It is believed that overexpression of a gene parallel to gene amplification is proportional to the number of copies made.
Proto-oncogenes encoding growth factors and growth factor receptors have been identified as playing an important role in the causes of various human malignancies, including breast cancer. For example, the 185-kd transmembrane glycoprotein receptor related to epidermal growth factor receptor (EGFR) (p185^HER2, HER2), the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2) was found to be overexpressed in about 25% to 30% of human breast cancers (Slamon et al., Science 235: 177-182 [1987]; Slamon et al., Science 244: 707-712 [1989]).
[0006]
Protooncogene gene amplification is an event typically involved in more malignant forms of cancer and has been reported to be able to act as a predictor of clinical outcome (Schwab et al., Genes Chromosome Cancer 1, 181 -193 [1990]; Alitalo et al., Supra). That is, overexpression of erbB2 is commonly seen as a precursor of incomplete prognosis, particularly in patients with primary disease, including axillary lymph nodes (slamon et al., [1987] and [1989], supra. Ravdin and Chamness, Gene 159: 19-27 [1995]; and Hynes and Stern, Biochem Biophys Acta 1198: 165-184 [1994]), including hormone therapy and CMF (cyclophosphamide, methotrexate, and fluorouracil) It was associated with sensitivity or resistance to chemotherapeutic drugs (Baselga et al., Oncology 11 (3 Suppl 1): 43-48 [1997]). However, despite the association between erbB2 overexpression and incomplete prognosis, HER2-positive patients were three times more likely to be clinically responsive to taxane treatment than HER2-negative patients. Recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (humanized form of murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or Herceptin®) has been subjected to a wide range of conventional anticancer therapies. Is clinically active in patients with metastatic breast cancer that overexpresses. (Baselga et al., J. Clin. Oncol. 14: 737-744 [1996]).
[0007]
(Summary of Invention)
  Applicants have identified cDNA clones (DNA30676, DNA40575, and DNA61601) encoding novel polypeptides named "Nsp1, Nsp2, and Nsp3" (SEQ ID NOs: 1, 3, and 6), respectively, according to the present invention (respectively, SEQ ID NO: 2, 4 and 6) were identified.
  In one embodiment, the invention provides (a) amino acids 1 to 576 of FIG1 (SEQ ID NO: 1), amino acids 1 to 501 of FIG2 (SEQ ID NO: 3), or amino acids 1 to 1 of FIG3 (SEQ ID NO: 5). A DNA molecule encoding a polypeptide comprising the sequence 703, or (b) a DNA molecule of (a)Complementary sequenceAn isolated nucleic acid having at least about 80% sequence identity to is provided. The sequence identity is preferably about 85%, more preferably about 90%, and most preferably about 95%. In one embodiment, the isolated nucleic acid comprises 1-576 of FIG. 1 (SEQ ID NO: 1), 1-501 of FIG. 2 (SEQ ID NO: 3), and 1-703 amino acid residues of FIG. 3 (SEQ ID NO: 5). It has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity to a polypeptide having a group. Preferably, the highest sequence identity is at the serine / proline enriched domain (ie, amino acid residues 145-299 of SEQ ID NO: 1, amino acid residues 28-210 of SEQ ID NO: 3, amino acid residues of SEQ ID NO: 5). In groups 181 to 415). Alternatively, the greatest degree of identity occurs in the SH2 domain (ie, amino acid residues 1-118 of SEQ ID NO: 1 and amino acid residues 50-166 of SEQ ID NO: 5). In further embodiments, the isolated nucleic acid molecule comprises an amino acid residue: (a) FIG. 1 (SEQ ID NO: 1) 1-576, (b) FIG. 2 (SEQ ID NO: 3) 1-501, or (c). A DNA encoding a PRO201, PRO308 or PRO309 polypeptide having 1 to 703 of FIG. 3 (SEQ ID NO: 5); or complementary to such an encoding nucleic acid sequence, at least moderately and in some cases highly It remains stably bound to it under stringent conditions. Preferably, the nucleic acid molecule is a region determined by amino acid residues: (a) FIG. 1 (SEQ ID NO: 1) 32 to 576 or (b) FIG. 3 (SEQ ID NO: 5) 1 to 424 or 506 to 703 Hybridize to DNA encoding the inner fragment. In another aspect, the invention relates to clone DNA 30676-1223 (SEQ ID NO: 2), DNA 40575-1223 (SEQ ID NO: 4) and DNA 61601-1223 (SEQ ID NO: 4) deposited under ATCC deposit numbers ATCC 209567, ATCC 209565 and ATCC 209713, respectively. The nucleic acid of the full-length protein of No. 6), or clone DNA 30676-1223 (SEQ ID NO: 2), DNA 40575-1223 (SEQ ID NO: 4) and DNA 61601-1223 deposited under the deposit numbers ATCC 209567, ATCC 209565 and ATCC 209713, respectively. The coding sequence of (SEQ ID NO: 6) is provided.
[0008]
In yet another embodiment, the present invention provides a vector comprising DNA encoding a PRO201, PRO308 or PRO309 polypeptide. Host cells containing such vectors are also provided. For example, the host cell may be a CHO cell, E. coli or a yeast cell. Further provided is a method for producing PRO201, PRO308 or PRO309 polypeptide comprising culturing a host cell under conditions suitable for expression of PRO201, PRO308 or PRO309 and recovering it from the cell culture medium.
In yet another embodiment, the invention provides isolated PRO201, PRO308 or PRO309 polypeptide. In particular, the present invention provides an isolated native sequence PRO201, PRO308 or PRO309 polypeptide, which in one embodiment, includes residues 1 to 576 of FIG1 (SEQ ID NO: 1), residues 1 to 501 of FIG2 (sequence No. 3) or an amino acid sequence comprising residues 1 to 703 (SEQ ID NO: 5) of FIG. Specifically included are native PRO201, PRO308 or PRO309 polypeptides that may or may not be initiated from methionine. The present invention also provides PRO201, PRO308 or PRO309 polypeptides encoded by nucleic acids deposited under accession numbers ATCC 209567, ATCC 209565, and ATCC 209713, respectively.
In yet another embodiment, the invention provides a chimeric molecule comprising a PRO201, PRO308, or PRO309 polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule is a chimeric molecule comprising a PRO201, PRO308 or PRO309 polypeptide fused to an epitope tag sequence or an Fc region of an immunoglobulin.
In still other embodiments, the present invention provides compounds and methods that produce antagonists that promote and promote PRO201, PRO308, or PRO309 mediated cell signaling. In particular, PRO201 (eg, Nsp1, SEQ ID NO: 1), PRO308 (eg, Nsp2, SEQ ID NO: 3) or PRO309 (eg, Nsp3, sequence) in cell signaling, including small bioorganic chemical molecules and antisense nucleotides No. 5) is an antagonist of PRO201, PRO308, or PRO309 that prevents, prevents, inhibits, or neutralizes the normal function expression.
In still other embodiments, the present invention provides alternatively spliced variants of PRO201, PRO308 or PRO309 (eg, DNA40556).
[0009]
The invention further relates to compositions and methods for the diagnosis and treatment of tumorigenic cell growth and proliferation in mammals, including humans. The present invention is based on the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to contribute to tumor formation with overexpression of the gene product. Thus, the protein encoded by the amplified gene appears to be a useful target for the diagnosis and treatment (including prevention) of certain types of cancer, or to act as a prognostic for tumor therapy. In one embodiment, the invention provides a polypeptide named PRO201 (eg, Nsp1, SEQ ID NO: 1), PRO308 (eg, Nsp2, SEQ ID NO: 3) or PRO309 (eg, Nsp3, SEQ ID NO: 5). It relates to an isolated antibody that binds to. In one aspect, the antibody induces death of cells that overexpress a PRO201, PRO308 or PRO309 polypeptide. In other embodiments, the antibody is a monoclonal antibody, which preferably has non-human complementarity determining region (CDR) residues and a human framework region (FR). The antibody may be labeled or may be immobilized on a solid support. In a further aspect, the antibody is an antibody fragment, a single chain antibody, or an anti-idiotype antibody.
In another embodiment, the invention relates to a composition comprising a mixture of an antibody that binds to a PRO201, PRO308 or PRO309 polypeptide and a pharmaceutically acceptable carrier. In one aspect, the composition contains additional active ingredients, which may be, for example, additional antibodies or cytotoxic or chemotherapeutic agents. Preferably the composition is sterile.
In a further embodiment, the invention relates to nucleic acids encoding anti-PRO201, anti-PRO308 or anti-PRO309 antibodies, vectors containing such nucleic acids and host cells.
In a further embodiment, the present invention relates to anti-PRO201, anti-PRO308 or anti-probe by transfecting host cells with nucleic acid encoding the antibody under conditions in which the antibody is expressed and recovering the antibody from the cell culture medium. The present invention relates to a method for producing a PRO309 antibody.
[0010]
  The invention further relates to antagonists and agonists of PRO201, PRO308 or PRO309 polypeptide that inhibit one or more functions or activities of PRO201, PRO308 or PRO309 polypeptide.
  In a further embodiment, the present invention provides a nucleic acid molecule encoding a PRO201, PRO308 or PRO309 polypeptide.Complementary sequenceRelates to an isolated nucleic acid molecule which hybridizes to The nucleic acid is preferably DNA and hybridization preferably occurs under stringent conditions. Preferably, the nucleic acid molecule is from (a) the FIG. 1 (SEQ ID NO: 2) from about 245 to 2413 or (b) the FIG. 3 (SEQ ID NO: 6) from 1 to about 1312 or from about 1555 to about 2150 nucleotide residues. Hybridize to the region. Such nucleic acid molecules can act as antisense molecules for the amplified genes identified herein, and in turn find use in modulating the corresponding amplified genes or as antisense primers for amplification reactions. In addition, such sequences can be used as part of ribozyme or triple helix sequences, and in turn can be used in the regulation of amplified genes.
  In another embodiment, the invention also relates to a method of detecting the presence of a PRO201, PRO308 or PRO309 polypeptide, which comprises treating cells suspected of containing PRO201, PRO308 or PRO309 polypeptide with anti-PRO201, anti-PRO308. Or exposure to an anti-PRO309 antibody and measuring the binding of the antibody to cells.
  In yet another embodiment, the invention relates to a method for diagnosing a tumor in a mammal, comprising (a) a test sample of tissue cells obtained from a mammal, and (b) of a known normal tissue cell of the same cell type. Detecting the expression level of a gene encoding PRO201, PRO308 or PRO309 polypeptide in a control sample, wherein a high level in the test sample indicates the presence of a tumor in the mammal from which the test tissue cells were obtained.
  In another embodiment, the invention relates to a method for diagnosing a tumor in a mammal, which comprises (a) anti-PRO201, anti-PRO308 or anti-PRO309.antibody(B) detecting the formation of a complex of anti-PRO201, anti-PRO308 or anti-PRO309 antibody and PRO201, PRO308 or PRO309 in the test sample of interest. Including doing. Detection may be qualitative or quantitative and is performed in comparison to monitoring complex formation in a control sample of known normal tissue cells of the same cell type. A large amount of complex formation in the test sample indicates the presence of a tumor in the mammal from which the test tissue cells were obtained. The antibody preferably has a detectable label. Complex formation is monitored, for example, by light microscopy, flow cytometry, fluorescence measurements, or other techniques known in the art.
[0011]
The test sample is usually obtained from an individual suspected of having tumorigenic cell growth or proliferation (eg, cancerous cells).
In another embodiment, the invention relates to a diagnostic kit comprising anti-PRO201, anti-PRO308 or anti-PRO309 antibody and a carrier (eg, buffer) in suitable packaging. The kit preferably comprises instructions for the use of antibodies against PRO201, PRO308 or PRO309 polypeptides.
In yet another embodiment, tumor cell growth comprising exposing a cell overexpressing PRO201, PRO308 or PRO309 polypeptide to an effective amount of an agent that inhibits expression or activity of PRO201, PRO308 or PRO309 polypeptide. Relates to a method for inhibiting. This agent is preferably an anti-PRO201, anti-PRO308 or anti-PRO309 antibody, small organic and inorganic molecules, peptides, phosphopeptides, antisense or ribozyme molecules, or triple helix molecules. In particular embodiments, the agent, eg, anti-PRO201, anti-PRO308 or anti-PRO309 antibody, induces cell death. In a further aspect, the tumor cells are further exposed to radiation therapy or cytotoxic or chemotherapeutic agents.
In a further embodiment, the invention provides:
container;
A label on the container; and
A composition comprising an active agent contained in a container, wherein the composition is effective in inhibiting tumor cell growth and the label on the container is labeled with a PRO201, PRO308 or PRO309 polypeptide. The active agent in the composition is an agent that inhibits the expression or activity of PRO201, PRO308 or PRO309 polypeptide, indicating that it can be used to treat conditions characterized by overexpression of. In a preferred embodiment, the active agent is an anti-PRO201, anti-PRO308 or anti-PRO309 antibody.
A method of identifying a compound capable of inhibiting the expression or activity of a PRO201, PRO308 or PRO309 polypeptide is sufficient to allow a candidate compound to interact with the PRO201, PRO308 or PRO309 polypeptide under conditions and these two components. Including contact for a long time. In a special embodiment, either the candidate compound or PRO201, PRO308 or PRO309 polypeptide is immobilized on a solid support.
[0012]
Detailed Description of Preferred Embodiments
I. Definition
As used herein, the terms “PRO201, PRO308, or PRO309” and “PRO201, PRO308, or PRO309 polypeptide” refer to the native sequence PRO201, PRO308, or PRO309, and variants of PRO201, PRO308, or PRO309, respectively (here further Defined in detail). These may be isolated from a variety of sources, such as human tissue types or other sources, or may be prepared by recombinant or synthetic methods.
“Native sequence PRO201, PRO308, or PRO309” includes a polypeptide having the same amino acid sequence as a naturally occurring PRO201, PRO308, or PRO309 polypeptide. Such native sequence PRO201, PRO308 or PRO309 can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence PRO201, PRO308 or PRO309” specifically includes naturally occurring truncated or secreted forms of PRO201, PRO308 or PRO309 (eg, extracellular domain sequences), naturally occurring mutated forms (eg, alternatively splicing). And naturally occurring allelic variants of PRO201, PRO308 or PRO309. Throughout this specification, the designations “PRO” and “Nsp” are both used to indicate the corresponding protein, where “Nsp” is the specific native human sequence of FIGS. 1, 2 and 3, or under ATCC 209567, 209565 and 209713, respectively. Shows the deposited cDNA sequence. “Nsp” indicates a specific listed species, while “PRO” indicates a native sequence (including Nsps) and variants thereof.
In one embodiment of the invention, the native sequence PRO201, PRO308 or PRO309 is
(A) includes amino acids 1 to 576 of FIG1 (SEQ ID NO: 1), (b) amino acids 1 to 501 of FIG2 (SEQ ID NO: 3), and (c) amino acids 1 to 703 of FIG3 (SEQ ID NO: 5). The mature or full-length native sequence PRO201, PRO308 or PRO309, with or without an N-terminal signal sequence and with or without an initiation methionine at position 1.
[0013]
  “PRO201, PRO308 or PRO309 variant”, as defined below, respectively, (a) a DNA molecule encoding a PRO201, PRO308 or PRO309 polypeptide with or without a native signal sequence, or (b) (a) Of DNA moleculesComplementary sequenceAnd biologically active PRO201, PRO308 or PRO309 having at least about 80% amino acid sequence identity. In a special embodiment, the PRO201, PRO308 or PRO309 variant is shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 3), FIG. 3 (SEQ ID NO: 5) for the full-length native sequence PRO201, PRO308 or PRO309. It has at least about 80% amino acid sequence homology with PRO201, PRO308 or PRO309 having the deduced amino acid sequence. Such a PRO201, PRO308 or PRO309 variant is not limited to these, but is, for example, N- or C of the sequence of FIG1 (SEQ ID NO: 1), FIG2 (SEQ ID NO: 3), or FIG3 (SEQ ID NO: 5). -Includes PRO201, PRO308 or PRO309 polypeptides with one or more amino acid residues added or deleted at the terminus. Preferably, the nucleic acid or amino acid sequence identity is at least about 85%, more preferably at least about 90%, even more preferably at least about 95%.
[0014]
"Percent (%) amino acid sequence identity" identified herein to a PRO201, PRO308 or PRO309 sequence introduces gaps if necessary to align the sequences and obtain maximum percent sequence identity; Defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues of the PRO201, PRO308, or PRO309 sequence, assuming that any conservative substitutions are not considered part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved by various methods within the skill of the art, such as publicly available computer software such as the BLAST-2 software set to the initial values. This can be achieved by using wear. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity value used here may be calculated by the program “Align-2”, which has no parameters and is authorized by Genentech on December 10, 1991 with the instructions for use. US Copyright Office, Washington, D.C. C. Filed in 20559. Align-2 was registered under US copyright registration number TXU-510087.
“Percent (%) nucleic acid sequence identity” relative to the PRO201, PRO308 or PRO309 sequences identified herein introduces gaps if necessary to align the sequences and obtain maximum percent sequence identity, and Or defined as the percentage of nucleotides in the candidate sequence that are identical to the nucleotides of the PRO309 sequence. Alignments for the purpose of determining percent nucleic acid sequence identity can be achieved by various methods within the purview of those skilled in the art, such as publicly available computer software such as BLAST-2 software set to initial parameters. Can be achieved by using. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity value used here may be calculated by the program “Align-2”, which has no parameters and is authorized by Genentech on December 10, 1991 with the instructions for use. US Copyright Office, Washington, D.C. C. Filed in 20559. Align-2 was registered under US copyright registration number TXU-510087.
[0015]
“Isolated”, when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated or recovered from a component of its natural environment. Contaminant components of the natural environment are substances that typically interfere with the diagnostic or therapeutic use of the polypeptide, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide is (1) sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a spinning cup sequenator, or (2) Coomassie blue or It is preferably purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using silver staining. Isolated polypeptide includes protein in situ within recombinant cells, since at least one component of the PRO201, PRO308, or PRO309 natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
An “isolated” DNA 30676 (SEQ ID NO: 2), DNA 40575 (SEQ ID NO: 4) or DNA 61601 (SEQ ID NO: 6) nucleic acid molecule has been identified and the DNA 30676, DNA 40575 or DNA 61601 nucleic acid (SEQ ID NO: 2, respectively) A nucleic acid molecule separated from at least one contaminating nucleic acid molecule usually associated with a natural source of 4 or 6). The isolated DNA30676, DNA40575 or DNA61601 nucleic acid molecule (SEQ ID NO: 2, 4, or 6 respectively) is in a form or setting other than that found in nature. Thus, an isolated DNA30676, DNA40575 or DNA61601 nucleic acid molecule (SEQ ID NO: 2, 4 or 6 respectively) is a DNA30676, DNA40575 or DNA61601 nucleic acid molecule (SEQ ID NO: 2, 4 or 4 respectively) present in natural cells. Or 6). However, an isolated DNA30676, DNA40575 or DNA61601 nucleic acid molecule (SEQ ID NO: 2, 4 or 6 respectively) is usually a DNA30676, DNA40575 or DNA61601 nucleic acid molecule (for example, where the nucleic acid molecule is at a chromosomal location different from that of natural cells). Each comprises a DNA30676, DNA40575 or DNA61601 nucleic acid molecule (SEQ ID NO: 2, 4, or 6 respectively) contained in a cell expressing SEQ ID NO: 2, 4 or 6).
[0016]
The “stringency” of a hybridization reaction is readily determined by those skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strand is present in an environment near its melting point (Tm) but lower. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures make the reaction conditions more stringent, while lower temperatures reduce stringency. Furthermore, stringency is inversely proportional to salt concentration. For further details and explanation of the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
“Stringent conditions” as defined herein are: (1) Low ionic strength and high temperature for washing, eg, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% at 50 ° C. (2) denaturing agents such as formamide during hybridization, for example 50% (vol / vol) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0 at 42 ° C. Using 1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5 and 750 mM sodium chloride, 75 mM sodium citrate; (3) 50% formamide at 42 ° C., 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0 0.1% sodium pyrophosphate, 5 × denhard solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate and 0.2 × SSC (sodium chloride / citric acid at 42 ° C. By using a high stringency wash consisting of 0.1x SSC with formamide at 55 ° C followed by EDTA at 55 ° C.
“Moderate stringency conditions” are identified as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, and include washing solutions and hybrids less than the above stringency. Including the use of formation conditions (eg, temperature, ionic strength and% SDS). Examples of moderate stringency conditions are 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 mg / Conditions such as overnight incubation at 37 ° C. in a solution containing mL denatured sheared salmon sperm DNA, followed by washing the filter at 37-50 ° C. in 1 × SSC. One skilled in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length. The term “epitope tag” as used herein is a chimeric polypeptide comprising a DNA30676, DNA40575 or DNA61601 polypeptide nucleotide (SEQ ID NO: 1, 3 or 5 respectively) fused to a “tag polypeptide”. Means. A tag polypeptide has sufficient residues to provide an epitope from which the antibody can be produced, but is short enough so that it does not inhibit the activity of the polypeptide to which it is fused. Also, the tag polypeptide is preferably fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, usually about 8 to about 50 amino acid residues (preferably about 10 to about 20 residues).
[0017]
The term “control sequence” refers to a DNA sequence necessary to express an operably linked coding sequence in a particular host organism. For example, suitable control sequences for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
A nucleic acid is “operably linked” when it is in a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA, if expressed as a preprotein that participates in polypeptide secretion, is operably linked to the polypeptide DNA; a promoter or enhancer is responsible for transcription of the sequence. If it does, it is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is in a position that facilitates translation. In general, “operably linked” means that the bound DNA sequences are in close proximity and, in the case of a secretory leader, in close proximity and in the reading phase. However, enhancers do not necessarily have to be close together. Binding is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional techniques.
The term “antibody” is used in the broadest sense, particularly a single anti-PRO201, anti-PRO308 or anti-PRO309 monoclonal antibody (including agonists, antagonists and neutralizing antibodies), and anti-antibodies with multi-epitope specificity. -PRO201, anti-PRO308 or anti-PRO309 antibody compositions are included. The term “monoclonal antibody” as used herein is identical except for a substantially homogeneous population of antibodies, ie, naturally occurring mutations in which the individual antibodies that make up may be present in minor amounts. Refers to an antibody obtained from a population.
[0018]
“Active” and “activity” in the content of a molecule identified based on a PRO201, PRO308 or PRO309 polypeptide (or coding sequence thereof) refers to the biological or immunity of a natural or naturally occurring PRO201, PRO308 or PRO309. It means a polypeptide (such as an antibody) or an organic or inorganic small molecule, a peptide or the like that retains a biological activity.
A “biological” activity is a biological function (inhibitory) caused by a natural or naturally occurring PRO201, PRO308 or PRO309, other than the ability to include antibody production against an antigenic epitope possessed by the natural or naturally occurring PRO201, PRO308 or PRO309. Or irritation). “Immunological activity” means the ability to include antibody production against an antigenic epitope possessed by a naturally occurring or naturally occurring PRO201, PRO308 or PRO309.
Preferred biological activities include, for example, biological activities identified by the screening assays identified herein, such as growth inhibition of target tumor cells. Another preferred biological activity is a cytotoxic activity that results in the death of the target tumor cell. Further preferred activities include modulation or tumorigenesis and response to stimulation, for example by integrin receptor ligands and epidermal growth factor (EGF) and through other receptor tyrosine (RTK) ligands. More preferably, the biological activity can be determined by measuring the activation of guanylate exchange, in particular c-jun kinase (JNK).
The term “modulation” means an effect on the level of a signal transduction pathway (eg, up-regulation, down-regulation or other control). Cellular processes under the control of signal transduction include, but are not limited to, transcription of specific genes; normal cellular functions such as metabolism, proliferation, differentiation, adhesion, apoptosis and survival, and transformation, differentiation and metastasis. It can include abnormal processes such as blocking.
[0019]
As used herein, the term “antagonist”, in its broadest sense, refers to a process by a PRO201, PRO308 or PRO309 molecule of the invention that interferes with the interaction of any protein domain of PRO201, PRO308 or PRO309 with various target proteins. Includes any molecule that blocks, prevents, inhibits, neutralizes. Such an interaction is generally achieved at the C-terminus of PRO201, PRO308 or PRO309, or in particular the polypeptide proline with a SH2 or proline / serine (P / S) enriched region with a phosphotyrosyl residue and a target binding site. Can occur with motifs. Similarly, the term “agonist” as used herein refers to PRO201, PRO308, or PRO201, PRO308, which contain SH2 or proline / serine (P / S) enriched regions, each with a phosphotyrosyl residue and a polypeptide proline motif on various target proteins. Any molecule that promotes, enhances or stimulates the interaction of the protein domains of PRO309 (eg, Nsp1, Nsp2, and Nsp3, respectively). Preferred molecules that affect the interaction of PRO201, PRO308 or PRO309 and phosphotyrosyl residues and polypeptide proline motifs, respectively, or target proteins include the latter fragments or small bioorganic small molecules, such as peptide analogs, in some cases It will prevent or promote interaction accordingly. Non-limiting examples include proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptide analogs, pharmaceutical formulations or their metabolites, transcriptional and translational control sequences, etc. Including. Other preferred forms of antagonists include antisense nucleotides that inhibit PRO201, PRO308 or PRO309 modulated signaling. Preferred forms bind to PRO201, PRO308 or PRO309, or a specific region of the target with which PRO201, PRO308 or PRO309 interacts.
“Small molecule” is defined herein as having a molecular weight of less than about 500 Daltons. “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit specificity for a particular antigen, immunoglobulins include both antibodies and other antibody-like molecules that do not have antigen specificity. The latter type of polypeptide is produced, for example, at low levels by the lymphatic system and at improved levels by myeloma. The term “antibody” is used in the broadest sense and is not limited to an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody (eg, a bispecific antibody) formed from at least two intact antibodies, And antibody fragments so long as they have the desired biological activity.
[0020]
The terms “gene amplification” and “gene replication” are used interchangeably and refer to the process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The replication region (strand of amplified DNA) is often referred to as the “amplicon”. Usually, the amount of messenger RNA (mRNA) produced, ie, the level of gene expression, also increases in proportion to the copy number made with a particular gene expression. “Tumor” as used herein means the growth and proliferation of all tumorigenic cells, either benign or malignant, and all precancerous and cancerous cells and tissues.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include breast cancer, prostate cancer, rectal cancer, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, blastoma, cervical cancer, ovarian cancer , Liver cancer, bladder cancer, hepatocytoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, liver cancer, spawning cancer, thyroid cancer, hepatic carcinoma and various types of head and neck Some cancers are included.
[0021]
“Treatment” is a treatment intended to prevent the progression and change of disease pathology. "Treatment" thus refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already suffering from the disease as well as those that should protect against the disease. In tumor (eg, cancer) treatment, the therapeutic agent directly reduces the pathology of the tumor cells or renders the tumor cells susceptible to other therapeutic agents such as radiation or chemotherapy.
The “pathology” of cancer includes all phenomena that compromise the health of the patient. This includes, but is not limited to, abnormal and uncontrolled cell growth, metastasis, disruption of normal functioning of neighboring cells, release of cytokines or other secretions at abnormal levels, suppression or exacerbation of inflammation or immune response, etc. .
“Mammal” for therapeutic purposes means any animal classified as a mammal, humans, livestock and agricultural animals, and zoo, sports or pet animals such as dogs, horses, cats, hamsters Including rabbits, mice, cows, pigs, goats, sheep and the like. Preferably the mammal is a human.
[0022]
As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to it at the dosages and concentrations employed. is there. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include phosphate, citrate, and other organic acid buffers; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin , Gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides such as glucose, mannose or dextran; disaccharides and other carbohydrates; chelation such as EDTA Agents; sugar alcohols such as mannitol or sorbitol; self-forming counterions such as sodium; or nonionic surfactants such as TWEEN (trade name), polyethylene glycol (PEG), and PLURONICS (trade name).
Administration of “in combination with” or “in combination with” one or more further therapeutic agents includes simultaneous (temporary) and sequential administration in any order.
The term “cytotoxic agent” as used herein refers to a substance that inhibits or suppresses the function of cells or causes destruction of cells. This term refers to radioisotopes (eg, I¹³¹, I¹²⁵, Y⁹⁰And Re¹⁸⁶), Chemotherapeutic agents, and toxins such as enzymatically active toxins from bacteria, fungi, plants or animals, or fragments thereof.
[0023]
A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids such as paclitaxel (trade name ), Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere (trade name), Rhone-Poulenc Rorer, Antony, France), Toxoter, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C Mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamicin (US Pat. No. 4,675,187), -Fu, 6 comprises thioguanine, 6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan, and other related nitrogen mustards. Also included in this definition are hormone-like drugs that act to regulate or inhibit hormonal effects on tumors such as tamoxifen and onapristone.
“Growth inhibitory agent” as used herein means a compound or composition that inhibits the growth of cells, particularly cancer cells that overexpress any gene identified herein, in vitro or in vivo. That is, growth inhibitors significantly reduce the proportion of cells that overexpress such genes in the S phase. Examples of growth inhibitory agents include agents that inhibit the cell cycle (at a position other than S phase), such as agents that induce G1 arrest or M phase arrest. Classic M-phase blockers include vincas (vincristine and vinblastine), taxol, and topo II, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. These agents that cause G1 arrest also overflow S phase arrest, for example DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. More information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, ed., Chapter 1, Title `` Cell cycle reguration, oncogene, and antineoplastic drugs '', Murakami et al. (WB Saunders: Philadelphia, 1995), especially p13. it can.
“Doxorubicin” is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis) -10-[(3-amino-2,3,6-trideoxy-α-L-lyxohexapyranosyl) oxy] -7,8,9, 10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5,12-naphthacenedione.
[0024]
The term “cytokine” is a general term for proteins that are released from one cell population and act as intercellular mediators to other cells. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein such as follicle stimulating hormone (FSH) ), Thyrotropin (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Mueller inhibitor; mouse gonadotropin-related peptide Activin; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factor such as NGF-β; Platelet growth factor; Transforming growth factor such as TGF-α and TGF-β; Insulin-like growth factor-I And I Erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-) CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL -7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factors such as TGF-α and TGF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and is the biologically active equivalent of native sequence cytokines.
The term “prodrug” as used in this application is a precursor or derivative of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is enzymatically activated or converted to a more active parent form Means form. For example, Wilman, “Prodrugs in Cancer Chemotherapy”, Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting, Belfast (1986), and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery”, Directed Drug delivery. , Borchardt et al. (Ed.), Pp.147--267, Human Press (1985). The prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs Drugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to more active, non-cytotoxic drugs including. Non-limiting examples of cytotoxic agents that can be derivatized to the prodrug form used in the present invention include the chemotherapeutic agents listed above.
[0025]
“Natural antibodies” and “natural immunoglobulins” are heterotetrameric glycoproteins of approximately 150,000 daltons, usually composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain has regularly spaced interchain disulfide bonds. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is the heavy chain Aligned with the variable domain. Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains.
The term “variable” refers to the fact that certain sites in the variable domain vary widely in sequence within an antibody and are used for the binding and specificity of each particular antibody to that particular antigen. To do. However, variability is not uniformly distributed across the variable domains of antibodies. It is enriched in three or four segments called hypervariable regions or complementarity determining regions (CDS) of both the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The natural heavy and light chain variable domains are four-fold mainly in a β-sheet arrangement linked by a CDR that forms a loop bond that binds and in some cases forms a β-sheet structure. Each of the five FR regions is included. The CDRs of each chain are bound closer to the FR and, together with the CDRs of the other chains, contribute to the formation of the antigen binding site of the antibody (Kabat et al., NIH Publ. No. 91-3242, Vol. I Pp. 647-669 [1991]). The constant domain is not directly related to the binding of the antibody to the antigen, but indicates the involvement of the antibody in various effector functions, such as antibody-dependent cytotoxic activity.
`` High frequency variable region ''
As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that produce antigen binding. Hypervariable regions are amino acid residues from the “complementarity determining region” or “CDR” (ie, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain). And heavy chain variable domains 31-35 (H1), 50-65 (H2) and 95-102 (H3); Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda , MD. (1991)) or “hypervariable loop” (ie residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) of the light chain variable domain) and heavy Chain variable domain residues 26-32 (H1), 53-55 (L2) and 96-101 (L3); Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
[0026]
“Antibody fragments” comprise a portion of a native antibody, preferably the antigen binding or variable region of the native antibody. Examples of antibody fragments are Fab, Fab ′, F (ab ′) 2, and Fv fragments; diabody; linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]. ); Single chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies, called “Fab” fragments, two identical antigen-binding fragments, each with a single antigen-binding site, and the remaining “Fc” fragments, whose names reflect the ability to easily crystallize Is generated. Pepsin treatment produces an F (ab ′) 2 fragment that has two antigen binding sites but can be a cross-linked antigen.
“Fv” is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain in tight, non-covalent association. In this arrangement, V_H-V_LThe three CDRs of each variable region interact to determine the antigen binding site on the surface of the dimer. To be precise, six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only three CDRs that are antigen specific) has the ability to recognize and bind antigen, but has a lower affinity than the entire binding site.
[0027]
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the symbol for Fab ′ where the cysteine residue of the constant domain has a free thiol group. F (ab ′) 2 antibody fragments originally were produced as pairs of Fab ′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
“Light chains” of antibodies (immunoglobulins) from any species are classified into one or two distinct types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domains it can.
Based on the amino acid sequences of the constant domains of their heavy chains, immunoglobulins are divided into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
[0028]
The term “monoclonal antibody” as used herein is derived from a substantially homogeneous population of antibodies, ie, a population that is identical except for naturally occurring mutations in which the individual antibodies comprising the population are present in small amounts. Refers to antibody. Monoclonal antibodies are highly specific and are directed to a single antigenic site. Furthermore, unlike conventional (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. . In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and not being construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 [1975] or by recombinant DNA methods (eg, US Pat. No. 4,816,567). You may create it by reference. “Monoclonal antibodies” are also described from phage antibody libraries, for example, in Clackson et al., Nature, 352: 624-628 [1991] and Marks et al., J. Mol. Biol., 222: 581-597 (1991). May be isolated using different techniques.
Here, monoclonal antibodies specifically include “chimeric” antibodies (immunoglobulins), which correspond to antibodies whose heavy or light chain portions are derived from a particular species or belong to a particular antibody class or subclass. Antibodies that are identical or homologous to the sequence, but the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to a particular antibody class or subclass, as well as those desired Fragments of those antibodies as long as they exhibit biological activity (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 [1984]).
[0029]
“Humanized” forms of non-human (eg, murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′) that contain minimal sequence derived from non-human immunoglobulin. , F (ab ')₂Or other antigen-binding sequence of the antibody). For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) whose complementarity determining regions (CDRs) are CDRs (donor antibodies) of species other than humans such as mice, rats, goats, etc. Substituted with residues having the desired specificity, affinity and capacity derived from In some cases, FvFR frame residues of human immunoglobulins are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or frame sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody has at least one, typically all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin consensus sequence, typically Will contain substantially all of the two variable domains. An optimal humanized antibody will also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See Humanized antibodies include pre-primated PRIMATIZED (trade name) antibodies whose antigen-binding regions are derived from antibodies produced by immunizing macaques with an antigen of interest.
“Single-chain Fv” or “sFv” antibody fragments_HAnd V_LAntibody fragments that contain domains are present in a single polypeptide chain. Preferably, the Fv polypeptide is V_HAnd V_LFurther comprising a polypeptide linker between the domains, which allows sFV to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).
[0030]
The term “diabody” refers to a small antibody fragment having two antigen-binding sites, which fragments are identical polypeptide chains (V_H-V_L) Within the light chain variable domain (V_L) In the heavy chain variable domain (V_H) Are combined. Using a linker that is so short that it allows pairing of two domains on the same chain, the domain is forced to pair with the complementary domain of the other chain, creating two antigen binding sites. Diabodies are described in further detail, for example, in EP 404097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
An “isolated” antibody is one that has been identified and separated or recovered from a component of its natural environment. Contaminants of the natural environment are substances that interfere with the use of antibodies for diagnosis or therapy, and include enzymes, hormones, and other non-proteinaceous solutes. In a preferred embodiment, the antibody is (1) at least 95% by weight or more preferably 99% by weight antibody as determined by the Lowry method, and (2) by using a spinning cup sequenator. To the extent sufficient to obtain 15 N-terminal or internal amino acid sequence residues, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining Purified. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
The term “label” when used herein refers to a detectable compound or composition that binds directly or indirectly to the antibody to produce a “labeled” antibody. The label may be detectable by itself (eg, a radioisotope label or a fluorescent label), or in the case of an enzyme label, it may catalyze chemical conversion of the detectable substrate compound or composition. Radionucleotides that can provide a detectable label include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, And Pd-109.
“Solid phase” means a non-aqueous matrix to which an antibody of the invention can adhere. Examples of solid phases encompassed herein are those partially or wholly formed of glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicone. including. In some embodiments, depending on the context, the solid phase can include the wells of an assay plate; otherwise, a purification column (eg, an affinity chromatography column). The term also includes a discrete solid phase of discrete particles as described in US Pat. No. 4,275,149.
[0031]
II. Compositions and Methods of the Invention
A. Full length PRO201, PRO308 or PRO309
The present invention provides newly identified and isolated nucleic acid sequences encoding polypeptides referred to in the present application as human and PRO201, PRO308 or PRO309. In particular, Applicants have identified and isolated cDNA encoding a PRO201, PRO308, or PRO309 polypeptide, as disclosed in further detail in the Examples below. Using the BLAST-2 sequence alignment computer program set to the initial parameters, Applicants have determined that the full-length native sequences Nsp1, Nsp2, and Nsp3 (shown in FIGS. 6A and 6B and SEQ ID NOs: 1 and 3, respectively) show the SH2 homology region But Nsp1, Nsp2 and Nsp3 (SEQ ID NOs: 1, 3 and 5) were found to have proline / serine enriched (P / S) region homology. While the SH2 domain specifically binds to phosphotyrosyl residues, the P / S region is a potential SH3 interaction domain. Thus, it is now believed that human PRO201, PRO308 or PRO309 disclosed in this application are newly identified members of the adapter protein and can modulate intracellular signaling pathways.
[0032]
B. PRO201, PRO308 or PRO309 variant
In addition to the full-length native sequence PRO201, PRO308, or PRO309 described herein, it is contemplated that PRO201, PRO308, or PRO309 variants can be prepared. PRO201, PRO308 or PRO309 variants can be prepared by introducing appropriate nucleotide changes into the known PRO201, PRO308 or PRO309 DNA, or by synthesizing the desired PRO201, PRO308 or PRO309 polypeptide. One skilled in the art will appreciate that appropriate amino acid changes can alter the post-translational process of PRO201, PRO308 or PRO309 polypeptides. Mutations in the native full-length sequence PRO201, PRO308 or PRO309 or the various domains of PRO201, PRO308 or PRO309 described herein can be achieved by any technique for conservative and non-conservative mutations described, for example, in US Pat. No. 5,364,934. And using guidelines. A mutation is a substitution, deletion or insertion of one or more codons encoding PRO201, PRO308 or PRO309 that results in a change in the amino acid sequence of PRO201, PRO308 or PRO309 compared to the native sequence PRO201, PRO308 or PRO309. Good. In some cases, the mutation is a substitution of at least one amino acid by PRO201, PRO308 or PRO309 with any other amino acid in one or more domains. Guidance on which amino acid residues are inserted, substituted or deleted without adversely affecting the desired activity is compared to the sequence of a protein molecule of known homology with the sequence of PRO201, PRO308 or PRO309; It is found by minimizing amino acid sequence changes made within regions of high homology. The amino acid substitution can be, for example, a substitution of leucine with serine, that is, a conservative amino acid substitution, as a result of substitution of one amino acid with another amino acid having similar structural or chemical properties. Insertions and deletions can optionally be in the range of 1 to 5 amino acids. Acceptable mutations are determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting mutants for activity in any of the in vitro assays described in the Examples below. Is done.
[0033]
Mutations can be made using techniques well known in the art such as oligonucleotide-mediated (site-specific) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34: 315 (1985)], restricted selective mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)] or other DNA cloning techniques known in the art. To PRO201, PRO308 or PRO309 mutant DNA.
Scanning amino acid analysis can also be used to identify one or more amino acids along adjacent sequences. Among them, preferred scanning amino acids are relatively small and neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is a typically preferred scanning amino acid in this group because it eliminates side chains beyond the beta carbon and is unlikely to change the backbone structure of the mutant. Alanine is also typically preferred because it is the most common amino acid. Furthermore, it is often found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)]. If alanine substitution does not result in a sufficient amount of mutants, isoteric amino acids can be used.
[0034]
C. Modification of PRO201, PRO308 or PRO309
Covalent modifications of PRO201, PRO308 or PRO309 are included within the scope of the present invention. One type of covalent modification reacts with PRO201, PRO308 or PRO309 targeted amino acid residues with an organic derivatization reagent capable of reacting with a selected side chain or N or C terminal residue of PRO201, PRO308 or PRO309. It is to let you. Derivatization with a bifunctional reagent is useful, for example, to cross-link PRO201, PRO308 or PRO309 to a surface for use in a water-insoluble support matrix or anti-PRO201, PRO308 or PRO309 antibody purification method or vice versa. . Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide ester, such as 4-azidosalicylic acid, 3,3′-dithiobis (succinimidyl) Homobifunctional imidoesters including disuccinimidyl esters such as propionate), bifunctional maleimides such as bis-N-maleimide-1,8-octane, and methyl-3-[(p-azidophenyl)- Reagents such as dithio] propioimidate.
Other modifications include deamination of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, lysine, arginine, and histidine side chains, respectively. Methylation of the α-amino group of [TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of N-terminal amine, and optional C-terminus Includes amidation of carboxyl groups.
[0035]
Another type of covalent modification of a PRO201, PRO308 or PRO309 polypeptide included within the scope of the invention involves altering the native glycosylation pattern of the polypeptide. “Modification of native glycosylation pattern” refers to the deletion of one or more carbohydrate moieties found in the native sequence PRO201, PRO308 or PRO309, or one or more glycosylation not present in the native sequence PRO201, PRO308 or PRO309. It is intended herein to mean adding a site and / or altering the ratio and / or composition of sugar residues that bind to a glycosylation site.
Addition of glycosylation sites to PRO201, PRO308 or PRO309 polypeptides can be achieved by altering the amino acid sequence. This change is also made, for example, by the addition or substitution of one or more serine or threonine residues to the native sequence PRO201, PRO308 or PRO309 (for O-linked glycosylation sites). In some cases, the PRO201, PRO308, or PRO309 amino acid encodes a PRO201, PRO308, or PRO309 polypeptide with a preselected base such that a codon that translates into the desired amino acid is produced by changes at the DNA level. Altered by mutating DNA.
Another means of increasing the number of carbohydrate moieties on the PRO201, PRO308 or PRO309 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. These methods are described in International Patent Application No. WO 87/05330 published September 11, 1987 and Aplin and Wriston, CRC Crit. Rev. Biochem., Pp 259-306 (1981).
Removal of the carbohydrate moiety present on the PRO201, PRO308 or PRO309 polypeptide can be done either chemically or enzymatically or by mutational substitution of codons encoding the amino acid residues targeted for glycosylation. For example, chemical deglycosylation techniques are known in the art, for example, Hakimuddin et al., Arch. Biochem Biophys., 259: 52 (1987) and Edge et al., Anal. Biochem., 118: 131 (1981) It is described by. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved using various endo and exoglycosidases, as described in Thotakura et al., Meth. Enzymol., 138: 350 (1987).
Other types of covalent modification of PRO201, PRO308, or PRO309 include PRO201, PRO308, or PRO309 polypeptide to one of a variety of non-proteinaceous polymers such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
[0036]
Alternatively, PRO201, PRO308 or PRO309 of the present invention may be modified by a method of forming a chimeric molecule comprising PRO201, PRO308 or PRO309 fused to another heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of PRO201, PRO308, or PRO309 with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally located at the amino or carboxyl terminus of PRO201, PRO308 or PRO309. The presence of such an epitope tag form of PRO201, PRO308 or PRO309 can be detected using an antibody against the tag polypeptide. Providing the epitope tag also allows PRO201, PRO308 or PRO309 to be easily purified by affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the epitope tag. In another embodiment, the chimeric molecule comprises a fusion of PRO201, PRO308 or PRO309 with an immunoglobulin or a specific region of an immunoglobulin. In the bivalent form of the chimeric molecule, such a fusion can be the Fc region of an IgG molecule.
Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8: 2159. -2165 (1988)]; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)]; and herpes simplex virus sugars A protein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides include flag peptides [Hopp et al., BioTechnology, 6: 1204-1210 (1988)]; KT3 epitope peptides [Martin et al., Science, 255: 192-194 (1992)]; α-tubulin epitope peptides [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)]; and T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 ( 1990)].
[0037]
D. Preparation of PRO201, PRO308 or PRO309
The following description primarily relates to a method of producing a particular PRO201, PRO308 or PRO309 by culturing cells transformed or transfected with a vector comprising a PRO201, PRO308 or PRO309 nucleic acid. Of course, it is believed that PRO201, PRO308 or PRO309 can be prepared using other methods well known in the art. For example, PRO201, PRO308 or PRO309 sequences, or portions thereof, may be produced by direct peptide synthesis using solid phase techniques [eg, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963)]. In vitro protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. Various portions of PRO201, PRO308, or PRO309 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce full-length PRO201, PRO308, or PRO309.
[0038]
1. Isolation of DNA encoding PRO201, PRO308, or PRO309 DNA encoding PRO201, PRO308, or PRO309 is cDNA prepared from a tissue that possesses PRO201, PRO308, or PRO309 mRNA and is believed to express it at a detectable level Can be obtained from the library. Accordingly, human PRO201, PRO308 or PRO309 DNA can be conveniently obtained from a cDNA library prepared from human tissue, as described in the Examples. The PRO201, PRO308 or PRO309-encoding gene can also be obtained from a genomic library or by oligonucleotide synthesis.
The library can be screened with probes (such as antibodies to PRO201, PRO308 or PRO309 or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by that gene. Screening of cDNA or genomic libraries with selected probes is performed using standard procedures described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). be able to. Another method for isolating the gene encoding PRO201, PRO308 or PRO309 uses the PCR method [Sambrook et al., Supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995). ].
[0039]
The following examples describe screening techniques for cDNA libraries. The oligonucleotide sequence selected as the probe must be long enough and clear enough to minimize false positives. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Labeling methods are well known in the art,³²This includes the use of radiolabels such as P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions including moderate stringency and high stringency are given by Sambrook et al., Supra.
Sequences identified in such library screening methods can be compared and aligned with well-known sequences deposited in public databases such as Genbank or personal sequence databases and made available to the public. Sequence identity (either at the amino acid or nucleic acid level) within a certain region or over the entire length of the molecule is determined by BLAST, BLAST-2, ALIGN, DNAstar, and various algorithms for measuring homology. It can be determined through sequence alignment using a computer software program such as INHERIT.
Nucleic acids having protein coding sequences use the deduced amino acid sequences disclosed herein for the first time and, if necessary, Sambrook et al., Supra, which detects intermediates and precursors of mRNA that was not reverse transcribed into cDNA. Obtained by screening a selected cDNA or genomic library using conventional primer extension methods as described in.
[0040]
2. Host cell selection and transformation
A host cell is transfected or transformed with the expression or cloning vectors for production of PRO201, PRO308 or PRO309 described herein, a promoter is induced, a transformant is selected, or a gene encoding the desired sequence is Incubate in a conventional nutrient medium appropriately modified for amplification. Culture conditions, such as medium, temperature, pH, etc. can be selected by one skilled in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach, edited by M. Butler (IRL Press, 1991) and Sambrook et al., Supra. it can.
Methods of transfection, eg CaPO₄Electroporation is known to those skilled in the art. Depending on the host cell used, transformation is done using standard methods appropriate to that cell. Calcium treatment or electroporation with calcium chloride as described in Sambrook et al., Supra, is used for prokaryotes or other cells that contain a substantial cell wall barrier. Infection with Agrobacterium tumefaciens has been reported in certain plant cells as described in Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 published 29 June 1989. Used for transformation. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 (1978) is preferred. General aspects of mammalian cell host system transformations are described in US Pat. No. 4,399,216. Transformation into yeast is typically performed by Van solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. USA, 76: 3829 (1979). Implemented according to the method. However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, intact cells, or bacterial protoplast fusion using polycations such as polybrene, polyornithine, etc. can also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (1988).
[0041]
Suitable host cells for cloning or expressing DNA in the vectors described herein are prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, eubacteria such as Gram negative or Gram positive organisms such as Enterobacteriaceae such as E. coli. Various E. coli strains are available to the public, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strains W3110 (ATCC 27,325) and K5772 (ATCC 53,635).
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO201, PRO308 or PRO309-encoding vectors. Saccharomyces cerevisia is a commonly used lower eukaryotic host microorganism.
Suitable host cells for the expression of PRO201, PRO308 or PRO309 are derived from multicellular organisms. Examples of cell-free include insect cells such as Drosophila S2 and Spodospera Sf9, and plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More detailed examples include monkey kidney CV1 strain transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney strain (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse breast tumor cells (MMT 060562, ATTC CCL51). The selection of an appropriate host cell is within the common general knowledge of this field.
[0042]
3. Selection and use of replicable vectors
Nucleic acid (eg, cDNA or genomic DNA) encoding PRO201, PRO308, or PRO309 is inserted into a replicable vector for cloning (amplification of DNA) or expression. Various vectors are publicly available. The vector can be, for example, in the form of a plasmid, cosmid, virus particle, or phage. The appropriate nucleic acid sequence is inserted into the vector by a variety of techniques. In general, DNA is inserted into an appropriate restriction endonuclease site using techniques well known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Standard ligation techniques known to those skilled in the art are used to make suitable vectors containing one or more of these components.
PRO201, PRO308 or PRO309 is not only directly recombinantly produced but also a fusion peptide with a heterologous polypeptide that is a signal sequence or a mature protein or other polypeptide having a specific cleavage site at the N-terminus of the polypeptide Also produced. In general, the signal sequence is a component of the vector or a part of PRO201, PRO308 or PRO309 DNA that is inserted into the vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or heat stable enterotoxin II leader. For yeast secretion, signal sequences include, for example, the yeast invertase leader, alpha factor leader (Saccharomyces and Kluyveromyces alpha factor leader, the latter being described in US Pat. No. 5,010,182. ), Or acid phosphatase leader, C. albicans glucoamylase leader (EP362179, issued April 4, 1990), or international patent application WO 90/13646 published on November 15, 1990. Can be the signals described in. For mammalian cell expression, signal sequences from secreted polypeptides of the same or related species, other mammalian signal sequences such as viral secretion leaders may be used for direct secretion of the protein.
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for many bacteria, yeasts and viruses. The origin of replication derived from plasmid pBR322 is suitable for most gram negative bacteria, the 2u plasmid origin is suitable for yeast, and the various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are Useful for cloning vectors in mammalian cells. A preferred replicable vector is plasmid pRK5. Holmes et al., Science, 253: 1278-1280 (1991).
[0043]
Expression and cloning vectors typically contain a selection gene, also termed a selectable marker. Typical selection genes are (a) conferring resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) compensate for auxotrophic defects, or (c) the genetic code for eg Basili It encodes a protein that supplies important nutrients not available from complex media, such as D-alanine racemase.
Other examples of markers that can be appropriately selected for mammalian cells are those that can identify cellular components that can take up PRO201, PRO308, or PRO309 nucleic acids, such as DHFR or thymidine kinase. Suitable host cells when using wild-type DHFR are DHFR activity prepared and expanded as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). It is a defective CHO cell line. A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282: 39 (1979); Kingman et al., Gene, 7: 141 (1979); Tschemper et al. Gene, 10: 157 (1980)]. The trp1 gene provides a selectable marker for mutant strains of yeast lacking the ability to grow in tryptophan, such as, for example, ATCC 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
[0044]
Expression and cloning vectors usually contain a promoter operably linked to the PRO201, PRO308 or PRO309 nucleic acid sequences and which regulates mRNA synthesis. Suitable promoters that are recognized by a variety of possible host cells are known. Suitable promoters for use in prokaryotic hosts are the β-lactamase and lactose promoter systems [Cahng et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)], alkaline phosphatase, tryptophan (trp ) Promoter system [Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983). )]including. Promoters used in bacterial systems also have a Shine Dalgarno (S.D.) sequence operably linked to DNA encoding PRO201, PRO308, or PRO309.
Examples of suitable promoter sequences for use with yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)] or other glycolytic enzymes [Hess et al., J Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900 (1978)], eg enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase Glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, trioselate isomerase, phosphoglucose isomerase, and glucokinase.
[0045]
Other yeast promoters are inducible promoters that have the additional effect that transcription is controlled by growth conditions, such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glycine. There is a promoter region of seraldehyde-3-phosphate dehydrogenase and the enzyme that governs the use of maltose and galactose. Vectors and promoters suitably used for expression in yeast are further described in EP 73,657.
Transcription of PRO201, PRO308 or PRO309 from a vector in a mammalian host cell is, for example, polyoma virus, infectious epithelioma virus (UK 2,211,504 published July 5, 1989), adenovirus (eg adenovirus 2), bovine papilla Promoters derived from the genomes of viruses such as tumor virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40), promoters derived from heterologous mammals such as actin promoter or immunity Such promoters are controlled by globulin promoters and promoters derived from heat shock promoters as long as they are compatible with the host cell system.
[0046]
Transcription of DNA encoding PRO201, PRO308 or PRO309 by higher eukaryotes can be enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to enhance its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the late SV40 enhancer (100-270 base pairs) of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer and the adenovirus enhancer late of the origin of replication. Enhancers can be spliced into the vector at the 5 'or 3' position of the PRO201, PRO308 or PRO309 coding sequence, but are preferably located 5 'from the promoter.
Also, expression vectors used in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) are required for transcription termination and mRNA stabilization. Also includes sequences. Such sequences can be obtained from the normally 5 'and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO201, PRO308, or PRO309.
Still other methods, vectors and host cells suitable for adapting to the synthesis of PRO201, PRO308 or PRO309 in recombinant cell culture are described in Gething et al., Nature, 293: 620-625 (1981); Mantei et al., Nature 281: 40-46 (1979); EP 117,060; and EP 117,058.
[0047]
4). Gene amplification / expression detection
Gene amplification or expression is based on the sequences provided herein, using appropriately labeled probes, eg, conventional Southern blotting, Northern blotting to quantify mRNA transcription [Thomas, Proc. Natl Acad. Sci. USA, 77: 5201-5205 (1980)], dot blot (DNA analysis), or in situ hybridization, can be measured directly in the sample. Alternatively, antibodies capable of recognizing specific double strands, including DNA double strands, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes, can also be used. The antibody can then be labeled and an assay can be performed, where the duplex is bound to the surface, so that the antibody bound to the duplex at the time of formation on the surface of the duplex is bound. The presence can be detected.
Alternatively, gene expression can be measured by immunological methods that directly quantify gene product expression, such as immunohistochemical staining of cells or tissue sections and cell culture or body fluid assays. Antibodies useful for immunohistochemical staining or assay of sample fluids can be monoclonal or polyclonal and can be prepared in any mammal. Conveniently, the antibody is a specific antibody fused to the native sequence PRO201, PRO308 or PRO309 polypeptide, or to a synthetic peptide based on the DNA sequence provided herein, or to PRO201, PRO308 or PRO309 DNA. It can be prepared against an exogenous sequence encoding the epitope.
[0048]
5. Purification of polypeptides
Forms of PRO201, PRO308, or PRO309 can be recovered from media or lysates of host cells. If membrane-bound, it can be detached from the membrane using an appropriate wash solution (eg Triton-X100) or enzymatic cleavage. Cells used for expression of PRO201, PRO308, or PRO309 can be disrupted by various chemical or physical means such as freeze-thaw cycles, sonication, mechanical disruption, or cytolytic agents.
It may be desirable to purify PRO201, PRO308 or PRO309 from recombinant cell proteins or polypeptides. Purified by the following procedure, which is an example of a suitable purification procedure: fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or a cation exchange resin such as DEAE; chromatofocusing; PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A sepharose column to remove contaminants such as IgG; and metal chelation column to bind the epitope tag form of PRO201, PRO308 or PRO309. It is possible to use many protein purification methods known in the art and described, for example, in Deutcher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). it can. The purification process chosen depends, for example, on the production method used and in particular the nature of the PRO201, PRO308 or PRO309 produced.
[0049]
  E. Use of PRO201, PRO308 or PRO309
  Nucleic acid sequences encoding PRO201, PRO308 or PRO309 (or theirComplementary sequence) Has a variety of uses in the field of molecular biology including use as hybridization probes, in chromosome and gene mapping, and in the production of antisense RNA and DNA. PRO201, PRO308 or PRO309 nucleic acids will also be useful in the preparation of PRO201, PRO308 or PRO309 polypeptides by the recombinant techniques described herein.
  The full-length native sequence PRO201, PRO308 or PRO309 (SEQ ID NO: 1, 3 and 5) gene, or a portion thereof, is isolated from the full-length gene or FIG. 1-3 (SEQ ID NO: 1, 3 and 5) PRO201 A single other gene having the desired sequence identity to the PRO308 or PRO309 sequence (eg, a naturally occurring variant of PRO201, PRO308 or PRO309 or encoding PRO201, PRO308 or PRO309 from other species) It can be used as a hybridization probe for a cDNA library for separation. In some cases, the length of the probe is from about 20 to about 50 bases. The hybridization probe can be derived from the nucleic acid sequence of SEQ ID NO: 1, 3, or 5 or from a genomic sequence comprising the promoter, enhancer component and intron of the native sequence PRO201, PRO308 or PRO309. For example, the screening method involves synthesizing a selected probe of about 40 bases by isolating the coding region of the PRO201, PRO308 or PRO309 gene using a well-known DNA sequence. Hybridization probes can be labeled with a variety of labels including radioactive nucleotides such as 32P or 35S, or enzyme labels such as alkaline phosphatase attached to the probe via an avidin / biotin binding system. A labeled probe having a sequence complementary to the PRO201, PRO308 or PRO309 gene of the present invention screens a library of human cDNA, genomic DNA or mRNA, and any member of those libraries hybridizes to the probe. Can be used to determine. Hybridization techniques are described in further detail in the examples below.
  Probes can also be used in PCR techniques to create a pool of sequences for the identification of closely related PRO201, PRO308 or PRO309 sequences.
[0050]
The nucleic acid sequence encoding PRO201, PRO308, or PRO309 should also be used to create hybridization probes for mapping the gene encoding PRO201, PRO308, or PRO309, and for genetic analysis of individuals with genetic disorders. Can do. The nucleic acid sequences provided herein can be mapped to specific regions of chromosomes and chromosomes using well known techniques such as in situ hybridization, binding analysis to known chromosomal markers, and hybridization screening in libraries. .
When the coding sequence of PRO201, PRO308, or PRO309 encodes a protein that binds to another protein (for example, when PRO201, PRO308, or PRO309 is a receptor), PRO201, PRO308, or PRO309 is included in the binding interaction Can be used in assays to identify other proteins or molecules.
By such methods, inhibitors of receptor / ligand binding interactions can be identified. Proteins involved in such binding interactions can also be used to screen for peptides or small molecule inhibitors or agonists of binding interactions. PRO201, PRO308 or PRO309 can also be used to isolate related ligands. Screening assays are designed for the discovery of lead compounds that mimic the biological activity of native PRO201, PRO308, or PRO309, or for those that act as receptors for PRO201, PRO308, or PRO309. Such screening assays are also used for high-throughput screening of chemical libraries, making them particularly suitable for the identification of small molecule candidate drugs. Small molecules considered include synthetic organic or inorganic compounds. Assays are performed in a variety of formats including protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays that are well known and characterized in the art.
[0051]
  DNA 30676 (SEQ ID NO: 2) contains a single long open reading frame, which encodes a 576 amino acid protein, herein designated Nsp1 (a novel SH2-containing protein) (FIG. 1). Nsp1 (SEQ ID NO: 1) is related to Sck and Shc (FIG. 8, SEQ ID NOs: 16 and 17) as determined by the indicated N-terminal homology. Nsp1 (SEQ ID NO: 1) has a proline-serine enriched domain (P / S) in the middle of the protein, which can function as an SH3 interaction domain. The C-terminus of the protein has no apparent homology associated with known mammalian proteins. Nsp1 (SEQ ID NO: 1), Nsp2 (SEQ ID NO: 3) and Nsp3 (SEQ ID NO: 5) occupy an overall homology of 33 to 47%. Nsp3 (SEQ ID NO: 5) has an SH2 domain and a potential SH3 interaction domain, and Nsp2 (SEQ ID NO: 3) lacks an SH2 domain but has a potential SH3 domain. The absence of the SH2 domain in Nsp2 (SEQ ID NO: 3) suggests that this protein can act as a negative regulator of the other two Nsps. The lack of apparent kinase or phosphatase domains suggests that they correspond to a novel family of adapter proteins.
  Adapter proteins are thought to play an important role in the integration of multiple signaling cascades and in determining specific responses to extracellular stimuli. Signals generated by growth factors such as EGF or IGF-1 through receptor tyrosine kinases, or by extracellular matrix components acting through integrin receptors can induce cytoskeletal changes. Applicants have shown that the EGF receptor co-immunoprecipitates with Nsp1 and is phosphorylated for EGF signaling. That is, Nsp1 antagonists inhibit cellular responses (eg, tumorigenicity) by stimulation with growth factors such as EGF and IGF-1To doExpected to be useful.
[0052]
Several features suggest that Nsp1 plays an important role in modulating the response to external stimuli. Nsp1 (SEQ ID NO: 1) is phosphorylated upon EGF stimulation to form a complex comprising EGF receptor, PI3 kinase and Cas. The Nsp1 / Cas complex also responds to signaling that drives the fibronectin receptor. However, the stoichiometry of the interaction and the phosphorylation state of the components differ between the two stimuli. It is implied that the biological consequences for these extracellular signals are completely different with and without Nsp1. For example, FAK is associated with the SH3 region of Cas via the PXXP region at the C-terminus of FAKP (715) SRP-mouse nomenclature (Harte et al., J. BIOL. CHEM. 271: 13649-55 (1996) ). There are six PXXPs in Nsp1 (SEQ ID NO: 1). This creates the possibility that Nsp1 competes with SH3 on Cas, reducing the amount of Fak bound to Cas and altering Fak-dependent events. This data is consistent with EGF mediating a modest decrease in the phosphorylation of Cas associated with Nsp1 (SEQ ID NO: 1). This complex then reduces the number of proteins associated with Cas phosphorylated tyrosine, leading to changes in downstream events. Since Nsp1 (SEQ ID NO: 1) expression is highest in fetal tissue, this protein could potentially play an important role in mediating the developmental readout of extracellular signals.
[0053]
F. Principal elemental expression of genes encoding PRO201, PRO308 or PRO309 polypeptides and amplification in cell lines
This invention is based in part on the identification and characterization of genes that are amplified in certain cancer cells.
Two apparently contradictory requirements were imposed on prokaryotic and eukaryotic genomes. One is the preservation and propagation of DNA as genetic information in its original form, ensuring stable inheritance through multiple generations. On the other hand, cells or organisms must adopt recent environmental changes. Adaptation mechanisms can include qualitative or quantitative modification of genetic material. A qualitative modification includes a DNA mutation in which the coding sequence is altered to produce a structurally or functionally different protein. Gene amplification is a quantitative modification that increases the number of actual complete coding sequences, ie, genes, increases the number of templates available for transcription, increases the number of translatable transcripts, and ultimately amplifies Resulting in an increase in the amount of protein encoded by the encoded gene.
The phenomenon of gene amplification and the mechanisms there have been studied in vitro in several prokaryotic and eukaryotic cell culture systems. The most characterized example of gene amplification involves culturing eukaryotic cells in media containing various concentrations of the cytotoxic drug methotrexate (MTX). MTX is a folic acid analog that interferes with DNA synthesis by blocking the enzyme dehydrofolate reductase (DHFR). Most cells (> 99.9%) die upon first exposure to low concentrations of MTX. Small amounts of cells survive and can grow even with increasing MTX concentrations by producing large amounts of DHFR-RNA and protein. The basis for this overproduction is the amplification of a single DHFR gene. Additional copies of the gene are found as extrachromosomal copies in the form of small excess chromosomes (double micro) or as integrated chromosomal copies.
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Gene amplification occurs most commonly in the progression of resistance to cytotoxic drugs (antibiotics against recent and chemotherapeutic drugs against eukaryotes) and oncogenic transformation. Eukaryotic transformation as a spontaneous event or by viral or chemical / environmental invasion typically involves changes in the genetic material of the cell. The most common genetic change observed in human malignant housing is a mutation in the p53 protein. p53 controls the transition of cells from the stationary (G1) to replication (S) phase and prevents this transition in the presence of DNA damage. In other words, one of the main consequences of p53 mutation deficiency is the accumulation and growth of DNA damage, ie genetic changes. A common type of genetic change in tumorigenic cells is amplification and overall structural modification, eg, translocation, in addition to point mutations.
Amplification of the DNA sequence exhibits specific functional requirements as exemplified in the DHFR experimental system. Thus, certain oncogene amplifications in malignancy indicate a causative role for these genes in the process of malignant transformation and maintenance of transformed phenotypes. This hypothesis is supported by recent studies. For example, bcl-2 protein has been found to be amplified in certain types of non-Hodgkin lymphoma. This protein inhibits apoptosis and promotes the accumulation of tumorigenic cells. It has been found that members of the growth factor receptor gene family are amplified in various types of cancer, and overexpression of these receptors reduces the sensitivity of tumor cells to limited amounts of available growth factors. Examples include amplification of androgen receptor in recurrent prostate cancer during androgen deficiency treatment, and amplification of growth factor receptor homolog ERB2 in breast cancer. Recently, genes involved in intercellular signaling and cell cycle progression can undergo amplification during malignant transformation. This is exemplified by the amplification of bcl-I and ras genes in various epithelial and lymphoid tumorigenesis.
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These early studies exemplify the possibility of identifying amplified DNA sequences in tumorigenesis because these methods allow the identification of genes important for malignant transformation. In the case of ERB2, the transforming protein represents a new and specific target for tumor therapy, thus showing potential from a therapeutic standpoint.
Several different techniques can be used to indicate the amplified genomic sequence. Classical cytogenetic analysis of chromosomal expansions prepared from cancer cells is sufficient to identify global structural changes such as translocations, deletions and transformations. Amplified genomic regions are only visible if they contain high copy numbers or exist as extrachromosomal material. Cell development is the first technique to show a consistent relationship of specific chromosomal changes with specific tumorigenesis, but is insufficient to identify and isolate manageable DNA sequences. A more recently developed technique, competitive genomic hybridization (CGH), illustrates the widespread phenomenon of genome amplification in tumorigenesis. Tumor and normal DNA hybridize simultaneously during metaphase of normal cells, and the entire genome is screened by image analysis for DNA sequences that are frequently present in the tumor (WO 93 / 18,186; Gray et al., Radiation Res. 137: 275 -289 [1994]). As a screening method, this type of analysis revealed many of the recurrent amplicons (amplification of amplified DNA) in various human tumors. Although CGH is more sensitive in identifying amplified stretches of DNA than classical cytogenetic analysis, it does not allow rapid identification and isolation of coding sequences within amplicons by standard genetic techniques.
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The most sensitive method for detecting gene amplification is the polymerase chain reaction (PCR) based assay. These assays use very small amounts of tumor DNA as starting material, provide elaborate and sensitive DNA that can be used for further analysis such as sequencing, and are suitable for high volume throughput analysis.
The above assays are not mutually exclusive and are often used in combination to identify amplification in tumorigenesis. Cytogenetic analysis and CGH represent screening methods for overview of the entire genome of the amplified region, and PCR-based assays are best suited to ultimately identify the coding sequence, ie the gene for the amplified region.
According to the present invention, such genes can be obtained by quantitative PCR (S. Gelmini et al., Clin, Chem. 43: 752 [1997]), breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, testis. Identified by comparing DNA from various primary tumors, including ovarian, uterus, etc., tumors, or tumor cell lines, with pooled DNA from healthy donors. Quantitative PCR was performed using a Taqman instrument (ABI). Gene specific primers and fluorogenic probes are designed based on the coding sequence of the DNA.
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Human lung cancer cell lines are A549 (SRC768), Calu-1 (SRC769), Calu-6 (SRC770), H157 (SRC771), H441 (SRC772), H460 (SRC773), H522 (SRC832), H810 (SRC833), Including SKMES-1 (SRC774) and SW900 (SRC775), all from ATCC. Primary human lung tumor cells are usually derived from adenocarcinoma, squamous cell carcinoma, large cell carcinoma, non-small cell carcinoma, small cell carcinoma, and bronchoalveolar carcinoma, eg, SRC724 (abbreviated as “SqCCa”) Squamous cell carcinoma) (LT1), SRC725 (non-small cell carcinoma abbreviated as “NSCCa”) (LT1a), SRC726 (adenocarcinoma abbreviated as “AdenoCa”) (LT2), SRC 727 (adenocarcinoma) (LT3), SRC728 (squamous cell carcinoma) (LT4), SRC729 (adenocarcinoma) (LT6), SRC730 (gland / squamous cell carcinoma) (LT7), SRC731 (adenocarcinoma) (LT9), SRC732 (squamous epithelium) Cell carcinoma) (LT10), SRC733 (LT11), SRC734 (adenocarcinoma) (LT12), SRC735 (bronchoalveolar carcinoma, “abbreviated as BAC”) (LT13), SRC736 (squamous cell carcinoma) (LT15), SRC737 (Flat (Skin cell carcinoma) (LT16), SRC738 (squamous cell carcinoma) (LT17), SRC739 (squamous cell carcinoma) (LT18), SRC740 (squamous cell carcinoma) (LT19), SRC741 (pulmonary cell carcinoma, “LCCa” (Abbreviated) (LT21), SRC811 (adenocarcinoma) (LT22).
Colon cancer cell lines are, for example, ATCC cell lines SW480 (adenocarcinoma, SRCC776), SW620 (colon adenocarcinoma lymph node metastasis, SRC777), Colo320 (cancer, SRCC778), Colo205 (cancer, SRC828), HCC2998 (cancer, SRC830), HT29 (cancer, SRC779), HM7 (cancer, SRC780), KM12 (cancer, SRC831), CaWiDr (adenocarcinoma, SRC781), HCT15 (cancer, SRC829), HCT116 (cancer, SRC782), SKCO1 (adenocarcinoma) SRC783), SW403 (adenocarcinoma, SRC784), LS174T (cancer, SRC785), and HM7 (ATCC colon adenocarcinoma cell line LS174T high mucin producing mutant, obtained from Dr. Robert Warren, UCSF). Primary colon tumors are CT1 (SRC751), CT2 (SRC742), CT3 (SRC743), CT4 (SRC752), CT5 (SRC753), CT6 (SRC754), CT7 (SRC755), CT8 (SRC744), CT9 (SRC756), Includes colon adenocarcinoma designated CT10 (SRC745), CT11 (SRC757), CT12 (SRC746), CT14 (SRC747), CT15 (SRC748), CT16 (SRC749), CT17 (SRC750), CT18 (SRC758).
Human breast cancer cell lines are, for example, HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468 (SRCC762), MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), SKBR3 ( SRCC767).
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  G. Tissue distribution
  The results of the gene amplification assay here can be confirmed by further experiments such as measurement of mRNA expression in various human tissues.
  As described above, gene amplification or gene expression in various tissues can be performed using conventional Southern and Northern blots (Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205) for quantifying mRNA transcription. [1980]), dot blot (DNA analysis), or in situ hybridization, can be measured using an appropriate labeled probe based on the sequences provided herein. Alternatively, antibodies that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, may be used.
  Alternatively, gene expression in various tissues can also be measured by immunological methods such as immunohistological staining of tissue fragments and cell culture media or body fluids to directly quantify gene products. Antibodies useful for immunohistological staining or sample liquid assays may be monoclonal or polyclonal and are prepared from any animal. Conveniently, the antibody is a native sequencePRO201, PRO308 or PRO309Against a polypeptide, or against a synthetic peptide based on the DNA sequence provided herein, orPRO201, PRO308 or PRO309Prepared against an extracellular sequence fused to a DNA sequence and encoding a specific antibody epitope. General techniques for generating antibodies, and protocols for Northern blots and in situ hybridization are provided below.
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H. Chromosome mapping
If amplification of a given gene is functionally related, that gene should be amplified more than adjacent genomic regions that are not important for tumor survival. To test this, genes can be mapped to specific chromosomes, for example by radiohybrid analysis. The amplification level is then measured at the identified location and adjacent genomic regions. Selective or preferential amplification in the genomic region to which the gene is mapped is consistent with the possibility that the observed gene amplification promotes tumor growth or survival. Chromosome mapping includes both framework and epicenter mapping. For further details see, for example, Stewart et al., Genome Research 7, 422-433 (1997).
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I. Antibody binding experiments
The results of gene amplification experiments are further confirmed by antibody binding experiments in which the ability of anti-PRO201, anti-PRO308 or anti-PRO309 to inhibit the expression of PRO201, PRO308 or PRO309 polypeptide on tumor (cancer) cells is tested. it can. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which is described.
Antibody binding experiments may be performed in known assay methods such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).
Competitive binding assays rely on the ability of a labeled standard to compete with a test analyte for binding to a limited amount of antibody. The amount of target protein (encoded by the gene amplified in tumor cells) in the test sample is inversely proportional to the amount of standard that begins to bind to the antibody. To facilitate the measurement of the amount of standard that begins to bind, the antibody is preferably immobilized before or after competition, and easily from standards and analytes where the standard and analyte bound to the antibody remain unbound. So that it can be separated.
The sandwich assay involves the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In the sandwich assay, the test sample analyte binds to the first antibody immobilized on the solid support, after which the second antibody binds to the analyte, thus forming an insoluble ternary complex. See, for example, US Pat. No. 4,376,110. The second antibody may be labeled with a detectable moiety (direct sandwich assay) or measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one form of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
For immunohistology, the tumor sample may be fresh or frozen, embedded in paraffin, and fixed with a preservative such as formalin, for example.
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J. et al. Cell-based tumor assay
Cell-based assays and animal models of tumors (eg, cancer) can be used to confirm findings in gene amplification assays to further understand the relationship between gene amplification here and the progression and pathology of tumorigenic cell growth . The role of the gene product identified here in tumor or cancer progression and pathology can be tested using primary tumor cells or cell lines identified here as amplifying the gene. Such cells include, for example, the breast, colon and lung cancer cells and cell lines described above.
In a different method, cells of a cell type known to be included in a specific tumor are transfected with the cDNAs here and the ability of these cDNAs to induce overgrowth is analyzed. Suitable cells include, for example, stable tumor cell lines such as B104-1-1 (stable NIH-3T3 cell fluid transfected with neu proto-oncogene) and ras-transfected NIH-3T3 cells, which are It can be transfected with the desired gene and monitored for oncogenic growth. Such transfected cell lines are then poly- or monoclonal antibodies or antibodies by exerting cytostatic or cytotoxic activity on the growth of transformed cells or by mediating antibody-dependent cellular cytotoxicity (ADCC). It can be used to test the ability of the composition to inhibit tumorigenic cell growth. Cells transfected with the coding sequences of the genes identified here can further be used to identify candidate drugs for cancer treatment.
Furthermore, although primary media derived from tumors of transgenic animals (below) can be used for cell-based assays herein, stable cell lines are preferred. Techniques for deriving continuous cell lines from transgenic animals are well known in the art (see Small et al., Mol. Cell. Biol. 5, 642-648 [1985]).
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K. Animal model for anti-tumor activity
To further understand the role of the genes identified herein in tumor progression and cause, and to test the efficacy of candidate therapeutics, including antibodies and other agonists of natural polypeptides, including small molecule agonists Various well-known animal models can be used. The in vivo nature of these models can predict response, particularly in human patients. Animal models of tumors and cancers (eg, breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodents such as mouse models. Such models are based on standard techniques such as subcutaneous injection, tail vein injection, spleen transplantation, intraperitoneal transplantation, subrenal transplantation, or orthopin transplantation, eg, colon cancer cells transplanted into colon tissue. It is created by introducing tumor cells into syngeneic mice. (See PCT publication WO 97/33551 issued on September 18, 1997.)
Probably the most frequently used animal species for oncogene research is immunodeficient mice, especially nude mice. The observation that hypo mice / hypoplastic nude mice act as hosts for human tumor xenografts has led to widespread use for this purpose. An autosomal recessive nu gene is, for example, ASW, A / He, AKR, BALB / c, B10. A large number of different nude mice including LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I / st, NC, NFR, NFS, NFS / N, NZB, NZC, NZW, P, RIII and SJL It was introduced into a common gene line. In addition, a wide range of other animals other than nude mice with genetic immunodeficiency were grown and used as recipients for tumor xenografts. For further details, see The Nude Mouse in Oncology Research, E. Boven and B. Winograd, CRC Press, Inc., 1991.
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Cells introduced into these animals include well-known tumor / cancer cell lines, such as the tumor cell lines listed above, and for example, the B104-1-1 cell line (stable NIH-3T3 cells transfected with the neu proto-oncogene. Ras-transfected NIH-3T3 cells: Caco-2 (ATCC HTB-37), moderately well-differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or tumor And can be derived from cancer. Tumor or cancer cell samples can be obtained from patients undergoing surgery using standard conditions including freezing and storage in liquid nitrogen (Karmali et al., Br. J. Cancer 48, 689- 696 [1983]).
Tumor cells can be introduced into animals such as nude mice by various techniques. The subcutaneous (sc) space in mice is highly preferred for tumor transplantation. Tumors are obtained as a solid block, as a needle biopsy using a trochar, and as a cell suspension. c. Can be transplanted. For solid block or trochar transplantation, appropriately sized tumor tissue fragments are s. c. Introduced into space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines and injected subcutaneously. Tumor cells can also be injected as subcutaneous implants. In this position, the inoculum is s. c. It is deposited between the tissues. Boven and Winograd (1991), supra. Animal models of breast cancer are basically based on, for example, Drebin et al., PNAS by transplanting neuroblastoma cells (from which the neu oncogene is first isolated) or neu transfected NIH-3T3 cells into nude mice. Produced as described in USA 83, 9129-9133 (1986).
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Similarly, animal models of colon cancer are generated by passaging colon cancer cells to animals, such as nude mice, and leading to the development of tumors in these animals. Orthotopic transplantation models of human colon cancer in nude mice are described, for example, in Wang et al., Cancer Research 54, 4726-4728 (1994) and Too et al., Cancer Research 55, 681-684 (1995). This model is based on “METAMOUSE” commercially available from the so-called AntiCancer, Inc. (SanDiego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from in vitro media can then be passaged to animals. These tumors can be provided as targets for further testing and drug screening. Alternatively, tumors obtained from passages can be isolated and RNA of pre-passage cells and cells isolated after one or more passages are analyzed for identifiable expression of the gene of interest. Such passaging techniques can be performed on well-known tumor or cancer cell lines.
For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 have been introduced into the fibrosarcoma of BALB / c female mice (DeLeo et al., J. Exp. Med. 146, 720 [1977]), which contains various antigens. Provides a highly controllable model system for the study of the anti-tumor activity of (Palladino et al., J. Immunol. 138, 4023-4032 [1987]). Conveniently, tumor cells are grown in vitro in cell culture media. Prior to injection into animals, the cell lines are washed and about 10 × 10 × 10 in buffer.⁶To 10x10⁷Suspend at a cell density of cells / ml. Animals are then infected subcutaneously with 10-100 μl of cell suspension and left for 1-3 weeks until tumors appear.
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In addition, the mouse Lewis lung (3LL) carcinoma, one of the most fully studied experimental tumors, can be used as a research tumor model. Efficacy in this tumor model correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL). This tumor can be introduced into normal mice upon injection of tumor fragments from affected mice or cells remaining in the medium (Zupi et al., Br. J. Cancer 41, suppl. 4, 309 [1980]), with evidence , Showing that the tumor starts with an injection of only one cell and that a very high population of infected tumor cells survive. For further information on this tumor model, see Zacharski, Haemostasis 16, 300-320 [1986].
One way to evaluate the effectiveness of a test compound in an animal model of a transplanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of the transplanted tumor is measured with a two or three dimensional slide caliper. Measurements limited to two dimensions do not accurately reflect the size of the tumor and are therefore usually converted to the corresponding volume using mathematical formulas. However, measurement of tumor size is very inaccurate. The therapeutic effect of a candidate drug can be better described as treatment-induced growth delay and specific growth delay. Another important variable in the description of tumor growth is tumor volume doubling time. Computer programs for calculating and describing tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals, Wu and Sheng, Basel, 1989, 301 It is. However, it is noted that the necrosis and inflammatory response following the tumor can actually increase the size of the tumor at least initially. Therefore, these changes need to be carefully monitored using a combination of morphological methods and flow cytometry analysis.
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Recombinant (transgenic) animal models can be processed by introducing the coding portion of the genes identified herein into the genome of the animal of interest using standard techniques for the production of transgenic animals. A transgenic animal is one that has “transgene” or genetic material introduced into the genome at the prenatal stage (eg, embryonic stage) in the animal itself or in the ancestors of the animal. Animals that can be provided as targets for transgenic manipulation include, but are not limited to, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates such as baboons, chimpanzees and monkeys. Techniques known in the art for introducing transgenes into these animals include whole nuclear microinjection (Hoppe and Wanger, US Pat. No. 4,873,191); retrovirus-mediated gene transfer to the embryonic lineage (eg, Van der Putten et al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]); Gene targeting in embryonic hepatocytes (Thompson et al., Cell 56, 313-321 [1989]); (Lo, Mol. Cel. Biol. 3, 1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-73 [1989]). For review, see, for example, US Pat. No. 4,736,866.
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Typically, specific cells target the PRO201, PRO308 or PRO309 transgene inserts with tissue specific enhancers. A transgenic animal comprising a copy of a transgene encoding PRO201, PRO308 or PRO309 introduced into the animal's reproductive system at the embryonic stage can be used to test the effect of increased expression of DNA encoding PRO201, PRO308 or PRO309. Can be used. Such animals can be used, for example, as tester animals for reagents believed to confer protection from pathological conditions associated with their overexpression. According to this form of the invention, an animal is treated with a reagent, and if the symptoms of the pathological condition are alleviated compared to an animal with an untreated transgene, a potential therapeutic treatment for that pathological condition is performed. Show.
For the purposes of the present invention, transgenic animals include those that have the transgene only in part ("mosaic animals"). The transgene is integrated as a single transgene or as a concatamer, eg, head-to-head or head-to-tail tandem. Selective introduction of transgenes into specific cell types is also possible, for example, according to the technique of Lasko et al., Proc. Natl. Acad. Sci. USA 89, 6232-636 (1992).
Transgene expression in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification is used to confirm transgene integration. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunohistochemistry. The animals are further tested for signs of tumor or cancer development.
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  Alternatively, the non-human homologue of PRO201, PRO308 or PRO309 is between the modified genomic DNA encoding PRO201, PRO308 or PRO309 introduced into an animal embryonic cell and the endogenous gene encoding PRO201, PRO308 or PRO309. Can be used to create PRO201, PRO308 or PRO309 “knock-out” animals having a defective or altered gene encoding PRO201, PRO308 or PRO309. For example, cDNA encoding PRO201, PRO308 or PRO309 can be used for cloning genomic DNA encoding PRO201, PRO308 or PRO309 in accordance with established techniques. A portion of the genomic DNA encoding PRO201, PRO308, or PRO309 can be deleted or replaced with another gene, such as a gene encoding a selectable marker used to monitor integration. Typically, the vector contains several kilobases of intact flanking DNA (both 5 'and 3' ends) [eg, Thomas and Capecchi, for homologous recombination vectors,Cell,51 : 503 (1987)]. A vector is introduced into an embryonic stem cell (for example, by electroporation), and a cell in which the introduced DNA is homologously recombined with endogenous DNA is selected [eg, Li et al.,Cell,69 : 915 (1992)]. The selected cells are then injected into the blastocyst of the animal (eg mouse or rat) to form an aggregate chimera [eg Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, ed. , Oxford, 1987), pp. 113-152]. The chimeric embryo is then said to be transplanted into an appropriate pseudopregnant female nanny to create a “knockout” animal. Offspring with DNA homologously recombined into embryonic cells are identified by standard techniques and can be used to breed animals that contain DNA in which all cells of the animal are homologously recombined . Knockout animals are characterized by their ability to protect against certain pathological conditions and progression of pathological conditions due to the absence of PRO201, PRO308 or PRO309 polypeptides.
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The effectiveness of antibodies that specifically bind to the polypeptides identified herein and other candidate drugs in the treatment of naturally occurring animal tumors can also be tested. A suitable target for such studies is feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly invasive malignancy, the most common oral malignancy in cats, accounting for over 60% of oral tumors reported in this species. It hardly metastasizes to distant sites, but the low incidence of this metastasis only reflects the short survival time of the cat with this tumor. These tumors are usually inoperable, but mainly due to the cat's oral anatomy. There is currently no effective treatment for this tumor. Prior to entering the study, each cat was subjected to a complete clinical and biopsy and scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors were excluded from the study. The tongue will begin to paralyze due to this tumor, and the animal will not be able to feed itself even after treatment kills the tumor. Treat each cat repeatedly over time. Tumor photographs were taken every day during the treatment period and at the time of subsequent rechecks. After treatment, each cat was again CT scanned. CT scans and radiographs were evaluated every 8 weeks thereafter. Data were evaluated for differences in survival, reactivity and toxicity compared to the control group. A positive response requires shrinkage of the tumor, preferably improved quality of survival or prolonged survival.
In addition, other spontaneous animal tumors such as canine, feline and baboon fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma can also be tested. Breast cancer in these dogs and cats is a preferred model because its expression and behavior are very similar to those of humans. However, the use of this model is limited by the incidence of this type of tumor in animals.
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L. Screening assays for candidate drugs
Candidate drug screening assays are used to identify compounds that bind to or conjugate with the polypeptide encoded by the gene identified herein, or that inhibit the interaction of the encoded polypeptide with other cellular proteins. Designed. Such screening assays include assays that follow high-throughput screening of chemical libraries that make them particularly suitable for the identification of small molecule drug candidates. Small molecules are considered to include antibody synthetic organic or inorganic compounds, which are peptides, preferably soluble peptides, (poly) peptide-immunoglobulin fusions, particularly but not limited to poly- and monoclonal antibodies and antibody fragments. Single chain antibodies, anti-idiotype antibodies, and chimeric or humanized forms of those antibodies or fragments, as well as antibodies, including human antibodies and antibody fragments. The assays can be performed in a variety of formats and include well-characterized protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays in the field.
All assays are common in that they are contacted with the polypeptide encoded by the nucleic acid identified herein as a candidate drug for a time sufficient for the two components to interact.
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In binding assays, the interaction is binding and the complex formed is isolated or detected in the reaction mixture. In a specific embodiment, the polypeptide receptor or candidate drug encoded by the gene identified herein is immobilized to a solid phase, eg, a microtiter plate, either covalently or non-covalently. Non-covalent binding is generally achieved by coating a solid surface with a solution of the polypeptide and drying. Alternatively, an immobilized antibody specific for the peptide to be immobilized, such as a monoclonal antibody, can be used to anchor the peptide to a solid surface. The assay is performed by adding a non-immobilized component, which may be labeled with a detectable label, to a coated surface containing an immobilized component, such as an anchoring component. When the reaction is completed, unreacted components are removed, for example, by washing, and the complex adhered to the solid surface is detected. If the first non-immobilized component has a detectable label, detection of the label immobilized on the surface indicates that complex formation has occurred. If the initial non-immobilized component does not have a label, complex formation can be detected, for example, by a labeled antibody that specifically binds to the immobilized complex.
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If a candidate compound interacts but does not bind to a particular PRO201, PRO308 or PRO309 polypeptide encoded by the gene identified here, the interaction is well known for detecting protein-protein interactions. It can be assayed by the method. Such assays include traditional techniques such as cross-linking, co-immunoprecipitation, and co-purification through a gradient or chromatographic column. Furthermore, protein-protein interactions are described in Fields and co-workers [Fiels and Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88, 9578-9582. (1991)] is used to monitor as disclosed in Chevray and Nathans [Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991)]. be able to. Many transcriptional activators, such as yeast GAL4, consist of two physically distinct modular domains, one that acts as a DNA binding domain and the other that functions as a transcriptional activation domain. The yeast expression system described in the previous literature (commonly referred to as the “2-hybrid system”) takes advantage of this property and uses two hybrid proteins, while the target protein is the DNA binding domain of GAL4. On the other hand, the candidate activation protein is fused to the activation domain. Expression of the GAL1-lacZ reporter gene under the control of a GAL4-activated promoter depends on reconstitution of GAL4 activity through protein-protein interactions. Colonies containing interacting polypeptides are detected with a chromogenic substance for β-galactosidase. A complete kit (MATCHMAKER®) for identifying protein-protein interactions between two specific proteins using the two-hybrid technology is commercially available from Clontech. This system can also be extended to the mapping of protein domains involved in a particular protein interaction as well as the identification of amino acid residues important for this interaction.
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Compounds that inhibit the interaction of the PRO201, PRO308 or PRO309 coding sequences identified herein with other extracellular components can be tested as follows: usually the product of an amplified gene And a reaction mixture containing intracellular or extracellular components is prepared over the time that the two products interact and bind under conditions. In order to test the ability of the test compound to inhibit binding, the reaction is performed with or without the test compound. In addition, a placebo may be added to the third reaction mixture as a positive control. The binding (complex formation) between the test compound present in the mixture and the intracellular or external component is monitored as described above. A complex formed in the control reaction and not a reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
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M.M. Compositions and methods for tumor therapy
Compositions useful for the treatment of tumors with gene amplification identified herein include, but are not limited to, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc. Inhibits the expression or activity of a gene product.
For example, antisense RNA and RNA molecules directly block the translation of mRNA by hybridizing to the target mRNA and preventing protein translation. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation initiation site, eg, between -10 and +10 positions of the target gene nucleotide sequence, are preferred.
Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to complementary target RNA, followed by nucleotide strand breaks. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, for example, Rossi, Current Biology 4: 469-471 (1994) and PCT Publication No. WO 97/33551 (issued September 18, 1997).
The nucleic acid molecule in triple helix formation used for transcription inhibition is single-stranded and composed of deoxynucleotides. The basic composition of these oligonucleotides is designed to promote triple helix formation via Hoogstin base pairing rules, which generally requires a resizable extension of purine or pyrimidine on one strand of the duplex . For further details see, for example, PCT Publication No. WO 97/33551, supra.
These molecules can be identified by any or any combination of the screening assays described above or by other screening techniques known to those skilled in the art.
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N. Anti-PRO201, PRO308 or PRO309 antibody
The present invention further provides anti-PRO201, PRO308 or PRO309 antibodies. Examples of antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies. Some of the more potent candidate drugs of the present invention are antibodies and antibody fragments, which inhibit gene production or products of the genes identified herein and / or reduce the activity of the gene products.
1. Polyclonal antibody
Anti-PRO201, PRO308 or PRO309 antibodies of the present invention include polyclonal antibodies. Methods for preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies in mammals can be generated by one or more injections of, for example, an immunizing agent and, if desired, an adjuvant. Typically, the mammal is injected with the immunizing agent or adjuvant by multiple subcutaneous or intraperitoneal injections. The immunizing agent may comprise PRO201, PRO308 or PRO309 polypeptide or a fusion protein thereof. It is useful to conjugate the immunizing agent to a protein that is known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol will be selected by one skilled in the art without undue experimentation.
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2. Monoclonal antibody
Alternatively, the anti-PRO201, PRO308 or PRO309 antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using the hybridoma method as described in Kohler and Milstein, Nature, 256: 495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to generate antibodies that specifically bind to the immunizing agent, or to generate Trigger. Lymphocytes can also be immunized in vitro.
The immunizing agent typically comprises a PRO201, PRO308 or PRO309 polypeptide or a fusion protein thereof. Generally, peripheral blood lymphocytes ("PBL") are used when cells derived from humans are desired, or spleen cells or lymph node cells are used when non-human mammalian sources are desired. Subsequently, lymphocytes are fused with immortalized cell lines using an appropriate fusion agent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells from rodents, cows and humans. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells are preferably cultured in a suitable medium containing one or more substances that inhibit the survival or growth of unfused, immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxatin, aminopetitin and thymidine ("HAT medium"). This substance prevents the growth of HGPRT-deficient cells.
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Preferred immortalized cell lines are those that fuse efficiently, support stable high levels of antibody expression by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. A more preferred immortalized cell line is the mouse myeloma line, which is available, for example, from the Salk Institute Cell Distribution Center in San Diego, California or the American Type Culture Collection in Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines for generating human monoclonal antibodies have also been disclosed [Kozbor, J. Immunol., 133: 3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured is then assayed for the presence of monoclonal antibodies to PRO201, PRO308, or PRO309. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is measured by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be measured, for example, by the Scatchard assay by Munson and Pollard, Anal. Biochem., 107: 220 (1980).
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After the desired hybridoma cells are identified, the clones can be subcloned by a limiting dilution step and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown as ascites in vivo in a mammal.
Monoclonal antibodies secreted by the subclone can be isolated from the culture medium or ascites fluid by conventional immunoglobulin purification methods such as protein A-Sepharose method, hydroxylapatite chromatography method, gel electrophoresis method, dialysis method or affinity chromatography. Separated or purified.
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Monoclonal antibodies can also be made by recombinant DNA methods such as those described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily obtained using a conventional method (for example, using an oligonucleotide probe capable of specifically binding to the gene encoding the heavy and light chains of the mouse antibody). Can be isolated and sequenced. The hybridoma cells of the present invention are a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector that transfects a host cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce immunoglobulin protein. The monoclonal antibody can be synthesized in a recombinant host cell. DNA can also be obtained, for example, by replacing the coding sequences of human heavy and light chain constant domains in place of homologous mouse sequences [US. Patent No. 4,816,567; Morrison et al., Supra], or non-coding immunoglobulin coding sequences. Modification can be made by covalently linking part or all of the coding sequence of an immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide substitutes for the constant domain of the antibody of the invention or substitutes for the variable domain of one antigen binding site of the antibody of the invention to produce a chimeric bivalent antibody. can do. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies of the invention, or can be substituted for the variable domains of one antigen binding site of the antibodies of the invention, producing chimeric bivalent antibodies.
The antibody may be a monovalent antibody. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is generally cleaved at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted or deleted with other amino acid residues to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies. Generation of fragments thereof, particularly Fab fragments, by antibody digestion can be accomplished using conventional techniques known in the art.
[0080]
3. Humanized antibody
The anti-PRO201, PRO308 or PRO309 antibody of the present invention further includes a humanized antibody or a human antibody. Humanized forms of non-human (eg mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg Fv, Fab, Fab ′, F (ab ′) 2 or other antigen binding subsequences of antibodies). And contains the minimum sequence derived from non-human immunoglobulin. A humanized antibody is a CDR residue of a non-human species (donor antibody) in which the complementarity determining region (CDR) of the recipient has the desired specificity, affinity and ability, such as mouse, rat or rabbit. Human immunoglobulin (recipient antibody) substituted by In some examples, human immunoglobulin Fv framework residues are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has at least one, typically all or almost all CDR regions corresponding to those of a non-human immunoglobulin and all or almost all FR regions of a human immunoglobulin consensus sequence, typically Contains substantially all of the two variable domains. Humanized antibodies optimally comprise at least part of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op Struct. Biol., 2: 593-596 (1992)].
[0081]
Methods for humanizing non-human antibodies are well known in the art. Generally, one or more amino acid residues derived from non-human are introduced into a humanized antibody. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization essentially involves winter and co-workers [Jones et al., Nature, 321: 522-525 (1986); Riechmann by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)] by replacing the rodent CDR or CDR sequence with the corresponding sequence of a human antibody. To be implemented. Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies also include phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1992); Marks et al., J. Mol. Biol., 222: 581 (1991)]. It can also be created using various known methods. Techniques such as Cole et al. And Boerner et al. Can also be used for the preparation of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss. P. 77 (1985) and Boerner et al., J. Immunol. , 147 (1): 86-95 (1991)].
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4). Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)
The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug activating enzyme that converts a prodrug (eg, a peptidyl chemotherapeutic agent, see WO81 / 01145) to an active anticancer agent. can do. See, for example, WO 88/07378 and US Pat. No. 4,975,278.
The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme that can act on the prodrug to convert it to a more active cytotoxic form.
Without limitation, enzymes useful in the methods of the present invention include glycosidases, glucose-placed sidases, human lysozymes, human glucoronidases, alkaline phosphatases useful for converting phosphate-containing prodrugs to free drugs; sulfates Aryl sulfatase useful for converting a prodrug-containing prodrug into a free drug; cytosine deaminase useful for converting non-toxic 5-fluorocytosine to the anticancer agent 5-fluorouracil; proteases such as Serratia protease, thermolysin, subtilisin, carboxypeptidase (Eg, carboxypeptidase G2 and carboxypeptidase A) and cathepsins (eg, cathepsins B and L) that are useful for converting peptide-containing prodrugs to free drugs; D-alanyl carboxypeptidase useful for the conversion of prodrugs containing mino acid substituents; carbohydrate cleaving enzymes such as neuraminidase and β-galactosidase useful for converting glycosylated prodrugs to free drugs; derivatives with β-lactams Β-lactamases useful for converting derivatized drugs to free drugs; and penicillin amidases, such as phenoxyacetyl or phenylacetyl groups, respectively, to convert drugs derivatized at their aminic nitrogen to free drugs Penicillin V amidase or penicillin G amidase useful for this purpose is included. Alternatively, antibodies having enzymatic activity, also known as “abzymes”, can be used to convert the prodrugs of the invention into free active agents (eg, Massey, Nature 328: 457-458 [1987 ]). Antibody-abzyme conjugates can be prepared to deliver the abzyme to a tumor cell population as described herein.
The enzymes of this invention are covalently linked to anti-PRO201, anti-PRO308 or anti-PRO309 antibodies by using techniques well known in the art, such as the heterobifunctional cross-linking reagents discussed above. Can be made. Alternatively, a fusion protein in which at least the binding region of the antibody of the present invention is bound to at least a functionally active site of the enzyme of the present invention is prepared using recombinant DNA technology well known in the art. (Neuberger et al., Nature 312: 604-608 [1984]).
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5. Bispecific antibody
Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for PRO201, PRO308 or PRO309 and the other is for any other antigen, preferably a cell surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are well known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs with different specificities of the two heavy chains [Milstein and Cuello, Nature, 305: 537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually accomplished by an affinity chromatography step. Similar procedures are disclosed in WO 93/08829 published May 13, 1993, and Traunecker et al., EMBO J., 10: 3655-3656 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. Desirably, at least one fusion has a first heavy chain constant region (CH1) containing the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and cotransfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).
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According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimers recovered from recombinant cell culture. it can. A suitable interface includes at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). A complementary “cavity” of the same or smaller size as the larger side chain is created at the interface of the second antibody molecule by replacing the larger amino acid side chain with a smaller one (alanine or threonine). This provides a mechanism to increase the yield of heterodimers over other unwanted end products such as homodimers.
Bispecific antibodies are full length antibodies or antibody fragments (eg F (ab ′)₂Bispecific antibodies). Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) proteolytically cleaves intact antibody to F (ab ')₂Describes the procedure for producing fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The produced Fab ′ fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab′-TNB derivatives is then reconverted to Fab′-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab′-TNB derivative to form a bispecific antibody. The produced bispecific antibody can be used as a drug for selective immobilization of an enzyme.
Fab ′ fragments can be recovered directly from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) is a fully humanized bispecific antibody F (ab ')₂Describes the production of molecules. Each Fab ′ fragment is secreted separately from E. coli and undergoes directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibody thus formed is capable of binding to normal human T cells and cells overexpressing ErbB2 receptor, and triggers cytolytic activity of human cytotoxic lymphocytes against human breast tumor targets. Become.
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Various methods for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab ′ portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The “diabody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provided an alternative mechanism for generating bispecific antibody fragments. A fragment consists of a heavy chain variable domain (VH) joined to a light chain variable domain (VL) by a linker that is short enough to allow pairing between two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of the other fragment to form two antigen binding sites. Other strategies for producing bispecific antibody fragments by use of single chain Fv (sFv) dimers have also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).
More than bivalent antibodies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on the “pro” protein provided herein. Alternatively, anti- "pro" protein arms can be trigger molecules on leukocytes such as T cell receptor molecules (eg CD2, CD3, CD28 or B7), or FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) It may bind to an arm that binds to the Fc receptor (FcγR) of IgG, concentrating the cellular defense mechanism on specific “pro” protein expressing cells. Bispecific antibodies may be used as local cytotoxic agents against cells that express a particular “pro” polypeptide. These antibodies have a “pro” binding arm and an arm that binds to a cytotoxic or chelating agent such as EOTUBE, DPTA, DOTA, or TETA. Other bispecific antibodies of interest bind to “pro” polypeptides and further bind to tissue factor (TF).
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6). Heteroconjugate antibody
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies are used, for example, to target immune system cells to unwanted cells [US Pat. No. 4,676,980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. Proposed. This antibody could be prepared in vitro using known methods in synthetic protein chemistry, including those related to crosslinkers. For example, immunotoxins can be made using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in US Pat. No. 4,6767,980.
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7). Effector function design
It may be desirable to modify the antibody of the present invention for effector function, for example to enhance the efficacy of the antibody in the treatment of cancer. For example, cysteine residues are introduced into the Fc region to allow the formation of interchain disulfide bonds in this region. The homodimeric antibody produced in this way may have improved internalization ability or increased complement-mediated cell killing and antibody dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking agents as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be designed that has a dual Fc region and thus may have enhanced complement lysis and ADCC ability. See Stevenson et al., Anti-cancer Drug Design 3: 219-230 (1989).
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8). Immune complex
The invention is also conjugated to chemotherapeutic agents, cytotoxic agents such as toxins (eg, enzymatically active toxins from bacteria, fungi, plants or animals, or fragments thereof), or radioisotopes (ie, radioconjugates). It also relates to immune complexes comprising antibodies.
Chemotherapeutic agents useful for the generation of such immune complexes have been described above. Enzyme-active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragment of diphtheria toxin, cholera toxin, botulinum toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, Modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPIII, and PAP-S), Momodica charantia ( momordica charantia inhibitor, curcin, crotin, crotin, sapaonaria oficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin ( enomycin) and trichothecene. A variety of radioactive nucleotides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.
[0089]
The conjugates of antibodies and cytotoxic agents are various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), imidoester bifunctionality. Derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium Using derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) Can be created. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026.
In other embodiments, the antibody may be conjugated to a “receptor” (such as streptavidin) for use in tumor pre-targeting, and the antibody-receptor complex is administered to the patient, followed by a clarifier. Used to remove unbound complexes from the circulation, followed by administration of a “ligand” (eg, avidin) conjugated to a cytotoxic agent (eg, a radionucleotide, etc.).
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9. Immunoliposome
The antibodies disclosed herein may also be prepared as immunoliposomes. Liposomes containing antibodies are described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); Prepared by methods known in the art, such as described in 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in US Pat. No. 5,013,556.
Particularly useful liposomes are generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of a predetermined size to produce liposomes having a desired diameter. Fab 'fragments of the antibodies of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982). A chemotherapeutic agent (such as dixorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19) 1484 (1989).
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O. Use of anti-PRO201, PRO308 or PRO309 antibody
The anti-PRO201, PRO308 or PRO309 antibody of the present invention has various utilities. For example, anti-PRO201, PRO308, or PRO309 antibodies are used to detect PRO201, PRO308, or PRO309 diagnostic assays, such as detection of expression in specific cells, tissues, or serum. Competitive binding assays, direct or indirect sandwich assays, and immunoprecipitation assays performed in heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158] Various diagnostic assay techniques known in the art are used. Antibodies used in diagnostic assays are labeled with a detectable moiety. The detectable site must generate a signal that can be detected directly or indirectly. For example, the detectable site is a radioisotope such as 3H, 14C, 32P, 35S or 125I, a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine or luciferin, or alkaline phosphatase, beta-galactosidase or horseradish peroxidase, etc. The enzyme may be Hunter et al. Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40: 219 (1981); and Nygren, J. Histochem. And Cytochem. 30: 407 (1982), any method known in the art may be used to conjugate the antibody to the detectable site.
Anti-PRO201, PRO308 or PRO309 antibodies are also useful for affinity purification of PRO201, PRO308 or PRO309 from recombinant cell culture or natural sources. In this method, antibodies against PRO201, PRO308 or PRO309 are immobilized on a suitable support such as Sephadex resin or filter paper using methods well known in the art. Next, after the immobilized antibody is brought into contact with a sample containing PRO201, PRO308 or PRO309 to be purified, substantially all substances in the sample other than PRO201, PRO308 or PRO309 bound to the immobilized antibody are removed. Wash the support with a suitable solvent. Finally, the support is washed with another suitable solvent that will release PRO201, PRO308 or PRO309 from the antibody.
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P. Fusion antagonist
Several methods can be suitably used to generate the fusion antagonist and agonist compounds of the present invention. Any method that allows the antagonist molecule to be targeted inside the cell and that inhibits or prevents wild-type PRO201, PRO308 or PRO309 from normal action is preferred. Additional properties of such antagonist or agonist molecules, such as charge, size and hydrophobicity suitable for transmembrane transport, are readily determined by those skilled in the art.
If a PRO201, PRO308 or PRO309 mimetic or other homologue is identified and evaluated, the cells are exposed to the test compound and exposed to human PRO201, PRO308 or PRO309 only, exposed to both the compound and the natural ligand Compare to the negative control. When an antagonist or agonist of PRO201, PRO308 or PRO309 signal modulation is identified and assessed for death, the cells are exposed to a compound of the invention in the presence of the natural ligand and compared to a control not exposed to the test compound.
[0093]
Detection assays are used as a primary screen for phosphatase inhibition / promoting activity of antagonist / agonist compounds of the present invention. This assay is tested at concentrations in the test range, for example in the range 100 mM to 1 pM, and the amount (ICC) that phosphorylation or signaling is reduced or increased by 50% compared to the control₅₀Can be used only to assess the relative potency of the compounds.
The assay may be performed for the identification of compounds that affect phosphorylation of the PRO201, PRO308 or PRO309 substrate. In particular, the assay can be performed to identify compounds that increase the phosphorylation activity of PRO201, PRO308, or PRO309, and the assay can be performed to identify compounds that decrease phosphorylation of the PRO201, PRO308, or PRO309 substrate. Assays can be performed in a variety of ways, including protein-protein binding assays, biochemical screening assays, immunoassays, cell-based assays, and the like. Such assay formats are known in the art.
The screening assay of the present invention can be applied to high-throughput screening of chemical libraries, and is particularly suitable for identifying small molecule drug candidates.
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(1) Antagonist and agonist molecules
To screen for antagonists or agonists of PRO201, PRO308, or PRO309 signaling, the assay mixture is in the presence of a candidate agent, but under conditions where PRO201, PRO308, or PRO309 induces signaling with reference activity. Incubated (eg, binding of Nsp1 (SEQ ID NO: 1) to EGF receptor). The mixture components can be added in any order to provide the required activity. Incubations are performed at any temperature that facilitates optimal binding, typically between 4 ° C and 40 ° C, more usually between 15 ° C and 40 ° C. Incubation times are also selected for optimal binding, but are minimized to facilitate rapid, high throughput screening, typically about 0.1 to 10 hours, preferably less than 5 hours, more preferably Is less than 2 hours. After incubation, the effect of the candidate drug on PRO201, PRO308 or PRO309 signaling is determined by any convenient method. In cell-free binding assays, a separation step is often used to separate bound and unbound components. Separation is done, for example, by precipitation (eg TCA precipitation, immunoprecipitation, etc.), immobilization (eg on a solid substrate) and then washed. Bound protein is detected by a detectable label attached thereto, for example by measuring radioactive emission, optical or electron density, or by indirect detection, for example using antibody conjugation.
[0095]
Suitable molecules that affect protein-protein interactions of PRO201, PRO308, or PRO309 and binding proteins thereof include the latter fragments or small molecules that inhibit the interaction and correct complex formation, such as peptidomimetics. Such small molecules are usually less than 10K molecular weight, and are suitable as therapeutic agents because they are likely to be equivalent to cells, are not susceptible to degradation by various cellular mechanisms, and are unlikely to immunize like proteins. Small molecules include, but are not limited to, synthetic organic or inorganic compounds. Many pharmaceutical companies have libraries of such molecules that can be conveniently screened using the assays of the present invention. Non-limiting examples are proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological reagents, and their metabolites, transcription and translation Includes control sequences and the like.
A preferred technique for identifying molecules that bind to PRO201, PRO308, or PRO309 utilizes a chimeric substrate (eg, epitope tag fusion or fusion immunoadhesin) bound to a solid phase such as the well of an assay plate. In some cases, the binding of a labeled (eg, radiolabeled) candidate molecule to an immobilized receptor can be measured. Alternatively, competition for the interaction of Nsp1 with the binding protein can be assayed. In addition, the molecule may be screened to affect the tumorigenic potential of PRO201, PRO308 or PRO309 in NIH3T3 cells of nude mice. In screening for antagonists and / or agonists, PRO201, PRO308 or PRO309 is exposed to PRO201, PRO308 or PRO309 substrate and then putative antagonists or agonists, or PRO201, PRO308 or PRO309 binding protein and antagonists or agonists are added simultaneously, antagonists or The ability of an agonist to block PRO201, PRO308 or PRO309 activation is assessed.
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(2) Detection assay
PRO201, PRO308 or PRO309 polypeptides are useful in assays to identify lead compounds for therapeutically active agents that modulate PRO201, PRO308 or PRO309 signaling. In particular, lead compounds that prevent the formation of PRO201, PRO308 or PRO309 signaling complexes, or prevent or attenuate PRO201, PRO308 or PRO309 modulation can be conveniently identified.
[0097]
(A) Biochemical detection technology
Biochemical analysis techniques can be evaluated by various techniques. One exemplary assay mixture that can be used in the present invention is PRO201, PRO308 or PRO309 and usually a protein associated with PRO201, PRO308 or PRO309 (eg Cas), usually isolated, partially pure or pure. Contained in various forms. One or both of these components may be fused to other peptides or polypeptides that provide or promote protein-protein binding, for example under assay conditions, and improve stability. In addition, one of the components usually includes or is bound to a detectable label. The label provides direct detection by measurement of radioactivity, fluorescence, optical or electron density, or indirect detection of epitope tags, enzymes, and the like. The assay mixture further includes a candidate agent, and in some cases, various other options that promote binding, improve stability, reduce non-specific or background interactions, or improve the efficiency or sensitivity of the assay. Ingredients such as salts, buffers, carrier proteins such as albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents and the like.
The following detection methods can be used in a cell-free system in which a cell lysate comprising a signaling substrate molecule and PRO201, PRO308 or PRO309 is mixed with a compound of the invention.
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(I) Whole cell detection
A common technique is to incubate cells with PRO201, PRO308 or PRO309 and radiolabeled phosphate and use either SDS or PAGE from the lysate to produce either primary or two-dimensional cells. Separating the protein component and exposing to X-ray film to detect the presence of phosphorylated protein. Detection can also be done without the use of radioactive labels. In such techniques, protein components (eg, isolated SDS-PAGE) are transferred to a nitrocellulose membrane where the presence of phosphorylated serine / threonine is detected using an anti-phosphotyrosine antibody (anti-pTyr).
Alternatively, anti-pTyr is detected by conjugating to an enzyme such as horseradish peroxidase and then adding a colorimetric substrate for the enzyme. A further alternative involves the detection of anti-pTyr by reacting with a secondary antibody that recognizes anti-pTyr, which is labeled with radioactivity or enzyme as already mentioned. These examples and similar techniques are described in Hansen et al., Electrophoresis 14: 112-126 (1993); Campbell et al., J. Biol. Chem. 268: 7427-7434 (1993); Donato et al., Cell Growth Diff. 3: 258 -268 (1992); Katagiri et al., J. Immunol. 150: 585-593 (1993). Furthermore, anti-pTyr can be detected by labeling it with a radioactive substance and then scanning the labeled nitrocellulose for radioactivity or detection or by exposure to X-ray film.
[0099]
(B) Biological detection technology
The ability of the antagonist / agonist compounds of the invention to modulate the activity of PRO201, PRO308 or PRO309, which itself modulates intercellular signaling, is measured by scoring for morphological or functional changes associated with ligand binding. Qualitative and quantitative techniques known in the art can be applied for observation and measurement of cellular processes occurring under the control of PRO201, PRO308 or PRO309. For example, expression of Nsp1 (SEQ ID NO: 1) in NIH3T3 cells causes the formation of morphological transformation of the cells. The presence or number of these lesions can be used as an indicator of the biological effectiveness of an antagonist or agonist of Nsp1 signaling.
The data obtained in these cell culture assays and animal studies is used in formulating a range of doses for use in humans. The dosage of the compounds of the invention is in the range of circulating concentrations that have little or no toxicity. The dose varies within this range depending on the dosage form employed and the route of administration.
[0100]
(2) Antisense nucleotide
Another preferred class of antagonists includes the use of gene therapy techniques and involves the administration of antisense nucleotides. Applicable gene therapy techniques include single or multiple administrations of therapeutically effective DNA or mRNA. Antisense RNA and DNA can be used as therapeutic agents to block the expression of certain genes in vivo. Short antisense oligonucleotides can be transferred into cells, which act as inhibitors despite the low intracellular concentrations caused by limited uptake by the cell membrane, Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146 (1986). Oligonucleotides can be modified to facilitate their incorporation, for example by replacing negatively charged phosphodiester groups with uncharged groups.
[0101]
There are a variety of techniques known for introducing nucleic acids into viable cells. These techniques vary depending on whether they are transferred in vivo, in vitro cultured cells, or in vivo to a control host cell. Suitable techniques for transferring nucleic acids to mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection, Dzau et al., Trends Biotech. 11: 205-210 (1993). In some situations, it is preferable to provide a nucleic acid source with a reagent that targets the target cell, for example, using an antibody specific for a cell surface membrane protein with endocytosis to promote targeting or uptake. For example, specific cell-type capsid proteins or fragments thereof, antibodies of proteins that undergo internalization upon cycling, and proteins that target intracellular localization and improve intracellular half-life. Techniques for receptor-mediated endocytosis are described, for example, in Wu et al., J. Biol. Chem. 262: 4429-2232 (1987); Wagner et al., Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990). Has been described. For review of well-known gene generation and gene therapy protocols, see Anderson et al., Science 256: 808-813 (1992).
[0102]
In one embodiment, a PRO201, PRO308 or PRO309 antagonist or agonist molecule is used to bind to an endogenous ligand of a cell, whereby in particular the level of PRO201, PRO308 or PRO309 in the cell exceeds normal physiological levels. In some cases, the cells are rendered non-responsive to PRO201, PRO308 or PRO309 wild type. It may also be advantageous to bind to an endogenous PRO201, PRO308 or PRO309 substrate, or a complexing agent that activates unwanted cellular responses (such as tumor cell growth).
In a further embodiment of the invention, PRO201, PRO308 or PRO309 expression is expressed with PRO201, PRO308 or PRO309 antisense RNA or DNA in an amount effective to reduce expression of PRO201, PRO308 or PRO309 protein. Can be reduced by providing cells.
In a further embodiment of the invention, the expression of the binding partner of PRO201, PRO308 or PRO309 is accompanied by a predetermined amount of antisense RNA or DNA that goes to reduced expression of the binding partner of PRO201, PRO308 or PRO309, PRO201, PRO308 or PRO309. Reduced by providing expression cells.
[0103]
Q. Diagnostic use
Other uses of the compounds of the invention described herein (eg, PRO201, PRO308 or PRO309, PRO201, PRO308 or PRO309 variants and anti-PRO201, PRO308 or PRO309 antibodies) may be used to treat diseases by modulating PRO201, PRO308 or PRO309 modulation signaling. Is to help diagnose whether some progress has been made.
A diagnostic assay to determine whether PRO201, PRO308 or PRO309 signaling leads to a specific disease is performed using the following steps: (1) cell or tissue culture; (2) Nsp1, Nsp2 or Nsp3 Administration of compounds capable of inhibiting modulation signaling; and (3) measurement of the degree of phosphorylation on PRO201, PRO308 or PRO309 substrate in cell lysates, or PRO201, PRO308 or PRO309 mediated phenotype effects in test cells. The process can be performed using standard techniques in light of this disclosure. For example, standard techniques can be used for cell or tissue isolation and culture or in vivo.
[0104]
Compounds with varying degrees of selectivity are useful for diagnosing the role of PRO201, PRO308 or PRO309. For example, compounds that inhibit PRO201, PRO308, or PRO309 in addition to other forms of adapter molecules can be used as initial test compounds to determine whether some adapter molecules lead to disease. The test compound can then be used to further evaluate other intracellular signaling activities in the induction of disease. The test compound should be more potent in inhibiting intracellular signaling activity than exerting a cytotoxic effect (eg, IC50 / ID50 is greater than 1). IC50 and ID50 can be measured by standard techniques such as MTT assay, or by measuring LDH release. The degree of IC50 / ID50 of a compound should take into account the evaluation of the diagnostic assay. In general, as the ratio increases, the information becomes more relative. Appropriate controls take into account the possible toxic effects of the compound, for example, treatment of a cell without a proliferative disorder (eg, a control cell) with a test compound can be used as part of a diagnostic assay. The diagnostic methods of the invention include screening for reagents that modulate the effect on hedgehog signaling. Exemplary detection techniques include radiolabeling and immunoprecipitation (US Pat. No. 5,385,915).
[0105]
Surface proteins such as growth factor receptors that are overexpressed in certain tumors are excellent targets for drug candidates or tumor (eg, cancer) treatment, but are encoded by the same protein as well as genes amplified in tumor cells Secreted proteins have found further use in tumor diagnosis and prevention. For example, antibodies against protein products of genes amplified in tumor cells can be used for tumor diagnosis or prevention.
For example, an antibody comprising an antibody fragment can be used for qualitative or quantitative detection of expression of a protein encoded by the amplified gene (“marker gene product”). The antibody preferably comprises a detectable, eg fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorometry, or other techniques known in the art. This technique is particularly preferred when the amplified gene encodes a cell surface protein, such as a growth factor. Such binding assays are performed substantially as described in item 5 above.
In situ detection of antibodies that bind to the marker gene product can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological sample is removed from the patient and the labeled antibody is applied to it, preferably by covering the biological sample with the antibody. This approach also allows the distribution of the marker gene product in the tissue being tested to be determined. It will be apparent to those skilled in the art that a wide variety of histological methods are readily available for in situ detection.
[0106]
R. Pharmaceutical composition
Agonist antibodies that specifically bind to the products of the amplified genes identified here, as well as other molecules identified in the screening assays disclosed above, are a variety of those discussed above, including tumors including cancer, viral diseases, etc. For the treatment of pathological conditions, it can be administered as an immunomodulator in the form of a pharmaceutical composition.
When the protein encoded by the amplified gene is intracellular and the whole antibody is used as an inhibitor, an internalizing antibody is preferred. However, lipofection or liposomes can also be used to introduce antibodies, or antibody fragments, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is usually preferred. For example, peptide molecules that retain the ability to bind to a target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be synthesized chemically or produced by recombinant DNA techniques (eg, Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 [1993]).
[0107]
A therapeutic preparation of an antibody is prepared by mixing an antibody of the desired degree of purity with any pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lipophilic formulation or an aqueous solution. (Remington's Pharmaceutical Science 16th edition, Osol, A. Ed. [1980]). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate, and other organic acids; ascorbic acid and methionine Antioxidant; Preservative (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl paraben such as methyl or propylparaben; catechol; resorcinol; cyclohexanol 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; glycine, glutami , Amino acids such as asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides including glucose, mannose, or dextrin, and other chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol; sodium, etc. Non-ionic surfactants such as metal complex (eg Zn-protein complex) or TWEEN (trade name), PLURONICS (trade name), and polyethylene glycol (PEG) Including.
[0108]
Non-antibody compounds identified in the screening assays of the invention are formulated in a similar manner using standard techniques known in the art.
The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary properties that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitor. These molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredient can also be used in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, eg hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems <BR (For example, liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules) or in microemulsions. These techniques are disclosed in Remington's Pharmaceutical Science 16th edition, Osol, A. Ed. [1980].
The formulation used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes.
[0109]
Sustained release formulations may be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies, which matrix is in the form of a molded article, such as a film or microcapsule. Examples of sustained release matrices are polyester hydrogels (eg poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polyactides (US Pat. No. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamate. , Non-degradable ethylene-vinyl acetate, LUPRON DEPOT (trade name) (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate), poly- (D) -3-hydroxy Contains butyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels release proteins in a shorter time. When encapsulated antibodies remain in the body for a long time, they denature or aggregate upon exposure to moisture at 37 ° C, resulting in reduced biological activity and possible changes in immunogenicity. . Reasonable methods can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S—S bond formation through thio-disulfide exchange, stabilization may include modification of sulfhydryl residues, lyophilization from acidic solutions, control of water content, appropriate Addition of various additives and the development of specific polymer matrix compositions.
[0110]
S. Method of treatment
The antibodies and other anti-tumor compounds of the present invention may be used in the treatment of various tumors such as cancer, viral infections, and generally conditions where immune modulation, such as up-regulation of the immune system, is desired.
Examples of conditions or diseases to be treated include benign or malignant tumors (eg, renal, liver, kidney, bladder, breast, stomach, ovary, colorectal, prostate, pancreas, lung, vulva, Thymus, hepatic carcinoma, sarcoma; glioblastoma; and various head and neck tumors); leukemia and lymphoid malignancies; neurons, glia, astrocytes, hypothalamus and glands, macrophages, epithelium, between Quality and blastocoel diseases; and inflammation, angiogenesis and immunological diseases.
The antitumor agent (eg, antibody) of the present invention is administered to a mammal, preferably a human, in a well-known manner, for example, intravenous administration by bolus or continuous infusion over a predetermined time, intramuscular, intraperitoneal, intracerebral spinal cord, Administration is subcutaneous, interarticular, intrasynovial, intrathecal, oral, topical, or by inhalation route. Intravenous administration is preferred.
[0111]
Other therapeutic regimens may be combined with the administration of the anticancer agents of the invention. For example, patients treated with such anti-cancer agents may receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. The preparation method and dosage schedule of such chemotherapeutic agents are either used according to the manufacturer's instructions or determined empirically by skilled practitioners. Preparation methods and dose schedules for such chemotherapy are also described in Chemotherapy Service M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may be administered prior to or subsequent to the administration of the anti-tumor agent of the present invention, or may be administered simultaneously therewith. The antibodies of the invention may be combined with doses known for these molecules of anti-estrogenic compounds such as tamoxifen or anti-progesterones such as onapristone (see EP 616812).
It is also preferred to administer antibodies against tumor-associated antigens, such as antibodies that bind to ErB2, EGFR, ErB3, ErB4, or vascular endothelial factor (VEGF). Sometimes it is also advantageous to administer cytokines to the patient. In a preferred embodiment, the anticancer agent herein is co-administered with a growth inhibitor. For example, the growth inhibitor is first administered, followed by the anticancer agent of the present invention. However, simultaneous administration or administration of the anticancer agent of the present invention first is also conceivable. A suitable dose for a growth inhibitor is the amount currently used, but can be reduced by the combined (synergistic) effect of the growth inhibitor and this antibody.
[0112]
The appropriate dose of anti-tumor agent here for prevention or treatment of disease is the type of disease to be treated, severity and course of disease, as defined above, administered by the drug for the purpose of prevention or treatment. Whether it is done, prior treatment, the patient's clinical history and response to medication, and the discretion of the attending physician. The drug is suitably administered to the patient at one time or over a series of treatments.
For example, depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg, 0.1-20 mg / kg) of anti-tumor agent can be administered, for example, in one or more separate doses This is the first candidate dose to be administered to a patient by infusion. A typical daily dose will be about 1 μg / kg to 100 mg / kg or more, depending on the above factors. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until a desired suppression of disease symptoms appears. However, other dose schedules may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
[0113]
T.A. Products
In another embodiment of the present invention, an article of manufacture containing materials useful for the diagnosis or treatment of the above diseases is provided. This article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a material such as glass or plastic. The container contains a composition effective to diagnose and treat the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). May be). The active agent in the composition is the IFN-ε of the present invention, or an agonist or antagonist thereof. The label on or on the container indicates that the composition is used for diagnosis or treatment of the selected condition. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
[0114]
The following examples are provided for purposes of illustration only and are not intended to limit the scope of the invention in any way.
All patents and references cited herein are hereby incorporated by reference in their entirety.
(Example)
All other commercial reagents mentioned in the examples were used according to manufacturer's instructions unless otherwise indicated. The cell source identified by the ATCC registration number throughout the examples and specification below is American Type Culture Collection, Rockville, Maryland.
[0115]
Example 1
Isolation of human PRO201, PRO308 or PRO309 cDNA clones
An expression sequence tag (EST) database (LIFESEQ (trade name), Incyte Pharmaceuticals, Palo Alto, Calif.) Was searched to identify ESTs in a fetal pancreatic library with significant identity to the adapter protein Shc (1328938, DNA28710, FIG4A ) (SEQ ID NO: 13). The full length cDNA corresponding to the isolated EST was cloned from a human fetal kidney library using the in vivo cloning technique in pRK5 (DNA30676, Nsp1) (SEQ ID NO: 1). There is a single long open reading frame, which encodes a 576 amino acid protein. Nsp1 (SEQ ID NO: 1) is an Sck that appears in the SH2 region that appears at the N-terminus of Nsp1 (SEQ ID NO: 1).ShcAlso associated with (Fig 8) (SEQ ID NOs: 17 and 16, respectively). Nsp1 (SEQ ID NO: 1) also has a proline-serine enriched domain (PS) in the middle of the protein, which can act as a SH3 interaction domain. The C-terminus of Nsp1 does not have significant identity with known mammalian proteins. This C-terminus was then used for re-screening of the EST database and two additional fragments (104191) (DNA38653) (Fig4B) (SEQ ID NO: 14) and (1651811) (DNA38654) (Fig4C) (SEQ ID NO: 15 ) From these cloning and enriching primers were made and the full-length sequences for Nsp2 (SEQ ID NO: 3) and Nsp3 (SEQ ID NO: 5) each were isolated from a human placenta library in pRK5 using in vivo cloning techniques. The probes used for cloning the full-length sequence are as follows:
Nsp1 (SEQ ID NO: 1):
Cloning: ACTGAGGCCTGTTGAAAGTGCAGAGCTCAG (SEQ ID NO: 7)
Enriched primer: GCTGAAGAAGAGCTTCAG (SEQ ID NO: 8)
Nsp2 (SEQ ID NO: 3):
Cloning: CAATGCCGATGGCCATTGTGTTGTGTCTTTCAATTATGTCCAGGCGCA (SEQ ID NO: 9)
Enriched primer: ATCCCAGAATGTCCACTG (SEQ ID NO: 10)
Nsp3(SEQ ID NO: 5):
Cloning: GGCCAGCATGATGGACATGGTGTGGAACCTTTCCAGCAGGTCTAGGCGTA (SEQ ID NO: 11)
Enriched primer: GGTGCAGCCCAGGATGTC (SEQ ID NO: 12)
[0116]
The three proteins (Nsp1, Nsp2, Nsp3) (SEQ ID NO: 1, 3 and 5) have a total identity of 33% to 47% (FIG. 6B). Nsp3 is an SH2 domain and potentialSHIt has 3 interaction domains (PS region), Nsp2 lacks the SH2 domain but has a potential SH3 interaction domain. Nsp2SHDue to the absence of two domains, this protein probably acts as the other two Nsps dominant negative regulators. All three proteins lack an apparent kinase or phosphatase domain.
The cDNA clones Nsp1, Nsp2 and Nsp3 (SEQ ID NO: 2, 4 and 6) were sequenced in their entirety. The total nucleic acid sequences of DNA30676, DNA40575, and DNA61601 are shown in FIG1 (SEQ ID NO: 2), FIG2 (SEQ ID NO: 4), and FIG3 (SEQ ID NO: 6), respectively. Clone DNA30676-1223, DNA40575-1223 and DNA61601-1223 were deposited with ATCC and assigned ATCC deposit numbers 209567, 209565 and 209713. In addition, the related clone DNA40556-1223 and DNA40554-1223 were also deposited with the ATCC and were assigned ATCC deposit numbers 209566 and 2096564.
[0117]
Example 2
Northern blot analysis
Expression of PRO201, PRO308 and PRO309 mRNA in human tissues was examined by Northern blot analysis. Human RNA blots were complementary to the nucleotides encoding amino acids: (a) DNA 30676 90-102; (b) DNA 40575 270-284, or (c) DNA 61601 475-491.³²Hybridized to P-labeled DNA probe. Endocrine and fetal II (clontech) were hybridized in ExpressHyb® hybridization solution (Clontech) according to the manufacturer's instructions. Blots were incubated with probes in hybridization buffer (5 × SSPE: 2 × denhard solution; 100 mg / ml denatured sheared salmon sperm DNA; 50% formamide; 2% SDS) at 42 ° C. for 16 hours. Blot was performed with 2x SSC;0.05Washing with% SDS at room temperature for 1 hour, followed by washing with 0.1 × SSC; 0.1% SDS at 50 ° C. for 30 minutes. Blots were developed after overnight exposure by phosphoimager analysis (Fuji).
As shown in FIG. 9A, significant expression of Nsp1 is detected only in the human fetal liver library and can also be expressed in other fetal tissues (eg, fetal kidney). This expression pattern suggests a role for Nsp1 in the synchronization of signal transduction pathways important for fetal development. In contrast, Nsp2 (SEQ ID NO: 4) and Nsp3 (SEQ ID NO: 6) were more widely expressed in many tissues. In the hematopoietic tissue, two Nsp2 transcripts (3.8 kb and 3.2 kb) were detected (FIG. 9B).
[0118]
Example 3
EGF, insulin and fibronectin-induced phosphorylation and p130^CasComplex formation with
Since Nsp1 has three potential phosphorylable tyrosines, experiments were performed to determine whether Nsp1 could be phosphorylated in the presence of various extracellular stimuli. Treatment with EGF immediately induced tyrosine phosphorylation of Nsp1 (SEQ ID NO: 1), which occurred within 2 minutes (Fig 10A). Nsp1 (SEQ ID NO: 1) was also phosphorylated in response to insulin, IGF-1 and heregulin (not shown). In contrast, fibronectin (FN) only stimulated weak Nsp1 phosphorylation (FIG. 10C).
In order to follow the pathway stimulated by Nsp1 (SEQ ID NO: 1), Applicants identified a protein associated with Nsp1 (SEQ ID NO: 1) in vivo in a co-immunoprecipitation experiment. Treatment with EGF leads to binding between Nsp1 and a tyrosine phosphorylated protein with a molecular weight of approximately 170 kD. This protein is immediately tyrosine phosphorylated to EGF and can be detected by mAB to the EGF receptor. Furthermore, Nsp1 (SEQ ID NO: 1) can be detected by Western blot following immunoprecipitation of the EGF receptor (FIG. 10B). There are remaining Nsp1 / EGF receptor interactions prior to EGF treatment, but the extent of interaction increases significantly following exposure to EGF.
Immunoprecipitation experiments also revealed that Nsp1 interacts with a 130 kD protein (p130). Although p130 is phosphorylated moderately in serum-starved cells (Fig 10A), loss of cell adhesion leads to complete p130 dephosphorylation (Fig 10C). By Western blot analysis, this p130 is the adapter protein p130.^CasI found out that In FIG. 10A, anti- (P) Tyr antibody detected two bands at about 130 kD, whereas anti-Cas antibody recognized only the bottom band. We have not yet identified the upper band. Cas was first found as a hyperphosphorylated protein following induced expression of the virus Crk (V-Crk) [Sakai et al., EMBO J. 13:
3748-56 (1994)], phosphorylated in response to integrin interactions with the extracellular matrix as well as numerous other stimuli. Chen et al., J. Biol. Chem. 272: 27401-10 (1997); Casamassima and Rozengurt, J. Biol. Chem. 272: 9363-70 (1997); Nojima et al., J. Biol. Chem. 270: 15398- 402 (1995). Cas interacts directly with focal adhesion kinase (FAK) [Polte and Hanks, Proc. Natl. Acad. Sci. USA 92: 10678-82 (1995)], which allows extracellular events to It was found to be an important factor affecting survival. Daniel and Reynolds, Mol. & Cell. Biol. 15: 4819-24 (1995); Mo & Reynolds, Cancer Res. 56: 2633-40 (1996); Nakamoto et al., Mol. Cell. Biol. 17: 3884-97 (1997).
[0119]
The phosphorylation status of Nsp1 (SEQ ID NO: 1) and Cas, the relative amount of Cas associated with Nsp1 (SEQ ID NO: 1) depends on signal transduction through EGF or integrin receptors. Although EGF increases Nsp1 phosphorylation, dephosphorylation of both global (data not shown) and Nsp1 (SEQ ID NO: 1) is associated with Cas (FIG. 10A). The amount of Cas associated with Nsp1 after EGF treatment also increases (FIG. 10A). In contrast, fibronectin has only a minor effect on Nsp1 (SEQ ID NO: 1) phosphorylation, but increases Cas associated with Nsp1 and at the same time temporarily reduces the amount of Cas associated with Nsp1 (FIG. 10C). . An increase in Cas phosphorylation in response to integrins has already been reported. Nojima et al., J. Biol. Chem. 270: 15398--4029 (1995). This decrease in Nsp1 / Cas complex reached a minimum after about 30 minutes and then returned to baseline conditions around about 4 hours.
In FIG. 11, insulin-stimulated Nsp1 phosphorylation peaked at 2 hours and then decreased after 14.5 hours. The same blot was reprobed with anti-FLAG antibody to show equal loading. In FIG. 11, IGF-1 also stimulates phosphorylation of Nsp1, but the level of phosphorylation for IGF-1 has been shown to be lower than that seen for insulin.
IGF-1 results:
Nsp1 is associated with Cas in the absence of insulin (Fig 13) or EGF (not shown). Cas phosphorylation was observed to decrease after insulin treatment. This is shown in both FIG12 and FIG13. Remove the film of the sample tested in FIG.^CasReprobing with the antibody also showed that the amount of Cas associated with Nsp1 decreased following insulin treatment.
[0120]
Materials and methods
EGF / fibronectin:
Transfected and serum starved COS (A, B) were treated with 25 ng / ml EGF for the indicated times or left untreated. Transfected or serum starved 293 cells were allowed to adhere to plastic (on the dish), suspended (outside the dish), or placed on a 10 mg / ml FN-coated dish for the time indicated in FIG. 10C (C). In FIGS. 10A and 10C, anti-flag immunoprecipitations were blotted with anti-flag, anti- (P) Tyr or anti-Cas antibodies as indicated. In FIG. 10B, anti-EGF receptor (CalBiochem) immunoprecipitation was blotted as indicated with anti-flag or anti- (P) Tyr antibody. Transfected cells were treated with a buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton) containing a newly added protease inhibitor (1 mM AEBSF, 10 mM leupeptin, 2 mg / ml aprotinin, 1 mM pepstatin). X-100, 2 mM EDTA, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM orthovanadate). Anti-flag (Kodak), IBI immunoprecipitation and related proteins were visualized with the anti- (P) Tyr antibody PY20 (Transduction Lab). Remove the same blot and anti-p130^Cas(Transduction Lab) or replotted with anti-flag antibody and detected with ECL system (Pierce). A flap epitope (DYKDDDDK (SEQ ID NO: 19)) was added in frame to the N-terminus of the Nsp1 cDNA construct using in vitro mutagenesis to generate pRK. Nsp1. FLAG was created.
[0121]
insulin:
The FLAG epitope (DYKDDDDK (SEQ ID NO: 19)) was inserted into the N-terminus of the Nsp1 cDNA construct using standard in vitro mutagenesis and pRK.Nsp.FLAG was created. CHO cells overexpressing the insulin receptor (CHO-IR) were cultured in F12-DMEM containing 10% serum, 2 mM L-glutamine, 100 units / ml penicillin, and 100 μg / ml streptomycin. Liposome mediated transfection using DOSPER (Boehringer Mannheim) or superfect (Qiagen) was performed on CHO cells according to the manufacturer's instructions. CHO-IR cells were emptied with pRK Nsp1. Transiently transfected with any of the FLAG and serum starved for 16 hours. Cells were treated for different times with or without 100 nM insulin, then freshly added protease inhibitors (1μMAEBSF, 10μM Leupeptin, 2μg / ml aprotinin, 1μ1 ml immunoprecipitation assay (IPA) buffer (10 mM Tris, pH 7.5, 150 mM CaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, 10 mM sodium pyrophosphate, 10 mM Sodium fluoride, 2 mM orthovanadate). Samples were immunoprecipitated with anti-FLAG affinity gel (IBI, Kodak). Following SDS-polyacrylonitrile gel electrophoresis, the protein was transferred to a nitrocellulose membrane (Novex) and anti-PhosphoWestern blotting was carried out with tyrosine antibody PY-20 (Transduction Lab) or anti-FLAG antibody and detected with ECL system (Pierce).
IGF-1
PRK or pRK. Transfected with CHO-IR or CHO-IGFIR (IGF-1 receptor) cells. Nsp1. FLAG is serum starved, treated with 100 nM insulin or 100 ng / ml IGF-1, and buffer for co-immunoprecipitation assay (CoIPA) containing protease inhibitors (20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, 2 mM EDTA, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM orthovanadate). Samples were anti-FLAG or anti-p130^CasAnti-phosphotyrosine antibody PY-20 or anti-p130^CasWestern blotting with (Santa Cruz Biotechnology).
[0122]
Example 4
Mapping of phosphorylated tyrosine residues in Nsp1
In order to map phosphorylated residues in Nsp1, Applicants independently converted each of the three tyrosines of Nsp1 (SEQ ID NO: 1) to phenylalanine. The transfected cells were then stimulated with EGF and immunoprecipitated with Nsp1 (SEQ ID NO: 1). In all three cases, non-phosphorylated Nsp1 (SEQ ID NO: 1) immunoprecipitated from unstimulated cells was not associated with both Cas and EGF receptors. These results indicate that amino acid changes are not overall detrimental to the overall protein structure. Mutant Nsp1_Y61F(SEQ ID NO: 21) was normally phosphorylated on EGF, but Nsp1_Y95FNo phosphorylation of (SEQ ID NO: 22) was detected and Nsp1_Y231F(SEQ ID NO: 23) was weakly phosphorylated. This data suggests that there is first Y95 phosphorylation followed by Y231 phosphorylation. Y16 may or may not be phosphorylated, but is not required for phosphorylation of Y95 or Y231. further,EGF co-immunoprecipitated with Nsp1 (SEQ ID NO: 1)ReceptorSince Nsp1 (SEQ ID NO: 1) with Cas is Nsp1 (SEQ ID NO: 1) and Cas phosphorylation is increased by receptor phosphorylation but largely independent of phosphorylation of Nsp1 (SEQ ID NO: 1) State independent (Fig 10), this interaction was found to be mediated through the SH3 domain of Cas and the SH3 interaction domain of Nsp1 (SEQ ID NO: 1).
Materials and methods:
All three tyrosine residues of Nsp1 (SEQ ID NO: 1) were converted to phenylalanine using standard mutagenesis methods. Mutant (Y16F, Y95F and Y231F) (SEQ ID NO: 21-23, respectively) and wild type Nsp1 (SEQ ID NO: 1) DNA are transfected into COS cells and treated with 25 ng / ml EGF for 10 minutes or untreated It was left as it was. Cell lysate is anti-flagantibodyAnti-P (Tyr) antibody, anti-p130^CasWestern blotting with antibody or anti-flag antibody.
[0123]
Example 5
Transformation and tumorigenicity in nude mice
preface:
Since Cas was included in c-src mediated events [Sakai et al., EMBO J. 13: 3748-56 (1994); Sakai et al., Oncogene 14: 1419-26 (1997)], the applicant is responsible for the NIH3T3 transformation assay. The effect of Nsp1 (SEQ ID NO: 1) on was tested.
NIH3T3 is a cell line that normally grows in a monolayer even when cells are over confluent. These are used to determine whether a candidate gene has potential carcinogenicity when the candidate is transfected into the vector MSCV through retrovirus-mediated infection. Transfected cells are generated in the lesion, harvested, cultured to 10 million cells, and injected into nude mice. If the transfected cell is carcinogenic, it grows and forms a tumor without being inhibited by the defective immune system of nude mice. See Winograd et al., In Vivo 1 (1): 1-13 (1987).
[0124]
Discussion and results:
Over 100 morphologically transformed lesions were observed on 100 mm plates of NIH3T3 cells following transformation with Nsp1-expressing retrovirus and G418 selection, but did not appear with the subject (neo) vector. Transformed cells (Fig 14B) became more rounded and dense compared to the normally elongated fibroblast shape (Fig 14A) of control transfected NIH3T3 cells. To test whether the transformed Nsp1-expressing cells are also tumorigenic, three independent lesions were taken and expanded, and NIH3T3-MSCV. Nsp1-. sub1,-. sub2 and-. sub3 was generated. Controls are from a cell line that expresses only neo (NIH3T3-neo) and a pool of transfected cells that express low levels of Nsp1 (SEQ ID NO: 2) but are not transformed (NIH3T3-Nsp1.non-trans). Configured. Nsp1 (SEQ ID NO: 2) expressing, non-transformed cells were induced by infecting NIH3T3 cells with an Nsp1 (SEQ ID NO: 2) expressing retroviral vector. These bulk media were selected for neomycin resistance but did not progress through post-confluent growth that selected for lesion formation.
Each cell line was injected into 5 mice. Neo control cells or NIH3T3-Nsp1. Tumor growth was not observed in mice injected with non-trans cells. NIH3T3-MSCV. Nsp1-. sub1,-. sub2 and-. All 5 mice in each group injected with sub3 had obvious tumor growth within 3 weeks. Histological analysis showed that the tumor was a large, heterogeneous, moderate undifferentiated epithelial cell with a high mitotic index (Fig 14C). There was no clear evidence of metastasis.
The formed tumor is a well-extended, expansible substance consisting mainly of large, heterogeneous, medium undifferentiated epithelial cells, has a high mitotic index, and is dispersed in the periphery by a small region of spindle cell proliferation (FIG. 14).
[0125]
Materials and methods:
pRK. Nsp1. The FLAG plasmid was digested with EcoRI and SalI. The Nsp1 cDNA fragment containing the FLAG epitope was purified and subcloned into the EcoRI and XhoI sites of the retroviral vector MSCVneo and MSCVneo. Nsp1. FLAG. Mouse embryonic fibroblasts (ATCC) and retrovirus maker BOSC23 cells were maintained in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units / ml penicillin, and 100 μg / ml streptomycin.
Retroviral vectors MSCVneo and MSCVneo. Nsp1. FLAG was transfected into BOSC23 cells using calcium phosphate mediated transfection. The supernatant was used for 72 hours to infect NIH3T3 seeded on a 6-well plate. Infected cells were selected and pooled in 400 μg / ml G418 (Gobco), and NIH3T3-MSCV and -MSCV. An Nsp1 cell line was generated. NIH3T3-MSCV and -MSCV. Nsp1 cells were grown to confluence for 4 days, divided into one medium change, 1 to 5 ratio, and further grown for 4 days to confluence. After infection, over 100 morphologically transformed cells were observed on 100 mm plates of NIH3T3 cells, but not the control (neo) vector. FIG. 14A shows an ultrathin section of either control cell (FIG. 14A), and FIG. 14B shows an Nsp1 transformed lesion.
NIH3T3-MSCV. Three transformed foci of Nsp1 were harvested and expanded, subline NIH3T3-MSCV. Nsp1-. sc1,-. sc2 and-. scb3 was generated. 10⁷Vector transfected control cells, untransformed NIH3T3-MSCV. Nsp1 and 3 sublines were injected subcutaneously into the sole of the hind paw of each nude mouse. Five mice were injected for each cell line. Tumor size was measured at 2 and 4 weeks. Resulting tumors (4 weeks after injection) were fixed, blocked, and sections were stained with hematoxylin and eosin (Fig 14C). Control cells or untransformed NIH3T3-MSCV. No tumor growth was observed in mice injected with the vector transfected with Nsp1 cells. NIH3T3-MSCV. Nsp1-. sc1,-. sc2 and-. Tumors grew in each mouse injected with sc3 (Fig 15).
[0126]
Example 6
Apoptosis resistance
Since the above example showed that Nsp1 (SEQ ID NO: 2) expression leads to NIH3T3 transformation and tumorigenesis, Applicants have determined whether Nsp1 expression protects cells from apoptosis induced by removal of growth factors I experimented. The subcloned cell line is a morphologically transformed cell NIH3T3-MSCV. Nsp1-. sub1 and-. derived from sub2 (Nsp1.-sub1.1 and Nsp1.sub2.1). These transformed cell lines, non-transformed cell cultures (NIH3T3-Nsp1.non-trans) and control cells NIH3T3-neo were serum starved for 48 hours in the presence or absence of the PI3-kinase inhibitor LY294002, and annexin V (Clontech) apoptosis assay (Fig. 16). NIH3T3-Nsp1. The non-trans cells were morphologically normal and did not form tumors in nude mice, but they were the control NIH3T3-neo.cellIt was more resistant to apoptosis induced by growth factor withdrawal. This small but significant increase in apoptosis resistance was attenuated by the PI3 kinase inhibitor LY294002. In vector control cells that do not express Nsp1 (SEQ ID NO: 2), LY294002 itself further induced apoptosis. In the Nsp1 (SEQ ID NO: 2) transformation subline there was almost complete protection from serum starvation-induced apoptosis, but this effect was not sensitive to treatment with PI3-kinase inhibitors. This dependence of Nsp1 (SEQ ID NO: 1) on PI3-kinase at low levels would be located downstream of Nsp1 (SEQ ID NO: 1) PI3-kinase. Contrary to the observation that growth factor independence at high Nsp1 levels is not inhibited by LY294002, it suggests that Nsp1 (SEQ ID NO: 1) stimulates an additional pathway independent of PI3-kinase. This PI3-kinase has already been reported to be necessary and sufficient for growth factor-mediated apoptosis resistance. Kulik et al., Mol. Cell Biol. 17: 1595-606 (1997); Parrizas et al., J. Biol. Chem. 272: 154-61 (1997); Vemuri et al., Development 122: 2529-37 (1996).
Materials and methods:
10 μg of control cells (NIH3T3-neo), non-transformed Nsp1 (SEQ ID NO: 2) expressing cells (NIH3T3-Nsp1.non-trans) and transformed sublines (Nsp1.-sub1.1 and Nsp1.-sub2.1) Serum starvation was performed for 48 hours with or without / ml LY294002. The presence of apoptotic cells was assayed on FACS using Annexin V-FITC (Clontech) according to manufacturer's instructions. Each cell line was tested in triplicate and showed the mean and standard deviation. In the transformation subline, Nsp1 (SEQ ID NO: 1) protected the cells from serum starvation-induced apoptosis.
[0127]
Example 7
PI3-kinase interaction
In order to determine whether Nsp1 (SEQ ID NO: 1) interacts with PI3-kinase, a GST fusion protein comprising a PI3-kinase N-terminal or C-terminal SH2 domain was designated as Nsp1 (SEQ ID NO: 2). Were incubated with transiently expressed EGF-treated or untreated COS cell lysates or controls (Fig 20). The C-terminal SH2 domain GST fusion protein interacted with Nsp1 (SEQ ID NO: 1). This interaction is dependent, at least in part, on the phosphorylation state of Nsp1 (SEQ ID NO: 1), increasing the amount of Nsp1 (SEQ ID NO: 1) interacting with PI3-kinase following EGF stimulation. all right. The N-terminal SH2 domain of PI3-kinase had no measurable interaction with Nsp1 (SEQ ID NO: 1).
Materials and methods:
COS cells transfected with pRK or Nsp1 (SEQ ID NO: 2) were treated with 25 ng / ml EGF or left untreated. Cells were lysed in CoIP buffer (supra) and incubated with PI3-kinase N-terminal or C-terminal SH2 domain-GST beads (UBI). Precipitated Nsp1 (SEQ ID NO: 1) was detected with an anti-flag antibody.
[0128]
Example 8
Use of PRO201, PRO308 or PRO309 as a hybridization probe
The following methods describe the use of nucleic acid sequences encoding PRO201, PRO308 or PRO309 polypeptides as hybridization probes.
DNA containing the coding sequence of full-length or mature PRO201 (eg Nsp1), PRO308 (eg Nsp2) or PRO309 (eg Nsp3) or a fragment thereof is homologous DNA (PRO201, PRO308 or PRO309 of human tissue cDNA library or human tissue genomic library). Can be used as probes for screening of naturally occurring mutants of
Hybridization and any libraryDNAThe filter containing is washed under the following high stringency conditions. Hybridization of radiolabeled PRO201 (eg, Nsp1), PRO308 (eg, Nsp2) or PRO309 (eg, Nsp3) derived probes to a filter consists of 50% formamide, 5 × SSc, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, The test was carried out at 42 ° C. for 20 hours in a solution of 2 × Denhardt's solution and 10% dextran sulfate. The filter was washed at 42 ° C. in 0.1 × SSC and 0.1% SDS aqueous solution.
full lengthNaturalDNA having the desired identity with the DNA encoding the sequence PRO201, PRO308 or PRO309 can then be identified using standard techniques known in the art.
[0129]
Example 9: Expression of PRO201, PRO308 or PRO309 in E. coli This example illustrates the preparation of non-glycosylated forms of PRO201, PRO308 or PRO309 by recombinant expression in E. coli.
The DNA sequence encoding PRO201, PRO308 or PRO309 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6) was first amplified using selected PCR primers. The primer must have a restriction enzyme site corresponding to the restriction enzyme site of the selected expression vector. Various expression vectors are used. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)), which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into a vector. The vector is preferably an antibiotic resistance gene, trp promoter, polyhis leader (including the first 6 STII codons, polyhis sequence, and enterokinase cleavage site), PRO201, PRO308 or PRO309 coding region, lambda transcription termination region, and contains the argU gene.
The ligation mixture is then used to transform selected E. coli using the method described in Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and then antibiotic resistant clones are selected. Plasmid DNA is isolated and confirmed by restriction analysis and DNA sequencing.
Selected clones can be grown overnight in liquid media such as LB broth supplemented with antibiotics. The overnight medium is then used for seeding the large-scale medium. The cells are then grown at optimal density during which the expression promoter is activated.
After further incubation for several hours, the cells can be collected and centrifuged. The cell pellet obtained by centrifugation is solubilized using various reagents known in the art, and the solubilized PRO201, PRO308 or PRO309 protein is subjected to a condition for tightly binding the protein using a metal chelation column. Purified with.
[0130]
Alternatively, the expression in E. coli may be usually performed by the following method. DNA sequences encoding PRO201 (eg, Nsp1), PRO308 (eg, Nsp2) or PRO309 (eg, Nsp3) were amplified using initially selected PCR primers. The primer must have a restriction enzyme site corresponding to the restriction enzyme site of the selected expression vector. Various expression vectors are used. An example of a suitable vector is pBR322 (derived from E. coli; Bolivar et al., Gene, 2:95 (1977)), which contains genes for ampicillin and tetracycline resistance. Vectors are designed with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into a vector. The vector is preferably an antibiotic resistance gene, trp promoter, polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), PRO201, PRO308 or PRO309 coding region, lambda transcription termination region, and contains the argU gene.
The ligation mixture is then used for transformation of the selected E. coli using the method described in Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and then antibiotic resistant clones are selected. Plasmid DNA is isolated and confirmed by restriction analysis and DNA sequence analysis.
Selected clones can be grown overnight in liquid media such as LB broth supplemented with antibiotics. The overnight medium is then used for seeding the large-scale medium. The cells are then grown at optimal density during which the expression promoter is activated.
After further incubation for several hours, the cells were collected and centrifuged. The cell pellet obtained by centrifugation is solubilized using various reagents known in the art, and the solubilized PRO201, PRO308, or PRO309 protein is used under conditions for tightly binding the protein using a metal chelation column. Purified with.
[0131]
Example 10
Expression of PRO201, PRO308 or PRO309 in mammalian cells
This example illustrates the preparation of glycosylated forms of PRO201, PRO308 or PRO309 by recombinant expression in mammalian cells.
The vector pRK5 (see EP 307,247 issued March 15, 1989) was used as an expression vector. In some cases, PRO201, PRO308, or PRO309 DNA is ligated to pRK5 with a selected restriction enzyme, and PRO201, PRO308, or PRO309 DNA is inserted using a ligation method such as that described in Sambrook et al., Supra. The resulting vector is called pRK5-PRO201, PRO308 or PRO309.
In one embodiment, the selected host cell can be a 293 cell. Human 293 cells (ATCC CCL 1573) were grown and confluent in tissue culture plates in media such as DMEM supplemented with fetal bovine serum and optionally nourishing ingredients or antibiotics. About 10 μg of pRK5-PRO201, PRO308 or PRO309 DNA is mixed with about 1 μg of DNA encoding VA RNA gene [Thimmappaya et al., Cell, 31: 543 (1982))] and 500 μl of 1 mM Tris-HCl, 0.1 mM Dissolved in EDTA, 0.227M CaCl2. To this mixture, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, and 1.5 mM NaPO 4 were added dropwise and a precipitate formed at 25 ° C. for 10 minutes. The precipitate was suspended and added to 293 cells and allowed to settle for about 4 hours at 37 ° C. The culture medium was aspirated and 2 ml of 20% glycerol in PBS was added for 30 seconds. The 293 cells were then washed with serum free medium, fresh medium was added and the cells were incubated for about 5 days.
[0132]
Approximately 24 hours after transfection, the culture medium was removed and replaced with culture medium (only) or culture medium containing 200 μCi / ml 35S-cysteine and 200 μCi / ml 35S-methionine. After 12 hours of incubation, the conditioned medium was collected, concentrated with a spin filter and added to a 15% SDS gel. The treated gel was dried and exposed to the film for a selected time manifesting the presence of PRO201, PRO308 or PRO309 polypeptide. The medium containing the transformed cells was further incubated (in serum-free medium) and the medium was tested in the selected bioassay.
In an alternative technique, PRO201, PRO308 or PRO309 is transiently introduced into 293 cells using the dextran sulfate method described in Somparyac et al., Proc. Natl. Acad. Sci., 12: 7575 (1981). The 293 cells are grown to maximal density in a spinner flask and 700 μg of pRK5-PRO201, PRO308 or PRO309 DNA is added. The cells were first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate was incubated on the cell pellet for 4 hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and reintroduced into a spinner flask containing tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml bovine transferrin. After about 4 days, the conditioned medium was centrifuged and filtered to remove cells and cell debris. Samples containing expressed PRO201, PRO308 or PRO309 were then concentrated and purified by selected methods such as dialysis or column chromatography.
[0133]
In other embodiments, PRO201, PRO308 or PRO309 can be expressed in CHO cells. pRK5-PRO201, PRO308 or PRO309 is a CaPO₄Alternatively, CHO cells can be transfected using known reagents such as DEAE-dextran. Incubate the cell culture medium as described above, and culture medium (only) or³⁵It can be replaced with a medium containing a radioactive label such as S-methionine. After identifying the presence of PRO201, PRO308, or PRO309 polypeptide, the culture medium may be replaced with a serum-free medium. Preferably, the medium is incubated for about 6 days and then the conditioned medium is collected. The medium containing the expressed PRO201, PRO308 or PRO309 can then be concentrated and purified for the selected method.
Further, the epitope tag PRO201, PRO308 or PRO309 may be expressed in the host CHO cell. PRO201, PRO308 or PRO309 was subcloned from the pRK5 vector. The subclone insert can then be subjected to PCR and fused to a frame with a selected epitope tag such as a poly-his tag in a baculovirus expression vector. The poly-his tag PRO201, PRO308 or PRO309 insert can then be subcloned into an SV40 derived vector containing a selectable marker such as DHFR for selection of stable clones. Finally, CHO cells were transfected with the SV40 derived vector (as described above). In order to confirm expression, labeling may be performed as described above. The culture medium containing the expressed poly-his tag PRO201, PRO308 or PRO309 can then be concentrated and purified by selected methods such as Ni2 + -chelate affinity chromatography.
[0134]
Example 11
Expression of PRO201, PRO308 or PRO309 in yeast
The following method describes recombinant expression of PRO201, PRO308 or PRO309 in yeast.
First, a yeast expression vector is produced for intracellular production or secretion of PRO201, PRO308 or PRO309 from the ADH2 / GAPDH promoter. DNA encoding PRO201, PRO308 or PRO309, a selected signal peptide and a promoter are inserted into appropriate restriction enzyme sites of the selected plasmid to direct intracellular expression of PRO201, PRO308 or PRO309. For secretion, a plasmid selected for DNA encoding PRO201, PRO308 or PRO309, DNA encoding the ADH2 / GAPDH promoter, yeast alpha factor secretion signal / leader sequence, and (if necessary) PRO201, PRO308 or PRO309. Can be cloned together with a linker sequence for the expression of
Yeast strains such as yeast strain AB110 can then be transformed with the above expression plasmid and cultured in the selected fermentation medium. Transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gel with Coomassie blue staining.
Subsequently, recombinant PRO201, PRO308 or PRO309 can be isolated and purified by removing yeast cells from the fermentation medium by centrifugation and then concentrating the medium using a selected cartridge filter. The concentrate containing PRO201, PRO308 or PRO309 may be further purified using a selected column chromatography resin.
[0135]
Example 12
Expression of PRO201, PRO308 or PRO309 in insect cells infected with baculovirus
The following method describes recombinant expression of PRO201, PRO308 or PRO309 in baculovirus infected insect cells.
PRO201, PRO308 or PRO309 was fused upstream of the epitope tag contained in the baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (such as the Fc region of IgG). Various plasmids can be used, including plasmids derived from commercially available plasmids such as pVL1393 (Navogen). Briefly, PRO201, PRO308 or PRO309 or a specific portion of PRO201, PRO308 or PRO309 (such as a sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5 ′ and 3 ′ regions. Is done. The 5 'primer may include flanking (selected) restriction enzyme sites. The product is then digested with selected restriction enzymes and subcloned into an expression vector.
Recombinant baculoviruses can be obtained by co-transfecting the above plasmid and BaculoGold ™ viral DNA (Pharmingen) into Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). Created. After incubation at 28 ° C. for 4-5 days, the released virus was collected and used for further amplification. Viral infection and protein expression were performed as described in O'Reilley et al., Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford University Press (1994).
[0136]
Subsequent to PCR amplification, the corresponding coding sequence was transformed into a baculovirus expression vector (pb.PH.IgG and polyHis tag for IgG fusion).proteinPH. His. c) Subcloning the vector and Baculogold® baculovirus DNA (Pharmingen) into 105 Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711) with lipofectin (Gibco BRL) And co-transfected. pb. PH. IgG andpb. PH. HisIs a modification of the commercially available baculovirus expression vector pV1393 (Pharmingen) and has a polylinker region modified to include a His or Fc tag sequence. Cells were grown in Hink's TNM-FM medium supplemented with 10% FBS (Hyclone). The cells were incubated for 5 days at 28 ° C. Supernatants were collected and used for initial virus amplification at approximately 10 infection efficiency (MOI) by Sf9 cell infection in Hink's TNM-FH medium supplemented with 10% FBS. Cells were incubated for 3 days at 28 ° C. The supernatant is collected and expression of the construct in the baculovirus expression vector is performed using 1 ml of supernatant of 25 mL of Ni-NTA beads (QIAGEN) for histidine tag protein or Protein A Sepharose CL-4B beads for IgG tag protein ( Pharmacia) and then by SDS-PAGE analysis compared to known concentrations of protein standards by Coomassie blue staining.
The first amplified virus supernatant was used for infection of spinner medium (500 ml) of Sf9 cells grown in ESF-92 medium (Expression System LLC) at an MOI of about 0.1. The cells were incubated at 28 ° C. for 3 days. The supernatant was collected and filtered. Batch binding and SDS-PAGE were repeated as necessary until expression of the spinner medium was confirmed.
[0137]
The expressed poly-his tag PRO201, PRO308 or PRO309 is then purified, for example, by Ni2 + -chelate affinity chromatography as follows. Extracts were prepared from virus-infected recombinant Sf9 cells as described in Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9 cells were washed and sonicated buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂Resuspended in 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl) and sonicated twice on ice for 20 seconds. The sonicated product is clarified by centrifugation, and the supernatant is diluted 50-fold with loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8), 0.45FmFiltered with a filter. Ni²⁺-An NTA agarose column (commercially available from Qiagen) was prepared with a total volume of 5 mL, washed with 25 mL water and equilibrated with 25 mL loading buffer. The filtered cell extract was loaded onto the column at 0.5 mL per minute. Column A is the point where fraction collection begins.₂₈₀Washed with loading buffer to baseline. The column was then washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. A₂₈₀After reaching baseline again, the column was developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer. 1 mL fractions were collected and Ni complexed with SDS-PAGE and silver staining or alkaline phosphatase (Qiagen)²⁺-Analyzed by Western blot with NTA. Eluted His₁₀-Fractions containing the tag PRO201, PRO308 or PRO309 were pooled and dialyzed against loading buffer.
Alternatively, purification of IgG tag (or Fc tag) PRO201, PRO308, or PRO309 can be performed using known chromatographic techniques including, for example, protein A or protein G column chromatography.
[0138]
Alternatively, the modified baculovirus method may be used for High5 cell uptake. In this method, DNA encoding the desired sequence may be amplified in an appropriate system such as Pfu (Stratagene) or fused upstream (5′-) of the epitope tag contained in the baculovirus expression vector. Good. Such epitope tags include poly-His tags and immunoglobulin tags (such as the Fc region of IgG). A variety of plasmids can be used, including plasmids derived from commercially available plasmids such as pIE-1 (Novagen). The pIE-1 and pIE-2 vectors are designed for constitutive expression of the recombinant protein from the baculovirus ie1 promoter in stably transformed insect cells (1). This plasmid differs only in the direction of multiple cloning sites, ie in uninfected insect cells.1Includes all promoter sequences known to be important for mediator gene expression as well as the hr5 enhancer component. pIE-1 and pIE-2 contain the IE translation initiation site and can be used to produce fusion proteins. In brief,Desired sequence or sequenceDesired portions (such as sequences encoding the extracellular domain of a transmembrane protein) are amplified by PCR with primers complementary to the 5 'and 3' regions. The 5 'primer may introduce flanking (selected) restriction enzyme sites. The product is then digested with the selected restriction enzyme and subcloned into an expression vector. For example,pIE1 - 1The Fc region of human IgG (pb.PH.IgG) orDesiredThe sequence may contain an 8 histidine (pb.PH.His) tag downstream (3′-).
[0139]
Hi5 cells were grown to 50% confluence under conditions of 27 ° C., no CO 2, no hexane / strept. 30 μg for each 150 mm plateArrayPIE-based vector containing 1 ml Ex-cell medium (medium: Ex-cell 401 + 1/100 L-Glu JRH Biosciences # 14401-78P (note: this medium is lightly sensitive)) in a separate tube, 100 μl of cellfectin (CellFECTIN (Gibco BRL # 10362-010) (mixed by vortex)) was mixed with 1 ml of Ex-cell medium. The two solutions were mixed and incubated for 15 minutes at room temperature. 8 ml Ex-cell medium was added to 2 ml DNA / cellfectin mixture and layered on Hi5 cells washed once with Ex-cell medium. The plates were then incubated in the dark at room temperature. The DNA / cellfectin mixture is then aspirated and the cells are washed once with Ex-cells.WashingExcess cellfectin was removed. 30 ml of fresh Ex-cell medium was added and the cells were incubated at 28 ° C. for 3 days. Collect the supernatant and baculovirusExpressionIn vectorArrayIs well known by batch binding of 1 ml supernatant to 25 mL Ni-NTA beads (QIAGEN) for histidine-tagged protein or Protein A Sepharose CL-4B beads (Pharmacia) for IgG-tagged protein followed by Coomassie blue staining Measured by SDS-PAGE analysis in comparison to protein standards at different concentrations.
Conditioned medium (0.5-3 L) from transfected cells was collected by removing the cells by centrifugation and filtering through a 0.22 micron filter. For poly-His tag creation,Protein containing sequenceWas purified using a Ni-NTA column (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM. Conditioned medium was applied to a 6 ml Ni-NTA column equilibrated with 20 mM Hepes, pH 7.4 buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml / min.48Pumped at ° C. After loading, the column was further washed with equilibration buffer and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted with 25 ml G25 Superfine (Pharmacia) in storage buffer, pH 6.8, containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, at −80 ° C. Stored in
Protein immunoadhesin (Fc-containing) constructs were purified from conditioned media as follows. The conditioned medium was loaded onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into a tube containing 275 mL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted in the storage buffer described above for the poly-His tag protein.ArrayThe homogeneity can be assessed by SDS polyacrylamide gel and N-terminal amino acid sequencing by Edman degradation and other analytical techniques as desired or required.
[0140]
Example 13
Preparation of antibodies that bind to PRO201, PRO308 or PRO309
  This example illustrates the preparation of monoclonal antibodies that can specifically bind to PRO201, PRO308, or PRO309.
  Techniques for the production of monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used are purified PRO201, PRO308 or PROIncluding 309Fusion proteins, including cells expressing recombinant PRO201, PRO308 or PRO309 on the cell surface. The selection of the immunogen can be made without undue experimentation by one skilled in the art.
  Mice such as Balb / c are immunized with PRO201, PRO308 or PRO309 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally at 1-100 micrograms. Alternatively, the immunogen may be emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind footpad. Immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice are boosted with additional immunization injections. Serum samples from retroorbital hemorrhage may be taken periodically from mice for testing in an ELISA assay for detection of PRO201, PRO308 or PRO309 antibodies.
[0141]
After a suitable antibody titer has been detected, animals “positive” for antibodies can be given a final infusion of PRO201, PRO308 or PRO309 intravenous injection. Three to four days later, the mice were sacrificed and the spleen was removed. The spleen cells were then (using 35% polyethylene glycol), P3X63AgU. Fused to 1st selected mouse myeloma cell line. Hybridoma cells were generated by fusion and then plated on 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit the growth of non-fused cells, myeloma hybrids, and spleen cell hybrids. Hybridoma cells are screened in an ELISA for reactivity against PRO201, PRO308 or PRO309. The determination of “positive” hybridoma cells that secrete monoclonal antibodies to the desired PRO201, PRO308 or PRO309 is within the common general knowledge.
Positive hybridoma cells are injected intraperitoneally into syngeneic Balb / c mice to produce ascites containing anti-PRO201, anti-PRO308 or anti-PRO309 monoclonal antibodies. Alternatively, hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of monoclonal antibodies produced in ascites can be performed using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the affinity of antibody for protein A or protein G can also be used.
[0142]
Example 14
Gene amplification
This example shows that the PRO201, PRO308 or PRO309 encoding gene is amplified in the genome of certain human lung, colon or breast cancer or cell lines. Amplification involves overexpression of the gene product and exhibits binding specificity for at least two different antigens. thisCaseOf binding specificityon the other handIs for PRO201, PRO308 or PRO309 protein and has shown to be a useful target for therapeutic treatment in certain cancers such as colon, lung, breast and other cancers. The therapeutic agent may take the form of an antagonist of binding specificity for at least two different antigens. In this case, one of the binding specificities is a mouse-human chimera, humanized or human antibody against the PRO201, PRO308 or PRO309 encoding gene, for example binding specificities for at least two different antigens. In this case, one of the binding specificities is for a PRO201, PRO308 or PRO309 polypeptide.
The starting material for the screening is genomic DNA isolated from various cancers. DNA is quantitatively, eg, fluorescently accurate. As a negative control, DNA was isolated from 10 normal healthy individuals and pooled and used as an assay control for gene copy in healthy individuals (not shown). 5 'nuclease assays (eg TaqMan ™) and real-time quantitative PCR (eg ABI Prizm 7700 Sequence Detection System (trade name) (Perkin Elmer, applied Biosystems Division, Foster City, CA)) Was used to discover genes that were amplified in an automated manner. The results were used to determine whether the DNA encodes binding specificity for at least two antigens. In this case, one of the binding specificities is for PRO201, PRO308 or PRO309 and is overexpressed in the screened primary lung or colon cancer or cancer cell line or breast cancer cell line. Primary lung cancer was obtained from individuals with tumors of the type and stage shown in Table 1. A description of the abbreviations used to represent the primary tumors listed in Table 1 and the primary tumors and cell lines referenced throughout this example is given above.
[0143]
Taqman results were reported in delta (Δ) CT units. One unit corresponds to about 2 times amplification with respect to one PCR cycle or normal, 2 units corresponds to 4 times, 3 units corresponds to 8 times amplification, and so on. Quantification was obtained using TaqMan (trade name) fluorescent probes derived from primers and binding specificities for at least two different antigens. In this case, one of the binding specificities is for the PRO201, PRO308 or PRO309 encoding gene. The region of binding specificity is for at least two different antigens. In this case, one of the binding specificities is for PRO201, PRO308 or PRO309, which appears to contain the most unique nucleic acid sequence and does not have the most spliced introns, and primer and probe induction, eg 3-Primers and probes (forward, reverse and probe) used for preferred PRO201, PRO308 or PRO309 gene amplification for untranslated regions are as follows:
PRO201 (DNA30676)
30676.tm.f
5'-CGCAGACACCCTTCTTCACA-3 '(SEQ ID NO: 24)
30676.tm.r
5'-CGACTCCTTTGGTCTCTTCTGG-3 '(SEQ ID NO: 25)
30676.tm.p
5'-CCGGGACCCCCAGGTTTTTGC-3 '(SEQ ID NO: 26)
DNA40556:
40556.tm.f
5'-AGGGTCCTGCGTGGACTCT-3 '(SEQ ID NO: 27)
40556.tm.r
5'-TCCTGTTCTTCCTCAATGGAGAC-3 '(SEQ ID NO: 28)
40556.tm.p
5'-CCATCCCACCTGCTACATGCTCACC-3 '(SEQ ID NO: 29)
[0144]
The 5 'nuclease assay reaction is a fluorescent PCR-based technique that uses the 5' exonuclease activity of the Taq DNA polymerase enzyme for real-time amplification monitoring. Two oligonucleotide primers were used to generate amplicons typical for PCR reactions. A third oligonucleotide, or probe, was designed to detect the nucleic acid sequence located between the two PCR primers. The probe is non-extensible by Taq DNA polymerase enzyme and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. When the two dyes are located close together on the probe, the laser-induced emission from the reporter dye is quenched by the quenching dye. During the amplification reaction, the probe is cleaved in a template-dependent manner by the TAQ DNA polymerase enzyme. The resulting probe fragment dissociates into solution and the signal from the released reporter dye is not subject to the quenching effect from the second fluorophore. One molecule of reporter dye is labeled for each newly synthesized molecule, and detection of the non-quenched reporter dye provides the basis for quantitative interpretation of the data.
The 5 'nuclease method is performed on a real-time quantitative PCR instrument such as ABI Prism 7700TM sequence detection. The system consists of a temperature cycler, laser, charge coupled device (CCD) camera and computer. The system amplifies the sample in 96-well on a temperature cycler. During amplification, the laser-induced fluorescence signal is collected in 96 wells with an optical fiber cable and detected with a CCD. The system includes software for device execution and data analysis.
5 'nuclease assay data is initially expressed in Ct or boundary cycles. This is defined as the cycle in which the reporter signal accumulates beyond the fluorescent background. The Ct value isWhen comparing cancer DNA results with normal DNA resultsUsed as a quantitative measure of the relative number of speciation copies of a particular target sequence in a nucleic acid sample.
Table 1 lists the stages, T stage and N stage of the various primary tumors used to screen for the PRO201, PRO308 or PRO309 compounds of the present invention.
[0145]

[0146]
DNA preparation:
DNA was prepared from cultured cell lines, primary tumors, and normal human blood. Isolation was performed using purification kits, buffer sets and proteases, all from Quiagen, according to manufacturer's instructions and below.
Cell culture lysis:
Wash cells, 7.5x10 per chip⁸Trypsinized, pelleted by centrifugation at 1000 rpm for 5 minutes at 4 ° C., then washed with 1/2 volume PBS re-centrifuge. The pellet was washed 3 times and the suspended cells were collected and washed with 2 × PBS. The cells were then suspended in 10 mL PBS. Buffer C1 was equilibrated at 4 ° C. Quiagen protease # 19155 was diluted with 6.25 ml of cold ddH2O to a final concentration of 20 mg / ml and equilibrated at 4 ° C. 10 mL of G2 buffer was prepared by diluting Quiagen RNAse A stock (100 mg / ml) to a final concentration of 200 μg / ml.
Buffer C1 (10 mL, 4 ° C.) andddH 2 O (40 mL, 4 ° C.) was then added to 10 mL of cell suspension, mixed by inversion, and incubated on ice for 10 minutes. Cell nuclei were pelleted by centrifuging at 2500 rpm for 15 minutes at 4 ° C. in a Beckman swing bucket rotor. Discard the supernatant and vortex the nuclei with 2 mL buffer C1 (4 ° C.) and 6 mL ddH₂Suspended in O, and after 1 second, centrifuged at 2500 rpm for 15 minutes at 4 ° C. The nuclei were then resuspended in the remaining buffer using 200 μl per chip. Loose vortexing was applied while adding G2 buffer (10 ml) to the suspended nuclei. When buffer addition was complete, a strong vortex was applied for 30 seconds. Quiagen protease (200 μl prepared as above) was added and incubated at 50 ° C. for 60 minutes. Incubation and centrifugation were repeated until the lysate was clear (eg, incubated for an additional 30-60 minutes and pelleted at 3000 × g for 10 minutes at 4 ° C.).
[0147]
Preparation and lysis of solid human tumor samples:
Tumor samples were weighed and placed in a 50 ml conical tube and kept on ice. Processing was limited to less than 250 mg tissue per preparation (1 chip / preparation). Protease solution 6.25 ml cold ddH₂Freshly prepared by diluting in O to a final concentration of 20 mg / ml and stored at 4 ° C. G2 buffer (20 ml) was prepared by diluting DNAseA to a final concentration of 200 mg / ml (from a 100 mg / ml stock). Laminar flow to avoid aerosol inhalationTCTumor tissue was homogenized in 19 ml of G2 buffer for 60 seconds using a large polytron tip in the hood and kept at room temperature. 2L ddH each between samples₂The polytron was cleared by spinning with O for 2 × 30 seconds and then with G2 buffer (50 ml). If tissue was present on the generator chip, the device was disassembled and cleaned.
Quiagen protease (prepared for above, 1.0 ml) was added, then vortexed and incubated at 50 ° C. for 3 hours. Incubation and centrifugation were repeated until the lysate was clear (eg, incubated for an additional 30-60 minutes and pelleted at 3000 × g for 10 minutes at 4 ° C.).
[0148]
Human blood preparation and lysis:
Blood was drawn from healthy volunteers using a standard infectious agent protocol and citrated to a 10 ml sample per chip. Quiagen protease 6.25ml cold ddH₂Freshly prepared by diluting in O to a final concentration of 20 mg / ml and stored at 4 ° C. DNAseA from a 100 mg / ml stock to a final concentration of 200μ gG2 buffer by diluting to / ml-Prepared. Blood (10 ml) is placed in a 50 ml conical tube, 10 ml C1 buffer and 30 ml ddH₂O (both equilibrated at 4 ° C.) was added, inverted and mixed and held on ice for 10 minutes. The nuclei were pelleted with a Beckman swinging bucket rotor at 2500 rpm at 4 ° C. for 15 minutes and the supernatant discarded. While vortexing, the nuclei were suspended in 2 ml C1 buffer (4 ° C.) and 6 ml ddH 2 O (4 ° C.). Vortexing was repeated until the pellet was white. The nuclei were then suspended in the remaining buffer using a 200 μl tip. Vortex gently with addition of G2 buffer (10 ml) to the suspension nuclei and then vortex vigorously for 30 seconds. Quiagen protease was added (200 μl) and incubated at 50 ° C. for 60 minutes. Incubation and centrifugation were repeated until the lysate was clear (eg, incubated for an additional 30-60 minutes and pelleted at 3000 × g for 10 minutes at 4 ° C.).
[0149]
Purification of clarified lysate:
(1) Isolation of genomic DNA:
Genomic DNA was equilibrated with 10 ml QBT buffer (1 sample per maximum chip preparation). QF elution buffer was equilibrated at 50 ° C. The sample was vortexed for 30 seconds, then loaded onto the equilibration tip and drained by gravity. The chip was washed with 2x15 ml QC buffer. DNA was eluted in a 30 ml silanized autoclave 30 ml Cortex tube with 15 ml QF buffer (50 ° C.). Isopropanol (10.5 ml) was added to each sample, the tube was covered with paraffin and mixed by inversion repeatedly until the DNA precipitated. Samples were pelleted by centrifugation for 10 minutes at 15,000 rpm at 4 ° C. in an SS-34 rotor. The pellet position was marked, the supernatant was discarded, and 10 ml of 70% ethanol (4 ° C.) was added. The sample was pelleted again by centrifugation at 10,000 rpm for 10 minutes at 4 ° C. in an SS-34 rotor. The pellet position was marked and the supernatant was discarded. The tubes were then placed on each side of the drying shelf and allowed to dry at 37 ° C. for 10 minutes, taking care to avoid over-drying.
After drying, the pellet was dissolved in 1.0 ml TE (pH 8.5) and placed at 50 ° C. for 1-2 hours. The sample was kept at 4 ° C. overnight to continue dissolution. The DNA solution was then transferred to a 1.5 ml tube equipped with a 26 gauge needle on a tuberculin syringe. The transition was repeated 5x to shear the DNA. The sample was then placed at 50 ° C. for 1-2 hours.
[0150]
(2) Preparation for genomic DNA quantification and gene amplification assay:
The DNA level in each tube was diluted 1:20 (5 μl DNA + 95 μl ddH₂The standard A260, A280 spectra at O) were quantified using a Beckman DU640 spectrophotometer 0.1 ml quartz cuvette. The A260 / A280 ratio was in the range of 1.8-1.9. Each DNA sample was then diluted to about 200 ng / ml in TE (pH 8.5). If the initial material was at a high concentration (about 700 ng / μl), the material was allowed to stand for several hours until resuspended at 50 ° C.
The diluted material (20-600 ng / ml) was then subjected to fluorescent DNA quantification with the manufacturer's instructions modified as follows. This was done by warming the Hoeffer DyNA Quant 200 fluorometer for about 15 minutes. Hoechst dye working solution (# H33258, 10 μl, prepared within 12 hours of use) was diluted in 100 ml 1 × TNE buffer. A 2 ml cuvette was filled with the fluorometer solution and placed on the machine, and the machine was zeroed. pGEM3Zf (+) (2 μl, lot # 360851026) was added to a 2 ml fluorometer solution and calibrated to 200 units. An additional 2 μl of pGEM3Zf (+) DNA was then tested and the reading was confirmed at 400 +/− 10 units. Each sample was then read at least three times. When 3 samples were found to be within 10% of each other, they were averaged and this value was used as the quantification value.
The concentration determined by fluorescence measurement was then determined for each sample by ddH.₂Used to dilute in O to 10 ng / μlμ. This was done on all template samples simultaneously for a single TaqMan plate assay and with enough material to perform a 500-1000 assay. Samples were tested in triplicate with Taqman (trade name) primers and probes and are on a single plate with normal human DNA for both B-actin and GAPDH and no template control. Although diluted samples were used, the CT value of normal human DNA subtracted from the test DNA was +/- 1 CT. Diluted, lot-qualified genomic DNA was stored at −80 ° C. in 1.0 ml aliquots. Aliquots subsequently inherited and used for amplification assays were stored at 4 ° C. Each 1 ml aliquot is sufficient for 8-9 plates or 64 tests.
[0151]
Gene amplification assay:
The PRO201, PRO308 or PRO309 compounds of the present invention are screened in the following primary tumors and the resulting ΔCt values are reported in Table 2.

[0152]
DNA 30676 (SEQ ID NO: 2) was re-tested for tumors selected from the initial screening above with framework mapping. FIG. 18 and Table 3 show the chromosomal mapping of the framework markers used in this example. Framework markers were located approximately every 20 megabase and were used for control aneuploidy.
DNA 30676 (SEQ ID NO: 2) was retested with epicenter mapping. The markers shown in Table 4 are used to assess the relative amplification in the immediate vicinity of chromosome 19 where DNA 30676 (SEQ ID NO: 2) is located immediately (in the genome) and the corresponding molecule is located. The distance between individual markers is measured in centimeters (cR), which is a radiolysis unit and is approximately equal to the 1% chance of degradation between the two markers. 1 cR is very roughly equal to 20 kilobases. Marker SHGC-35441 is the marker found closest to the location of chromosome 19 to which DNA 30676 is mapped.
[0153]

The ΔCt values of the above framework markers along chromosome 19 for PRO201 are shown for the selected tumors in Table 5.
[0154]

[0155]
Table 6 shows the ΔCt values for the results of epicenter mapping for DNA 30676 (SEQ ID NO: 2) and shows the relative amplification in the region closer to the actual position of DNA 30676 (SEQ ID NO: 2) along chromosome 19.

[0156]
Discussion and conclusion:
VariouslungAnd ΔCt values for DNA30676 (SEQ ID NO: 2) and DNA40556 (SEQ ID NO: 20) in colon tumors are reported in Table 2. ΔCt> 1 is typically used as a threshold for amplification scoring, which represents twice as many gene copies. Table 2 shows: Primary lung tumors LT1a, LT3, LT11, LT13, LT15, LT17, LT19; primary colon tumors CT2, CT3, CT8, CT10, CT12, CT14, CT15, CT16, CT17, CT1, CT4, CT5, CT6, CT7, CT9, CT11, CT18; significant amplification of DNA 30676 generated in lung tumor cell lines Calu-1, Calu-6, H157, H441, SKMES1, H522 and H810; and colon tumor cell lines Colo-320 and Coo-205 Indicates. Table 2 further shows the significant expression of DNA40556 in primary lung tumors LT1a, LT8, LT11, LT15, LT17, LT19; primary colon tumors CT14, CT5, CT11 and lung tumor cell line A549.
The ΔCt and mean ΔCt values of DNA30676 (SEQ ID NO: 2) correspond to primary lung tumors: 1.10, 2.05, 1.52, 2.13, 2.58, 1.63, 3. 43; primary colon tumors are: 3.81, 2.3, 1.97, 3.01, 2.76, 3.34, 2.58, 2.46, 2.64, 1.57 3.39, 2.95, 2.81, 2.77, 2.63, 3.0, 2.23; applicable to lung tumor cell lines: 2.37, 2.61, 2.39 2.07, 3.01, 2.91 and 2.07; colon tumor cell lines: 1.95 and 1.26. The ΔCt and mean ΔCt values for DNA40556 are 1.03, 1.14, 1.33, 1.18, 1.00 and 1.42 for primary lung; 12, 1.00 and 1.135; lung tumor cell line 1.26.
The ΔCt and average ΔCt values of DNA30676 (SEQ ID NO: 2) listed in the above paragraph are:
2.14, 4.14, 2.87, 4.38, 5.98, 3.09, 10.70 for primary lung tumors: 14.03, 4.92, 3.92, 8.70 for primary colon tumors. 06, 6.77, 10.13, 5.98, 5.50, 6.23, 2.97, 10.48, 7.73, 7.01, 6.82, 6.19, 8.0, 4.69; 5.17, 6.10, 5.24, 4.20 for lung tumor cell lines, 8.06, 7.51, 4.20; 3.86, 2.40 for colon tumor cell lines It represents an increase in gene copy relative to normal tissue. For DNA40556, these values are 2.04, 2.20, 2.51, 2.27, 2.0, 2.68 for lung tumors, 2.17, 2.0, 2.20 for colon tumors. Represents an increase in gene copy relative to normal tissue of 2.39.
[0157]
Amplification is the framework of DNA 30676 (SEQ ID NO: 2) in primary lung tumors LT3, LT15, LT16, LT17, LT18; and primary colon tumors CT2, CT3, CT8, CT9, CT10, CT12, CT14, CT15, CT16, CT17. Confirmed by mapping. The reported ΔCt values for primary lung tumors are 1.04, 1.43, 1.35, 1.51 and 1.22, and for primary colon tumors 2.81, 2.03, 1.39. 2.21, 1.93, 2.37, 1.27, 1.76 and 1.65 have been reported. For normal tissue, this is approximately 2.06 for lung tumors.,2.69,2.55, 2.85 and 2.32 fold increase, 7.01, 4.08, 2.62 for colon tumors,It represents an increase of 4.63, 3.81, 5.17, 2.41, 3.39, 3.14 times. Epicenter mapping of DNA3066 is shown in primary lung tumors LT3, LT13, LT15, LT16, LT18; and colon tumors CT1, CT2, CT3, CT4, CT5, CT6, CT7, CT8, CT9, Ct10, CT11, CT12, CT14, CT15 , CT16, CT17 and CT18 confirmed significant amplification. The reported ΔCt values for primary lung tumors are 1.87, 1.44, 1.99, 1.72 and 1.09, and primary colon tumors are 2.56, 3.51, 2.19, 3 .22, 2.83, 2.93, 2.43, 1.95, 2.72, 3.12, 3.26, 2.77, 2.88, 2.60, 2.61, 2.24 And a ΔCt of 2.48 and an average ΔCt value of 2.48. For normal tissuetheseAre 3.66, 2.71, 3.97, 3.29, 2.13 fold increase in gene copy for lung tumors and 5.90, 11.39, 4.56, 9.32 for colon tumors. 7.11, 7.62, 5.39, 3.86, 6.59, 8.69, 9.58, 6.82, 7.36, 6.06, 6.11, 4.72, 5 Represents a 58-fold increase in gene copy.
In contrast, amplification of the nearest framework marker (one, ie, excluding S50) (Table 5) or epicenter marker (Table 6) did not occur to a greater extent than DNA30676 (SEQ ID NO: 2). This strongly suggests that DNA 30676 (SEQ ID NO: 2) is a gene that causes amplification of a specific region on chromosome 19. Since amplification of DNA 30676 (SEQ ID NO: 2) occurs in various lung and colon tumors and cell lines (especially the colon), it is likely to play a significant role in tumor formation or growth. As a result, antagonists (eg, antibodies) to DNA 30676 (SEQ ID NO: 2) (ie, Nsp1, SEQ ID NO: 1) DNA 40575 (SEQ ID NO: 4) and DNA 61601 (SEQ ID NO: 6) have utility in cancer therapy. It is predicted.
[0158]
Example 15
In-situ hybrid formation
In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It is useful, for example, for identification of gene expression sites, analysis of transcript tissue distribution, identification and localization of viral infection, specific mRNA synthesis and chromosomal mapping.
In situ hybridization is performed by PCR according to the optimal variant of the protocol of Lu and Gillett, Cell Vision 1: 169-176 (1994).³³Performed with P-labeled riboprobe. Briefly, formalin-fixed, paraffin-embedded human tissue was sectioned, deparaffinized, deproteinized with proteinase K (20 g / ml) for 15 minutes at 37 ° C., and as described in Lu and Gillett, supra. In situ hybrid formation. [³³-P] UTP-labeled antisense riboprobe is generated from the PCR product and hybridized at 55 ° C overnight. The slides are immersed in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
³³P-riboprobe synthesis
6.0 μl (125 mCi)³³P-UTP (Amersham BF 1002, SA <2000 Ci / mmol) was speed-vacuum dried. Dry³³The following ingredients were added to the tube containing P-UTP:
2.0 μl of 5x transcription buffer
1.0 μl of DTT (100 mM)
2.0 μl of NTP mixture (2.5 mM: 10μ l; 10 mM GTP, CTP and ATP + 10 μl H₂O)
1.0 μl UTP (50 μM)
1.0 μl of Rnasin
1.0 μl DNA template (1 μg)
1.0 μl H₂O
1.0 μl RNA pomerase (T3 = AS, T7 = S, normal for PCR products)
[0159]
Tubes were incubated at 37 ° C. for 1 hour, 1.0 μl RQ1 DNase was added, and then incubated at 37 ° C. for 15 minutes. 90 μl TE (10 mM Tris pH 7.6 / 1 mM EDTA pH 8.0) was added and the mixture was pipetted onto DE81 paper. The remaining solution was loaded into a Microcon-50 ultrafiltration unit and spun using program 10 (6 minutes). The filtration unit was converted to a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 μl TE was added. 1 μl of final product was pipetted onto DE81 paper and counted with 6 ml of Biofluor II.
The probe was run on a TBE / urea gel. 1-3 μl probe or 5 μl RNA MrkIII was added to 3 μl loading buffer. After heating on a heating block to 95 ° C. for 3 minutes, the gel was immediately placed on ice. Gel wells were flushed, sample loaded and run at 180-250 volts for 45 minutes. The gel was wrapped with Saran wrap and exposed to an XAR film with a reinforcing screen for 1 hour to overnight in a −70 ° C. refrigerator.
[0160]
³³P-hybrid formation
Cryosection pretreatment
Slides were removed from the refrigerator, placed in an aluminum tray and thawed at room temperature for 5 minutes. The tray was placed in a 55 ° C. incubator for 5 minutes to reduce condensation. Slides were fixed in 4% paraformaldehyde in a steam hood for 5 minutes and washed with 0.5xSSC for 5 minutes at room temperature (25ml 20xSSC + 975ml SQ H₂O). 0.5 μg / ml proteinaseKAfter deproteinization for 10 minutes at 37 ° C. (12.5 μl of a 10 mg / ml stock in 250 ml preheated RNase-free RNase buffer), sections were washed with 0.5 × SSC for 10 minutes at room temperature. Sections were dehydrated in 70%, 95%, 100% ethanol for 2 minutes each.
Pretreatment of paraffin-embedded sections
Slide deparaffinized and SQ H₂Place in O and rinse twice with 2 × SSC for 5 minutes each at room temperature. Sections were 20 μg / ml proteinase K (500 μl 10 mg / ml in 250 ml RNase-free RNase buffer; 37 ° C., 15 minutes) —human embryo or 8 × proteinase K (100 μl in 250 ml RNase buffer, 37 ° C., 30 minutes) — Deproteinized with formalin tissue. Subsequent rinsing and dehydration with 0.5 × SSC was performed as described above.
Pre-hybridization
Slides were arranged in plastic boxes lined with Box buffer (4 × SSC, 50% formamide) -saturated filter paper. Tissues were coated with 50 μl of hybridization buffer (3.75 g dextran sulfate + 6 ml SQ H 2 O), vortexed, uncapped and heated in the microwave for 2 minutes. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 × SSC and 9 ml SQ H 2 O were added and the tissue was vortexed well and incubated at 42 ° C. for 1-4 hours.
Hybrid formation
1.0x10 per slide⁶The cpm probe and 1.0 μl tRNA (50 mg / ml stock) were heated at 95 ° C. for 3 minutes. Slides were chilled on ice and 48 μl of hybridization buffer was added per slide. After vortexing, 50 μl³³P mixture was added to 50 μl of prehybrid on slide. Slides were incubated overnight at 55 ° C.
Washing
Washing was performed for 2 × 10 minutes with 2 × SSC, EDTA at room temperature (400 ml 20 × SSC + 16 ml 0.25 M EDTA, Vf = 4 L) followed by RNase A treatment for 30 minutes at 37 ° C. (10 μg / ml in 250 ml RNase buffer 500 μl = 20 μg) / ml). Slide 2x10 minutes,2x SSC,Wash with EDTA at room temperature. Stringent wash conditions are as follows: 2 hours at 55 ° C., 0.1 × SSC, EDTA (20 ml 20 × SSC + 16 ml EDTA, Vf = 4 L).
[0161]
DNA 30676panc. In human tissue III. Shc (nsp-1) expression A comparable background with sense and antisense probes was observed in many tissues. The only sites where expression was found to be specific were fetal thymic medulla, fetal spleen, fetal small intestinal epithelium and new osteogenic bone cell regions. No specific signal was observed in the fetus and normal adult prostate. The fetal tissue was about 12-16 weeks of gestation. The oligos used were B-191D and B-191E.
[0162]
Colon tumors, fetal liver, and transfected cellssystemOf DNA30676 (SHChlog / nsp1) in Escherichia coli
The purpose of this experiment is to measure the expression of DNA30676 in colon adenocarcinoma. This DNA has been shown to be amplified in colon cancer. Expression was analyzed in control cell pellets and 10 colon cancers. The control cell pellet contains SHC transfected 293 cells and SW480 cells, which express SHC (cells have been submitted as H98-717).
Cell pellet testing makes this experiment problematic, as SHC transfected cells are positive for both sense and antisense probes. SW480 cells were negative for both probes.Colon cancerFor AS, only the AS probe was performed. A number of colon cancers showed slight expression, which was strongest in specimen 9727/98. However, from the cell pellet data, this signal is difficult to interpret in its entirety, and the signal seems insufficient to be called positive.
[0163]
The following oligonucleotides were used in the in situ hybridization described in this example.
B166A-GGATTCTAATACGACTCACTATAGGGCGCGGAGGCTGCTCTGGGGTAG (SEQ ID NO: 30)
B166B-CTATGAAATTAACCCTCACTAAAGGGATGTTGCCCTGGCTGGTCTTGA (SEQ ID NO: 31)
B166D-GGATTCTAATACGACTCACTATAGGGCATCTGCCTTGCCCCGAACGAG (SEQ ID NO: 32)
B166E-CTATGAAATTAACCCTCACTAAAGGGATCATCCAGAGCCCGCATCAGC (SEQ ID NO: 33)
A-322I-GGATTCTAATACGACTCACTATAGGGCAGATGTGGAAGACTGAGGCCT (SEQ ID NO: 34)
A-323J-CTATGAAATTAACCCTCACTAAAGGGAATATGTGCCAAATCTGCAGGCT (SEQ ID NO: 35)
[0164]
Deposit of materials
The following cell lines were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA (12301 Parklawn Drive, Rockville, MD, USA) (ATCC):
Material ATCC Deposit Number Deposit Date
DNA30676-1223 209567 December 23, 1997
DNA40575-1223 209565 December 23, 1997
DNA40556-1223 209566 December 23, 1997
DNA40554-1223 209564 December 23, 1997
DNA61601-1223 209713 March 31, 1998
[0165]
This deposit was made in accordance with the provisions of the Budapest Treaty and its regulations (Budapest Convention) on the international recognition of the deposit of microorganisms in the patent procedure. This ensures that the viable culture of the deposit is maintained for 30 years from the date of deposit. Deposits may be obtained from the ATCC in accordance with the terms of the Budapest Treaty and in accordance with the agreement between Genentech and ATCC, regardless of which is the first issue of the relevant U.S. patent or any Ensures that the progeny of the deposited culture are made available permanently and unrestricted at the time of publication of a US or foreign patent application, and is subject to 35 USC 122 and the Patent Office Commissioner Regulation (specifically 37 CFR of reference number 886OG638). Guarantees that the offspring will be made available to those who have been determined to have the rights in accordance with (including Section 1.14).
The assignee of this application agrees that if the deposited culture dies or is lost or destroyed when cultured under appropriate conditions, the material will be immediately replaced with the same other upon notification. To do. The availability of deposited materials is not to be considered a license to practice the present invention in violation of rights granted under any governmental authority in accordance with patent law.
The above written description is considered to be sufficient to enable one skilled in the art to practice the invention. The deposited embodiments are intended as an illustration of certain aspects of the invention, and since any functionally equivalent product is within the scope of the invention, the deposited product will limit the scope of the invention. It is not limited. The deposit of material herein does not admit that the written description contained herein is insufficient to allow implementation of any aspect of the present invention, including its best mode, and that It is not to be construed as limiting the scope of the claims for the particular illustrations represented. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
[Brief description of the drawings]
FIG. 1 shows the nucleic acid sequence of DNA30676 (SEQ ID NO: 2) and the encoded amino acid sequence of the native sequence PRO201 (Nsp1) polypeptide (SEQ ID NO: 1).
[FIG. 2] A diagram showing the nucleic acid sequence of DNA40575 (SEQ ID NO: 4) and the encoded amino acid sequence of the native sequence PRO308 (Nsp2) polypeptide (SEQ ID NO: 3).
[FIG. 3] A diagram showing the nucleic acid sequence of DNA61601 (SEQ ID NO: 6) and the encoded amino acid sequence of the native sequence PRO309 (Nsp3) polypeptide (SEQ ID NO: 5).
[FIG. 4A-C] Full-length DNA 30676 (SEQ ID NO: 2), DNA 40575 (SEQ ID NO: 4) and DNA 61601 (SEQ ID NO: 6) of the present invention were used for isolation of nucleic acid sequences (LIFESEQ® database, Incyte Pharmaceuticals) , Palo Alto, Ca), 1328938 (SEQ ID NO: 13), 104191 (SEQ ID NO: 14) and 165811 (SEQ ID NO: 15).
[FIG. 5A] This is a figure showing the oligonucleotide sequences (SEQ ID NO: 7, SEQ ID NO: 8) used for the isolation of DNA30676 (SEQ ID NO: 2).
[FIG. 5B] It is a figure showing the oligonucleotide sequences (SEQ ID NO: 9, SEQ ID NO: 10) used for the isolation of DNA40575 (SEQ ID NO: 4).
[FIG. 5C] A diagram showing oligonucleotide sequences (SEQ ID NO: 11, SEQ ID NO: 12) used for the isolation of DNA61601 (SEQ ID NO: 6).
FIG. 6A illustrates a comparison of various domains between Nsp1 (SEQ ID NO: 1), Nsp2 (SEQ ID NO: 3) and Nsp3 (SEQ ID NO: 5).
FIG. 6B shows the actual sequences themselves, with gaps inserted as necessary to maximize the degree of identity between the three sequences.
[FIG. 7] A diagram showing the sequence of Nsp1 (SEQ ID NO: 1), in which the SH2 region is bold, and the proline and serine of the P / S region are indicated by one and two underlines, respectively.
[FIG. 8] A diagram showing a comparison between Nsp1 (SEQ ID NO: 1) and human Shc (SEQ ID NO: 16), Sck (SEQ ID NO: 17), and Fes (SEQ ID NO: 18).
[FIG. 9A] Nsp1 (SEQ ID NO: 2) is significantly increased in human fetal liverExpressionHowever, Northern blots showing that Nsp2 (SEQ ID NO: 4) and Nsp3 (SEQ ID NO: 6) are more widely expressed.
[FIG. 9B] It is a diagram showing the expression of two Nsp3 transcripts in hematopoietic tissues.
FIG. 10A-10C. 10A and 10C were Western blots and blotted as indicated by anti-flag immunoprecipitation with anti-flag, anti- (P) Tyr or anti-Cas antibodies. In 10B, anti-EGF receptor (CalBiochem) immunoprecipitation was blotted as indicated by anti-flag or anti- (P) Tyr antibody.
[FIG. 11] Anti-Flag or anti-p130^CasWestern blot showing immunoprecipitation at and anti- (P) TyrAbPY-20 or anti-p130^CasIt is a figure which shows the blot in.
[Fig 12A] Western blot showing reduced phosphorylation of Nsp1 (SEQ ID NO: 1) upon insulin treatment.
[FIG. 12B] A Western blot purified by stripping the FIG. 12A blot, showing anti-p130^CasThe 130 kD protein is actually p130^CasIt was confirmed that.
FIG. 13 shows Western blots of various Nsp1 mutants, which were transfected with COS cells, treated with EGF, and immunoprecipitated with anti-FlagAb, lysates of anti- (P) Tyr. Anti-p130^CasAlternatively, Western blotted with anti-flagAb.
[FIG. 14] FIG. 14A is a photomicrograph of an ultrathin section of a retrovirus-infected vector MSCVNIH3T3 cell, and 14B is a NI3T3 cell transfected with Nsp1 (SEQ ID NO: 2). 14C is a micrograph of ultrathin sections of four weak tumors that were fixed, blocked, and cross-section stained with hematoxylin and eosin.
[Figure 15] Vector controlled transfection and Nsp1. sc1,. sc2 and. Figure 3 is a bar graph comparing the tumor size of sc3 cells.
[FIG. 16] Transformation subclone Nsp. sub1.1, Nsp. sub2.1,Of non-transformed cell cultures (NIH3T3-Nsp1.non-trans) and control cells NIH3T3-neogrowthfactorhungerIt is a bar graph which shows the apoptosis tolerance of NIH3T3.
[Fig 17] EGF transfected with pRK or Nsp1Process withFIG. 5 shows a Western blot of treated or untreated COS cells, which were lysed in CoIP buffer and incubated with pI3-kinase N-terminal or C-terminal SH2 domain-GST beads (UBI). Precipitated Nsp1 was detected with anti-flag antibody.
FIG. 18 is a graphic representation of chromosome 19 showing a rough approximate mapping of DNA 30676 (SEQ ID NO: 2) in the human genome.
[Fig19] VariouslungAnd DNA40556 (SEQ ID NO: 20), a splicing variant of DNA30676 (SEQ ID NO: 2) that is amplified in colon tumors.
[FIG. 20] A diagram showing comparison of protein sequences encoded by Nsp1 (SEQ ID NO: 2), Nsp2 (SEQ ID NO: 4) and DNA40556 (SEQ ID NO: 20).
[Sequence Listing]

Claims (30)

(A) DNA encoding a polypeptide comprising the sequence of amino acids 1 to 576 of SEQ ID NO: 1 , or (b) one to several substitutions, deletions, additions or insertions in the base sequence of the DNA of (a) And an isolated nucleic acid comprising DNA encoding a polypeptide that inhibits cellular apoptosis . The isolated nucleic acid of claim 1, comprising DNA encoding a polypeptide comprising amino acid residues 1 to 576 of SEQ ID NO: 1 . (A) DNA encoding the same mature polypeptide encoded by the cDNA of ATCC deposit no. An isolated nucleic acid comprising DNA encoding a polypeptide comprising substitutions, deletions, additions or insertions and which inhibits apoptosis of cells .   A vector comprising the nucleic acid according to claim 1. 5. The vector of claim 4 , operably linked to a control sequence recognized by a host cell transformed with the vector. A host cell comprising the vector of claim 5 . The host cell according to claim 6 , wherein the cell is a mammal. The host cell according to claim 7 , wherein the cell is a CHO cell. The host cell according to claim 6 , wherein the cell is a prokaryotic organism. The host cell according to claim 9 , wherein the cell is an E. coli cell. The host cell according to claim 6 , wherein the cell is a yeast cell. The host cell according to claim 11 , wherein the cell is Saccharomyces cerevisiae. An isolated native sequence polypeptide comprising amino acid residues 1 to 576 of SEQ ID NO: 1 . An isolated native sequence polypeptide encoded by a nucleotide deposited under ATCC accession number 209567. The host cell of claim 6, and cultured under conditions suitable for expression of the polypeptide, method for producing the polypeptide of claim 13 or 14 comprising recovering the polypeptide from the cell culture medium. An antibody that specifically binds to the polypeptide of claim 13 or 14 . The antibody according to claim 16 , wherein the antibody is a monoclonal antibody. 18. The antibody of claim 17 , which induces death of cells that overexpress the polypeptide of claim 13 or 14 . The antibody according to claim 18 , wherein the cell is a tumor cell. The antibody according to claim 17 , which is labeled. 21. The antibody of claim 20 , wherein the antibody is solidified on a solid support. 21. The antibody of claim 20 , which is an active fragment, a single chain antibody or an anti-idiotype antibody. A composition comprising the antibody of claim 16 together with a pharmaceutically acceptable carrier. 24. The composition of claim 23 , comprising a growth inhibitory amount of the antibody. 25. The composition of claim 24 further comprising a second antibody or cytotoxic or chemotherapeutic agent. The method for measuring a polypeptide according to claim 13 or 14, wherein a cell containing the polypeptide according to claim 13 or 14 or suspected of expressing a DNA encoding the polypeptide according to claim 13 or 14 is used. 17. A method comprising exposing to the antibody of claim 16 and measuring binding of the antibody to the cell. In a method for examining a tumor in a mammal in vitro ,
Detecting the expression level of a gene encoding the polypeptide of claim 13 or 14 in (a) a tissue cell test sample obtained from a mammal and (b) a known tissue cell control sample of the same cell type And the high expression level of the test sample indicates the presence of a tumor in the mammal from which the test tissue cells were obtained. In a method for examining a tumor in a mammal in vitro ,
(A) exposing the antibody of claim 16 to a test sample of tissue cells obtained from a mammal, and (b) a complex between the polypeptide of claim 13 or 14 and the antibody in the test sample. Detecting a body formation. 30. The method of claim 28 , wherein the test sample is obtained from a mammal suspected of having neoplastic cell growth or proliferation. A kit for diagnosing cancer comprising the antibody according to claim 16 and a carrier suitable for packaging.

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