JP2004502418A - Novel fibroblast growth factor and nucleic acid encoding the same - Google Patents

Abstract

The present invention provides novel isolated polypeptides and polynucleotides encoding them, and antibodies that immunospecifically bind to these polypeptides, or any of these polypeptides, polynucleotides or antibodies. Derivatives, variants, variants or fragments are provided. The present invention further provides methods for utilizing these polypeptides, polynucleotides and antibodies for the detection and treatment of a wide range of pathological conditions, as well as other uses.

Description

。
【０２８６】
５０μｌ容量中、総量５ｎｇのヒト前立腺ｃＤＮＡテンプレート、各１μＭのＦＧＦ−ＣＸ順方向プライマーおよびＦＧＦ−ＣＸ逆方向プライマー、５μＭ　ｄＮＴＰ（Ｃｌｏｎｔｅｃｈ　Ｌａｂｏｒａｔｏｒｉｅｓ，Ｐａｌｏ　Ａｌｔｏ　ＣＡ）および１μｌの５０×Ａｄｖａｎｔａｇｅ−ＨＦ　２ポリメラーゼ（Ｃｌｏｎｔｅｃｈ　Ｌａｂｏｒａｔｏｒｉｅｓ）を用いて、ＰＣＲ反応を実行した。以下のＰＣＲ反応条件を使用した：
ａ）９６℃　３分間
ｂ）９６℃　３０秒間　変性
ｃ）７０℃　３０秒間、プライマーアニーリング（この温度は、１℃／サイクルずつ徐々に低下させた。）
ｄ）７２℃　１分間　伸長。
【０２８７】
工程（ｂ）〜（ｄ）を１０回繰り返す。
【０２８８】
ｅ）９６℃　３０秒間　変性
ｆ）６０℃　３０秒間　アニーリング
ｇ）７２℃　１分間　伸長
工程（ｅ）〜（ｇ）を２５回繰り返す。
【０２８９】
ｈ）７２℃　５分間　最終伸長。
【０２９０】
約６４０ｂｐの予想サイズを有する単一ＰＣＲ産物を、アガロースゲル上での電気泳動後に単離し、そしてｐＣＲ２．１ベクター（Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＣＡ）に連結した。クローン化した挿入物を、ベクター特異的Ｍ１３順方向プライマー（−４０）およびＭ１３逆方向プライマーを用いて配列決定し、これによって、このヌクレオチド配列が、上流ＢｇｌＩＩクローニング部位と下流ＸｈｏＩクローニング部位との間に直接挿入された図１（配列番号１）の配列に１００％同一であることを確認した。クローン化された配列は、推定ＦＧＦ−ＣＸ全長タンパク質をコードするオープンリーディングフレームを構成する。このクローンを、ＴＡ−ＡＢ０２０８５−Ｓ２７４−Ｆｌ９と呼ぶ。
【０２９１】
（実施例４．哺乳動物発現ベクターｐＣＥＰ４／Ｓｅｃの調製）
オリゴヌクレオチドプライマー、ｐＳｅｃ−Ｖ５−Ｈｉｓ順方向（ＣＴＣＧＴ　ＣＣＴＣＧ　ＡＧＧＧＴ　ＡＡＧＣＣ　ＴＡＴＣＣ　ＣＴＡＡＣ（配列番号１４））およびｐＳｅｃ−Ｖ５−Ｈｉｓ逆方向（ＣＴＣＧＴ　ＣＧＧＧＣ　ＣＣＣＴＧ　ＡＴＣＡＧ　ＣＧＧＧＴ　ＴＴＡＡＡ　Ｃ（配列番号１５））を、Ｖ５およびＨｉｓ６を含むｐｃＤＮＡ３．１−Ｖ５Ｈｉｓ（Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＣＡ）発現ベクターからフラグメントを増幅するように設計した。ＰＣＲ産物を、ＸｈｏＩおよびＡｐａＩで消化し、そしてＸｈｏＩ／ＡｐａＩで消化したＩｇκリーダー配列を保有するｐＳｅｃＴａｇ２　Ｂベクター（Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ　ＣＡ）に連結した。インフレームでＩｇκリーダーおよびＶ５−Ｈｉｓ６を含む生じたベクター（ｐＳｅｃＶ５Ｈｉｓ）の正確な構造を、ＤＮＡ配列分析によって確認した。このベクターｐＳｅｃＶ５Ｈｉｓを、ＰｍｅＩおよびＮｈｅＩで消化して、正確なフレームで上記のエレメントを維持するフラグメントを提供した。ＰｍｅＩ−ＮｈｅＩフラグメントを、ＢａｍＨＩ／ＫｌｅｎｏｗおよびＮｈｅＩで処理したベクターｐＣＥＰ４（Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＣＡ）に連結した。生じるベクターをｐＣＥＰ４／Ｓｅｃと命名し、そしてこのベクターは、インフレームでＩｇκリーダー、目的のクローン挿入のための部位、ならびにＶ５エピトープおよび６×ＨｉｓをＰＣＭＶおよび／またはＰＴ７プロモーターの制御下で含む。ｐＣＥＰ４／Ｓｅｃは、任意のタンパク質をＩｇκ鎖シグナルペプチドに続くマルチクローニング部位に融合させることによって、異種タンパク質の発現および分泌を可能にする発現ベクターである。Ｃ末端のＶ５エピトープタグおよび６×Ｈｉｓ　ｔａｇ（Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＣＡ）の存在を補助として、発現されたタンパク質を検出および精製する。
【０２９２】
（実施例５．ヒト胚性腎臓（ＨＥＫ）２９３細胞におけるＦＧＦ−ＣＸの発現）
ＦＧＦ−ＣＸ配列を含むＢｇｌＩＩ−ＸｈｏＩフラグメントを、ＴＡ−ＡＢ０２０８５−Ｓ２７４−Ｆ１９（実施例３）から単離し、そしてＢａｍＨＩ−ＸｈｏＩで消化したｐＣＥＰ４／Ｓｅｃにサブクローニングして、発現ベクターｐＣＥＰ４／Ｓｅｃ−ＦＧＦ−ＣＸを作製した。このｐＣＥＰ４／Ｓｅｃ−ＦＧＦ−ＣＸベクターを、製造業者の指示書に従ってＬｉｐｏｆｅｃｔａｍｉｎｅＰｌｕｓ試薬（Ｇｉｂｃｏ／ＢＲＬ／Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｒｏｃｋｖｉｌｌｅ，ＭＤ）を用いて２９３細胞にトランスフェクトした。細胞ペレットおよび上清を、トランスフェクションの７２時間後に収集し、抗Ｖ５抗体を用いたウエスタンブロッティング（還元条件）によってＦＧＦ−ＣＸ発現について調べた。図１２は、ＦＧＦ−ＣＸが２９３細胞によって分泌された約３４ｋＤａの見かけの分子量（Ｍｒ）を有するポリペプチドとして発現されることを示す。さらに、主要でないバンドが、約３１ｋａに観察される。
【０２９３】
（実施例６．Ｅ．ｃｏｌｉ．におけるＦＧＦ−ＣＸの発現）
ベクターｐＲＳＥＴＡ（ＩｎＶｉｔｒｏｇｅｎ　Ｉｎｃ．，Ｃａｒｌｓｂａｄ，ＣＡ）を、ＸｈｏＩおよびＮｃｏＩ制限酵素で消化した。配列　５’　ＣＡＴＧＧＴＣＡＧＣＣＴＡＣ　３’（配列番号１６）および５’　ＴＣＧＡＧＴＡＧＧＣＴＧＡＣ　３’（配列番号１７）のオリゴヌクレオチドリンカーを、３７℃でアニーリングし、そしてＸｈｏＩ−ＮｃｏＩで処理したｐＲＳＥＴＡに連結した。生じるベクターを、制限分析および配列決定によって確認し、そしてｐＥＴＭＹと命名した。ＦＧＦ−ＣＸをコードする配列のＢｇｌＩＩ−ＸｈｏＩフラグメント（実施例３を参照のこと）を、ＢａｍＨＩおよびＸｈｏＩ制限酵素で消化したベクターｐＥＴＭＹに連結した。発現ベクターを、ｐＥＴＭＹ−ＦＧＦ−ＣＸと命名する。このベクターにおいて、ｈＦＧＦ−ＣＸは、そのＮ末端が６×ＨｉｓタグおよびＴ７エピトープに融合していた。次いで、プラスミドｐＥＴＭＹ−ＦＧＦ−ＣＸを、Ｅ．ｃｏｌｉ発現宿主ＢＬ２１（ＤＥ３，ｐＬｙｓ）（Ｎｏｖａｇｅｎ，Ｍａｄｉｓｏｎ，ＷＩ）にトランスフェクトし、タンパク質ＦＧＦ−ＣＸの発現を、製造業者の指示書に従って誘導した。誘導後、総細胞を収集し、そしてタンパク質を、抗ＨｉｓＧｌｙ抗体（Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＣＡ）を用いたウエスタンブロッティングによって分析した。図１４は、ＦＧＦ−ＣＸが約３２ｋＤａのタンパク質として発現されたことを示す。
【０２９４】
（実施例７．クローン化されたシグナルペプチド有りおよび無しでの、組み換えＦＧＦ−ＣＸタンパク質の発現比較）
ａ）シグナルペプチド無しでの発現
発明の詳細な説明中で記載したように、ＦＧＦ−ＣＸは古典的アミノ末端シグナル配列を明らかに欠く。ＦＧＦ−ＣＸが哺乳動物細胞から分泌されるか否かを決定するために、全長ＦＧＦ−ＣＸタンパク質をコードするＢｇｌＩＩ−ＸｈｏＩフラグメントとして得られるｃＤＮＡを、ＴＡ−ＡＢ０２０８５−Ｓ２７４−Ｆｌ９（実施例３）からＢａｍＨＩ／ＸｈｏＩ消化ｐｃＤＮＡ３．１（Ｉｎｖｉｔｒｏｇｅｎ）にサブクローニングした。これは、ｐＦＧＦ−ＣＸと称される哺乳動物発現ベクターを提供する。この構築物は、Ｖ５エピトープタグおよびポリヒスチジンタグをタンパク質のカルボキシ末端に組み込み、それぞれ、このタンパク質の同定および精製を補助し、そして約２７ｋＤａのポリペプチドを生成するはずである。２９３ヒト胚性腎臓細胞に一過性トランスフェクションした後、馴化培地をトランスフェクションの４８時間後に収集した。
【０２９５】
馴化培地へのＦＧＦ−ＣＸの分泌に加えて、ＦＧＦ−ＣＸが細胞ペレット／ＥＣＭと会合することもまた、見出された（データには示さず）。ＦＧＦは、細胞表面上および細胞外マトリックス（ＥＣＭ）中に存在する硫酸ヘパリンプロテオグリカン（ＨＳＰＧ）に結合することが公知であり、本発明者らは、ＦＧＦ−ＣＸがこのように隔離される可能性を調査した。このために、ＦＧＦ−ＣＸをトランスフェクトした細胞を、１００□Ｍ　スラミン（ｓｕｒａｍｉｎ）（増殖因子とＨＳＰＧとの間の低親和性相互作用を破壊することが公知の化合物（Ｌａ　Ｒｏｃｃａ，Ｒ．Ｖ．，Ｓｔｅｉｎ，Ｃ．Ａ．＆Ｍｙｅｒｓ，Ｃ．Ｅ．（１９９０）Ｃａｎｃｅｒ　Ｃｅｌｌｓ　２，１０６−１１５））を含む０．５ｍｌ　ＤＭＥＭで、３０分間４℃で処理することによって抽出した。次いで、スラミン抽出した馴化培地を、収集し、そして遠心分離（５分間；２０００×ｇ）で清澄した。
【０２９６】
次いで、馴化培地およびスラミン抽出物を等容量の２×ゲルローディング緩衝液と混合した。サンプルを１０分間煮沸し、還元条件下、４〜２０％勾配ポリアクリルアミドゲル（Ｎｏｖｅｘ，ＤａｎＤｉｅｇｏ，ＣＡ）上でのＳＤＳ−ＰＡＧＥによって分解し、そしてニトロセルロースフィルター（Ｎｏｖｅｘ）に転写した。ウエスタン分析を、ＨＲＰ結合体化抗Ｖ５抗体（Ｉｎｖｉｔｒｏｇｅｎ）およびＥＣＬ検出システム（Ａｍｅｒｓｈａｍ　Ｐｈａｎｎａｃｉａ　Ｂｉｏｔｅｃｈ，Ｐｉｓｃａｔａｗａｙ，ＮＪ）を用いて標準的な手順に従って実行した。
【０２９７】
予想Ｍｒを有する１つのバンドを、ｐＦＧＦ−ＣＸでトランスフェクトした２９３細胞からの馴化培地中で同定した（図１１Ａ、レーン１）。コントロールベクターでトランスフェクトした細胞からの馴化培地は、抗体と反応しなかった（図１１Ａ、レーン５）。スラミン処理後、有意な量のＦＧＦ−ＣＸが実際に細胞表面／ＥＣＭから放出され得たことが見出され、このことは、ＨＳＰＧがおそらくこのタンパク質の分離に役割を果たすことを示す（図１１Ａ、レーン２）。これらの結果は、ＦＧＦ−ＣＸが古典的シグナルペプチド無しで分泌され得ることを示す。
【０２９８】
組換えＦＧＦ−ＣＸタンパク質は、ＤＮＡ合成および細胞増殖（おそらく細胞表面レセプターへのＦＧＦ−ＣＸの高い親和性結合を介して媒介され、そしてＨＳＰＧとの低い親和性相互作用を介して調節される効果）を刺激する。スラミン抽出データは、ＦＧＦ−ＣＸが細胞表面上および／またはＥＣＭに存在するＨＳＰＧに結合することを示唆する。
【０２９９】
ｂ）シグナルペプチド有りでの発現
タンパク質分泌を増強させる目的のために、ＦＧＦ−ＣＸ　ｃＤＮＡがＩｇκ遺伝子由来の切断可能なアミノ末端分泌シグナル配列とインフレームで融合している構築物（ｐＣＥＰ４／Ｓｅｃ−ＦＧＦ−ＣＸ）を作製した。生じるタンパク質もまた、ｐＦＧＦ−ＣＸについて上記で記載したようなカルボキシ末端Ｖ５およびポリヒスチジンタグを含んだ。２９３細胞へのトランスフェクション後、約３１ｋＤａの予想Ｍｒを有するタンパク質産物を得て、そしてスラミンが有意な量の分離ＦＧＦ−ＣＸタンパク質を放出することをまた見出した（図１１Ａ；レーン３および４）。予想したように、ｐＣＥＰ４／Ｓｅｃ−ＦＧＦ−ＣＸは、ｐＦＧＦ−ＣＸよりも、多くの可溶性ＦＧＦ−ＣＸタンパク質を生成した。【０３００】
２９３細胞についての上記の結果と類似した結果を、ＮＩＨ　３Ｔ３細胞で得た（図１１Ｂ）。
【０３０１】
（実施例８．ＰＣＲによる、ＦＧＦ−ＣＸ核酸のリアルタイム定量発現分析）　種々のクローンの定量発現を、Ｐｅｒｋｉｎ−Ｅｌｍｅｒ　Ｂｉｏｓｙｓｔｅｍｓ　ＡＢＩ　ＰＲＩＳＭ（登録商標）　７７００　Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍで実行したリアルタイム定量ＰＣＲ（ＴＡＱＭＡＮ（登録商標）分析）によって、４１個の正常サンプルおよび５５個の腫瘍サンプルにおいて評価した（ほとんどの場合、図１５のパネルＡおよびＢに示すサンプルは、表３中に同定されるサンプルである）。表３において、以下の略語を使用する：
ｃａ．＝癌腫、
^＊＝転移から樹立された、
ｍｅｔ＝転移、
ｓ　ｃｅｌｌ　ｖａｒ＝小細胞変異体、
ｎｏｎ−ｓ＝ｎｏｎ−ｓｍ＝非小、
ｓｑｕａｍ＝扁平上皮、
ｐｌ．ｅｆｆ＝ｐｌ　ｅｆｆｕｓｉｏｎ＝胸水、
ｇｌｉｏ＝神経膠腫、
ａｓｔｒｏ＝星状細胞腫、および
ｎｅｕｒｏ＝神経芽細胞腫。
【０３０２】
第１に、９６ＲＮＡサンプルをβアクチンおよびグリセルアルデヒド−３−リン酸デヒドロゲナーゼ（ＧＡＰＤＨ）に対して正規化した。ＲＮＡ（合計約５０ｎｇまたは約１ｎｇポリＡ＋）を、ＴＡＱＭＡＮ（登録商標）逆転写酵素キット（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ，Ｆｏｓｔｅｒ　Ｃｉｔｙ，ＣＡ；カタログ番号Ｎ８０８−０２３４）および製造者のプロトコールに従ったランダムな６量体を使用してｃＤＮＡに変換した。反応を、２０μｌで実施し、そして３０分間４８℃でインキュベートした。次いで、ｃＤＮＡ（５μｌ）を、製造業者のプロトコールに従って、βアクチンおよびＧＡＰＤＨ　ＴＡＱＭＡＮ（登録商標）アッセイ試薬（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ；各々、カタログ番号４３１０８８１Ｅおよび４３１０８８４Ｅ）およびＴＡＱＭＡＮ（登録商標）ユニバーサルＰＣＲマスターミックス（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ；カタログ番号４３０４４４７）を使用するＴＡＱＭＡＮ（登録商標）反応のために、別々のプレートに移した。反応を、以下のパラメータを使用して２５μｌで実施した：５０℃で２分；９５℃で１０分；９５℃で１５秒／６０℃で１分（４０サイクル）。結果を、ｌｏｇスケールを使用するＣＴ値（所与のサンプルが、蛍光の閾値レベル超えるサイクル）として記録し、所与のサンプルと最も低いＣＴ値を有するサンプルとの間のＲＮＡ濃度の差は、２のΔＣＴ乗として示した。次いで、相対発現パーセントを、このＲＮＡの差の逆数をとって１００を掛けることにより得る。βアクチンおよびＧＡＰＤＨについて得られたＣＴの平均値は、ＲＮＡサンプルを正規化するために使用した。最も高いＣＴ値を生じるＲＮＡサンプルは、さらなる希釈を必要としないが、他の全てのサンプルは、β−アクチン／ＧＡＰＤＨの平均ＣＴ値に従ってこのサンプルと比較して希釈した。
【０３０３】
正規化ＲＮＡ（５μｌ）を、ｃＤＮＡに転換し、製造者の指示に従って、Ｏｎｅ　Ｓｔｅｐ　ＲＴ−ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ　Ｒｅａｇｅｎｔｓ（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ；カタログ番号４３０９１６９）および遺伝子特異的プライマーを使用するＴＡＱＭＡＮ（登録商標）によって分析した。プローブおよびプライマーを、インプットとしてクローン１０３２６２３０．０．３８の配列を使用する、Ｐｅｒｋｉｎ　Ｅｌｍｅｒ　ＢｉｏｓｙｓｔｅｍのＰｒｉｍｅｒ　Ｅｘｐｒｅｓｓ　Ｓｏｆｔｗａｒｅパッケージ（Ａｐｐｌｅ　ＣｏｍｐｕｔｅｒのＭａｃｉｎｔｏｓｈ　Ｐｏｗｅｒ　ＰＣ用のヴァージョンＩ）に従って、各アッセイに対して設定した。デフォルトの設定を、反応条件に対して使用し、そして以下のパラメータを、プライマーを選択する前に設計した：プライマーの濃度＝２５０ｎＭ、プライマーの融解温度（Ｔ_ｍ）範囲＝５８℃〜６０℃、プライマーの最適Ｔ_ｍ＝５９℃、プライマーの最大差＝２℃、プローブは、５’Ｇを有さず、プローブＴ_ｍは、プライマーＴ_ｍよりも１０℃高くなければならない、アンプリコンサイズ７５ｂｐ〜１００ｂｐ。選択されるプローブおよびプライマー（以下を参照のこと）を、Ｓｙｎｔｈｅｇｅｎ（Ｈｏｕｓｔｏｎ，ＴＸ，ＵＳＡ）によって合成した。プローブを、未反応の色素を除去するために２回ＨＰＬＣ精製し、プローブのそれぞれ５’末端および３’末端へのレポーター色素およびクエンチャー色素のカップリングを実証するために、質量分析法によって評価した。順方向プライマーおよび逆方向プライマーの最終濃度は、各々９００ｎＭであり、そしてプローブの濃度は、２００ｎＭであった。
【０３０４】
ＰＣＲについて、各組織および各細胞株からの正規化ＲＮＡを、９６ウェルＰＣＲプレート（Ｐｅｒｋｉｎ　Ｅｌｍｅｒ　Ｂｉｏｓｙｓｔｅｍｓ）の各ウェルにスポットした。２つのプローブ（ＦＧＦ−ＣＸに特異的なプローブおよび内部標準として作用するための第２の遺伝子特異的プローブ）を含むＰＣＲカクテルを、５ｍＭ　ＭｇＣｌ_２、ｄＮＴＰ（１：１：１：２の比率のｄＡ、ｄＧ、ｄＣ、ｄＵ）、０．２５　Ｕ／ｍｌ　ＡｍｐｌｉＴａｑ　Ｇｏｌｄ^ＴＭ（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）、および０．４Ｕ／μｌ　ＲＮａｓｅインヒビター、および０．２５Ｕ／μｌ　逆転写酵素と共に、１×ＴａｑＭａｎ^ＴＭ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘを用いて、ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ　７７００に対して設定した。逆転写を、４８℃で３０分間実施し、続いて、以下のようにＰＣＲ増幅した：９５℃　１０分間、次いで、９５℃　１５秒間、６０℃　１分間を４０サイクル。
【０３０５】
（表３．ＴａｑＭａｎ発現分析において使用した組織サンプル）
番号　組織サンプル
１　内皮細胞
２　内皮細胞（処理）
３　膵臓
４　膵臓ｃａ．　ＣＡＰＡＮ　２
５　脂肪
６　副腎
７　甲状腺
８　唾液腺
９　下垂体
１０　脳（胎児）
１１　脳（全体）
１２　脳（小脳扁桃）
１３　脳（小脳）
１４　脳（海馬）
１５　脳（視床下部）
１６　脳（黒質）
１７　脳（視床）
１８　脊髄
１９　ＣＮＳ　ｃａ．（ｇｌｉｏ／ａｓｔｒｏ）Ｕ８７−ＭＧ
ＣＮＳ　ｃａ．（ｇｌｉｏ／ａｓｔｒｏ）Ｕ−１１８−
２０　ＭＧ
２１　ＣＮＳ　ｃａ．（ａｓｔｒｏ）ＳＷ１７８３
ＣＮＳ　ｃａ．^＊（ｎｅｕｒｏ；ｍｅｔ）ＳＫ−Ｎ
２２　ＡＳ
２３　ＣＮＳ　ｃａ．（ａｓｔｒｏ）ＳＦ−５３９
２４　ＣＮＳ　ｃａ．（ａｓｔｒｏ）ＳＮＢ−７５
２５　ＣＮＳ　ｃａ．（ｇｌｉｏ）ＳＮＢ−１９
２６　ＣＮＳ　ｃａ．（ｇｌｉｏ）　Ｕ２５１
２７　ＣＮＳ　ｃａ．（ｇｌｉｏ）ＳＦ−２９５
２８　心臓
２９　骨格筋
３０　骨髄
３１　胸腺
３２　脾臓
３３　リンパ節
３４　結腸（上行）
３５　胃
３６　小腸
３７　結腸　ｃａ．ＳＷ４８０
結腸　ｃａ．^＊（ＳＷ４８０
３８　ｍｅｔ）ＳＷ６２０
３９　結腸　ｃａ．　ＨＴ２９
４０　結腸　ｃａ．ＨＣＴ−１１６
４１　結腸　ｃａ．ＣａＣｏ−２
４２　結腸　ｃａ．ＨＣＴ−１５
４３　結腸　ｃａ．ＨＣＣ−２９９８
胃　ｃａ．^＊（肝臓　ｍｅｔ）　ＮＣＩ−
４４　Ｎ８７
４５　膀胱
４６　気管
４７　腎臓
４８　腎臓（胎児）
４９　腎臓　ｃａ．７８６−０
５０　腎臓　ｃａ．Ａ４９８
５１　腎臓　ｃａ．ＲＸＦ　３９３
５２　腎臓　ｃａ．ＡＣＨＮ
５３　腎臓　ｃａ．ＵＯ−３１
５４　腎臓　ｃａ．ＴＫ−１０
５５　肝臓
５６　肝臓（胎児）
５７　肝臓　ｃａ．（肝芽腫）ＨｅｐＧ２
５８　肺
５９　肺（胎児）
６０　肺　ｃａ．（小細胞）ＬＸ−１
６１　肺　ｃａ．（小細胞）　ＮＣＩ−Ｈ６９
６２　肺　ｃａ．（ｓ．ｃｅｌｌ　ｖａｒ．）ＳＨＰ−７７
６３　肺　ｃａ．（大細胞）ＮＣＩ−Ｈ４６０
６４　肺　ｃａ．（ｎｏｎ−ｓｍ．ｃｅｌｌ）Ａ５４９
６５　肺　ｃａ．（ｎｏｎ−ｓ．ｃｅｌｌ）ＮＣＩ−Ｈ２３
６６　肺　ｃａ．（ｎｏｎ−ｓ．ｃｅｌｌ）ＨＯＰ−６２
６７　肺　ｃａ．（ｎｏｎ−ｓ．ｃｌ）ＮＣＩ−Ｈ５２２
６８　肺　ｃａ．（ｓｑｕａｍ．）ＳＷ　９００
６９　肺　ｃａ．（ｓｑｕａｍ．）ＮＣＩ−Ｈ５９６
７０　乳腺
７１　胸部　ｃａ．^＊（ｐｌ．ｅｆｆｕｓｉｏｎ）ＭＣＦ−７
７２　胸部　ｃａ．^＊（ｐｌ．ｅｆ）ＭＤＡ−ＭＢ−２３１
７３　胸部　ｃａ．^＊（ｐｌ．ｅｆｆｕｓｉｏｎ）　Ｔ４７Ｄ
７４　胸部　ｃａ．ＢＴ−５４９
７５　胸部　ｃａ．ＭＤＡ−Ｎ
７６　卵巣
７７　卵巣　ｃａ．ＯＶＣＡＲ−３
７８　卵巣　ｃａ．ＯＶＣＡＲ−４
７９　卵巣　ｃａ．ＯＶＣＡＲ−５
８０　卵巣　ｃａ．ＯＶＣＡＲ−８
８１　卵巣　ｃａ．ＩＧＲＯＶ−１
８２　卵巣　ｃａ．^＊（腹水）ＳＫ−ＯＶ−３
８３　子宮筋層
８４　子宮
８５　胎盤
８６　前立腺
８７　前立腺　ｃａ．^＊（骨ｍｅｔ）ＰＣ−３
８８　精巣
８９　黒色腫　Ｈｓ６８８（Ａ）．Ｔ
９０　黒色腫^＊（ｍｅｔ）Ｈｓ６８８（Ｂ）．Ｔ
９１　黒色腫　ＵＡＣＣ−６２
９２　黒色腫　Ｍ１４
９３　黒色腫　ＬＯＸ　ＩＭＶＩ
９４　黒色腫^＊（ｍｅｔ）ＳＫ−ＭＥＬ−５
９５　黒色腫　ＳＫ−ＭＥＬ−２８
９６　黒色腫　ＵＡＣＣ−２５７
以下のプライマーおよびプローブを設計した。各々は、ＦＧＦ−ＣＸに特異的であるように、非常に相同性であるヒトＦＧＦ−９遺伝子およびヒトＦＧＦ−１６遺伝子の対応する領域に、最少３つのミスマッチを保有する。セットＡｇ８１ｂは、図１（配列番号１）の塩基２７０〜塩基３４３の領域をカバーする。これらは、他の公知のＦＧＦファミリーのメンバーを検出すべきではない。使用したプライマーおよびプローブは以下であった。
Ａｇ８１ｂ（Ｆ）：５’−ＧＧＡＣＣＡＣＡＧＣＣＴＣＴＴＣＧＧＴＡ−３’（配列番号１８）；
Ａｇ８１ｂ（Ｒ）：５’−ＴＧＴＣＣＡＣＡＣＣＴＣＴＡＡＴＡＣＴＧＡＣＣＡＧ−３’（配列番号１９）；
Ａｇ８１ｂ（Ｐ）：５’−ＦＡＭ−ＣＣＣＡＣＴＧＣＣＡＣＡＣＴＧＡＴＧＡＡＴＴＣＣＡＡ−ＴＡＭＲＡ−３’（配列番号２０）。
【０３０６】
代表的な実験からの結果を、図１５パネルＡおよび図１５パネルＢに示す。発現を、最高レベルの発現を示すサンプルのパーセンテージとしてプロットする。四連の泳動を行い、これを、種々に陰影を付けたバーで表した。試験された３９個の正常ヒト組織において、ＦＧＦ−ＣＸは、脳、特に小脳において最も高度に発現されることが見出された（図１５パネルＡおよび図１５パネルＢ）。中枢神経系の他の組織は、はるかにより弱いレベルのＦＧＦ−ＣＸを発現した。試験された５４個のヒト腫瘍細胞株のうち、ＦＧＦ−ＣＸは、肺癌腫細胞株（ＬＸ−１）、結腸癌腫細胞株（ＳＷ−４８０）、結腸癌細胞株および転移（ＳＷ４８０）、ならびに胃癌腫細胞株（ＮＣＩ−Ｎ８７；図１５パネルＡおよび図１５パネルＢを参照のこと）において、最も高度に発現されることが見出された。
【０３０７】
さらなるリアルタイム（実時間）の発現分析を、外科手術の間に得られた腫瘍組織の広範なパネルにおいて実施した。これらの組織は、それ自体実際の腫瘍由来の部分、ならびに代表的に既に炎症しており、そして形成異常の組織学的証拠を示す、「正常隣接組織（ＮＡＴ）」と称される部分を含む。ＦＧＦ−ＣＸに特異的であるように選択されたプライマー−プローブセット（Ａｇ８１）を、このような外科組織サンプルと共にＴａｑＭａｎ実験において使用した。ここでは、二連の泳動を実施した：
Ａｇ８１（Ｆ）：５’−ＡＧＧＣＡＧＡＡＧＣＧＧＧＡＧＡＴＡＧＡＴ−３’（配列番号２１）；
Ａｇ８１（Ｒ）：５’−ＡＧＣＡＧＣＴＴＴＡＣＣＴＣＡＴＴＣＡＣＡＡＴＧ−３’（配列番号２２）；および
Ａｇ８１（Ｐ）：ＴＥＴ−５’−ＣＣＡＴＣＴＡＣＡＴＣＣＡＣＣＡＣＣＡＧＴＴＧＣＡＧＡＡ−３’−ＴＡＭＲＡ（配列番号２３）。
Ｓｅｔ　Ａｇ８１は、図１（配列番号１）の塩基４７７〜塩基５５４の領域をカバーする。この複写物は、図１５パネルＣおよび図１５パネルＤにおいて、灰色および黒の陰影をつけたバーとして示される。この結果は、腫瘍およびその形成異常性ＮＡＴサンプルの多くの一致した対について、ＦＧＦ−ＣＸが、腫瘍自体ではなくＮＡＴにおいて；より詳細には、腫瘍に隣接した実質細胞において、高度に発現されることを劇的に示す。この一致したパターンを生じる例としては、卵巣癌、膀胱癌、子宮癌、肺癌、前立腺癌、および肝臓癌が挙げられる。
【０３０８】
理論によって限定されないが、図１５パネルＣおよび図１５パネルＤの結果から、ＦＧＦ−ＣＸが、宿主組織（内皮細胞、間質線維芽細胞、浸潤リンパ球および類似の細胞型）における腫瘍上皮および／または他の成分のパラクリン刺激による腫瘍進行に寄与し得ると考えられる。同様に、ＦＧＦ−ＣＸは、オートクライン様式でＦＧＦ−ＣＸを合成または分泌する宿主組織中の成分を刺激するように機能し得る。これらの宿主成分細胞は、引き続いて、腫瘍画分において作用し得る。
【０３０９】
一致しない正常組織と比較した、ＦＧＦ−ＣＸの発現プロフィールの上昇によって、ＦＧＦ−ＣＸが、腫瘍進行における予定的（ｐｒｏｓｐｅｃｔｉｖｅ）または促進的な役割を果たすことが示唆される。従って、多くの標的化アプローチ（制限することのない例として、モノクローナル抗体、リボザイム、アンチセンスオリゴヌクレオチド、ＦＧＦ−ＣＸと同族（ｃｏｇｎａｔｅ）レセプターとの相互作用を中和するペプチド、およびＦＧＦ−ＣＸについての未同定レセプターを調節する小分子の薬物を含む）のいずれかを使用するＦＧＦ−ＣＸの治療的標的化は、疾患進行に対してポジティブな治療的影響を有すると予期される。同様に、腫瘍進行におけるＦＧＦ−ＣＸの生物活性を調節するためのこのような薬剤の使用は、従来の化学療法および放射線療法に相乗作用を与えるか、またはそれらを増強すると予期される。ＦＧＦ−ＣＸの治療的標的化が適用され得る特定の疾患適用としては、結腸、前立腺、肺、腎臓、子宮、乳房、膀胱、卵巣の腺癌が挙げられる。
【０３１０】
（実施例９．組換えＦＧＦ−ＣＸによって取りこまれるブロモデオキシウリジンの刺激）
２９３−ＥＢＮＡ細胞（Ｉｎｖｉｔｒｏｇｅｎ）を、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　２０００を製造業者のプロトコール（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｇａｉｔｈｅｒｓｂｕｒｇ，ＭＤ）に従って使用してトランスフェクトした。細胞に、トランスフェクションの５時間後、１０％胎仔ウシ血清（ＦＢＳ；Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）を補充した。ＢｒｄＵおよび増殖アッセイ（実施例１０）のためのタンパク質を生成するために、細胞を洗浄し、そしてトランスフェクションの１８時間後、Ｄｕｌｂｅｃｃｏ改変Ｅａｇｌｅ培地（ＤＭＥＭ；Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）で給餌した。４８時間後、この培地を捨て、そしてこの細胞単層を、０．５ｍｌのＤＭＥＤ中で３０分間４℃で、１００μＭスラミン（Ｓｉｇｍａ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）と共にインキュベートした。次いで、このスラミン抽出馴化培地を取り出し、遠心分離（５分間；２０００×ｇ）によって清澄化し、そしてカルボキシ末端ポリヒスチジンタグを利用してＴＡＬＯＮ金属アフィニティークロマトグラフィーに、製造業者の指示書（Ｃｌｏｎｔｅｃｈ，Ｐａｌｏ　Ａｌｔｏ，ＣＡ）に従って供した。保持された融合タンパク質を、イミダゾールでカラムを洗浄することによって放出した。
【０３１１】
ＦＧＦ−ＣＸタンパク質濃度を、既知の濃度のＶ５タグ化タンパク質を用いて作成された検量線を使用するウェスタン分析によって推定した。ウェスタン分析のために、馴化培地をトランスフェクションの４８時間後に収集し、次いで、細胞単層を、４℃で３０分間、１００μＭスラミンを含有する０．５ｍｌ　ＤＭＥＭでインキュベートした。次いで、このスラミン含有馴化培地を収集した。
【０３１２】
コントロールタンパク質を生成するために、２９３−ＥＢＮＡ細胞に、ｐＣＥＰ４プラスミド（Ｉｎｖｉｔｒｏｇｅｎ）をトランスフェクトし、そして上記に概要を述べた精製手順に供した。
【０３１３】
組換えＦＧＦ−ＣＸを、ブロモデオキシウリジン（ＢｒｄＵ）取り込みアッセイにおいてＤＮＡ合成を誘導するその能力について試験した。ＮＩＨ　３Ｔ３細胞（ＡＴＣＣ番号ＣＲＬ−１６５８，Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ，Ｍａｎａｓｓａｓ，ＶＡ）、ＣＣＤ−１０７０Ｓｋ細胞（ＡＴＣＣ番号ＣＲＬ−２０９１）、またはＭＧ−６３細胞（ＡＴＣＣ番号ＣＲＬ−１４２７）を、９６ウェルプレートにおいて、約１００％コンフルエントまで培養し、ＤＭＥＭで洗浄し、そして２４時間（ＮＩＨ　３Ｔ３）または４８時間（ＣＣＤ−１０７０ＳｋおよびＭＧ−６３）、ＤＭＥＭ中で血清飢餓（ｓｅｒｕｍ−ｓｔａｒｖｅｄ）させた。次いで、組換えＦＧＦ−ＣＸまたはコントロールタンパク質を、細胞に１８時間添加した。ＢｒｄＵアッセイは、５時間のＢｒｄＵ取り込み時間を使用して、製造業者の仕様書（Ｒｏｃｈｅ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｃｈｅｍｉｃａｌｓ，Ｉｎｄｉａｎａｐｏｌｉｓ，ＩＮ）に従って実施した。
【０３１４】
ＦＧＦ−ＣＸは、約５ｎｇ／ｍｌの最大半減濃度で、ＮＩＨ　３Ｔ３マウス線維芽細胞においてＤＮＡ合成を誘導することが見出された（図１６Ａ）。対照的に、コントロールベクターでトランスフェクトされた細胞から精製されたタンパク質は、ＤＮＡ合成を誘導しなかった。ＢｒｄＵ取り込みによって測定された場合、ＦＧＦ−ＣＸが種々のヒト細胞株（ＣＣＤ−１０７０Ｓｋ正常ヒト皮膚線維芽細胞（図１６Ｂ）、ＣＣＤ−１１０６ケラチノサイト（図１６Ｃ）、ＭＧ−６３骨肉腫細胞（データを示さず）および乳房上皮細胞を含む）において比較できる用量レベルで、ＤＮＡ合成を誘導することもまた見出された。
【０３１５】
（実施例１０．組換えＦＧＦ−ＣＸによる細胞増殖の誘導）
組換えＦＧＦ−ＣＸが細胞増殖を誘導するか否かを決定するために、ＮＩＨ３Ｔ３細胞を約５０％コンフルエンスまで６ウェルプレート中で培養し、ＤＭＥＭで洗浄し、そして組換えＦＧＦ−ＣＸまたはコントロールタンパク質を含有するＤＭＥＭで４８時間給餌し、次いでカウントした。細胞をトリプシン処理し、そしてそれらをＢｅｃｋｍａｎ　Ｃｏｕｌｔｅｒ　Ｚ１シリーズ計数器（Ｂｅｃｋｍａｎ　Ｃｏｕｌｔｅｒ，Ｆｕｌｌｅｒｔｏｎ，ＣＡ）でカウントすることによって、細胞数を決定した。ＦＧＦ−ＣＸは、このアッセイにおいて、コントロールタンパク質と相対的に、約３倍の細胞数増加を誘導することが見出された（図１７）。
【０３１６】
増殖における形態学的変化事象を詳述するために、ＮＩＨ　３Ｔ３細胞を、ＤＭＥＭ／２％仔ウシ血清中で組換えＦＧＦ−ＣＸまたはコントロールタンパク質で４８時間処理し、そしてＺｅｉｓｓ　Ａｘｉｏｖｅｒｔ　１００顕微鏡（Ｃａｒｌ　Ｚｅｉｓｓ，Ｉｎｃ．，Ｔｈｏｒｎｗｏｏｄ，ＮＹ）で写真撮影した。
【０３１７】
より高い細胞密度を達成することに加えて（図１７）、実施例９に記載のように調製されたＦＧＦ−ＣＸの存在下で培養されたＮＩＨ　３Ｔ３細胞は、無秩序な増殖パターンを示した。これは、接触阻害の欠損を示す（図１８）。さらに、個々の細胞は、細長くかつ屈曲性であることが見出された。これらの結果は、ＦＧＦ−ＣＸが、増殖因子として作用することを示し、そして組換えＦＧＦ−ＣＸが、ＮＩＨ　３Ｔ３細胞の形態学的トランスフォーメーションを媒介することを示唆する。
【０３１８】
（実施例１１．ヌードマウスにおける、異所性ＦＧＦ−ＣＸトランスフェクトＮＩＨ　３Ｔ３細胞による腫瘍形成）
ＮＩＨ　３Ｔ３細胞を、製造業者（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）のプロトコールに従ってＬｉｐｏｆｅｃｔａｍｉｎｅ　Ｐｌｕｓを用いて、ｐＣＥＰ４／Ｓｅｃ−ＦＧＦ−ＣＸまたはコントロールベクターでトランスフェクトした。細胞に、トランスフェクションの５時間後、１０％仔ウシ血清（ＣＳ；Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）を補充した。ｐＣＥＰ４／Ｓｅｃ−ＦＧＦ−ＣＸトランスフェクト細胞は、トランスフェクションの４８時間後までに形態学的にトランスフォーメーションされ、そしてその形態学的トランスフォーメーションを、ハイグロマイシン含有増殖培地中での２週間の選択後に維持した。対照的に、コントロールベクターでトランスフェクトされた細胞は、その正常な形態を維持した（データは示さず）。従って、トランスフェクトされた細胞は、例えば、実施例１０に報告された実験に基づいて予期されたように挙動する。
【０３１９】
異所性腫瘍の誘導を研究するために、ＮＩＨ　３Ｔ３細胞を、種々の実験ベクターおよびコントロールベクターでトランスフェクトした。トランスフェクションの２日後、細胞を、ＤＭＥＭ／５％ＣＳ（ｐＦＧＦ−ＣＸトランスフェクト細胞に対して）または５００μｇ／ｍｌハイグロマイシンＢを補充したＤＭＥＭ／１０％ＣＳ（ｐＣＥＰ４／Ｓｅｃ−ＦＧＦ−ＣＸトランスフェクト細胞に対して）のいずれかに配置した。２週間の培養後、サブコンフルエントな細胞をトリプシン処理し、ＤＭＥＭ／１０％ＣＳで中和し、ＰＢＳで洗浄し、そしてカウントした。ＰＢＳ中の１００万個の細胞を、雌性無胸腺症ヌードマウス（Ｊａｃｋｓｏｎ　Ｌａｂｏｒａｔｏｒｉｅｓ，Ｂａｒ　Ｈａｒｂｏｒ，ＭＥ）の側方皮下組織中に注射した。
【０３２０】
ＮＩＨ　３Ｔ３細胞に、ＦＧＦ−ＣＸ発現プラスミド（ｐＦＧＦ−ＣＸおよびｐＩｇκ−ＦＧＦ−ＣＸ）またはそれらの適切なコントロールベクターでトランスフェクトした。本発明者らは、いずれかのＦＧＦ−ＣＸ発現ベクターでトランスフェクトされた細胞が、トランスフェクションの４８時間後までに形態学的にトランスフォーメーションされ（データは示さず）、そして組換えＦＧＦ−ＣＸへのＮＩＨ　３Ｔ３細胞の曝露後に生じる表現型と類似の表現型を有すること（図１７）を見出した。対照的に、コントロールベクターでトランスフェクトされた細胞は、その正常な形態を維持した（データは示さず）。
【０３２１】
インビボでのＦＧＦ−ＣＸの異所性発現がＮＩＨ　３Ｔ３細胞の腫瘍形成性を誘導するか否かを決定するために、安定なトランスフェクト体を作製し、そしてヌードマウス中に皮下注射した。１１日目までに、ｐＦＧＦ−ＣＸトランスフェクト細胞またはｐＩｇκ−ＦＧＦ−ＣＸトランスフェクト細胞のいずれかを注射された全ての動物は、１４日目までサイズを増大させる急速増殖性腫瘍を有していたが、コントロール細胞を注射された動物はいずれも、２週間目までに腫瘍を発生しなかった（図１９）。これらの結果は、ＦＧＦ−ＣＸ遺伝子を保有するベクターでのトランスフェクションによって形質転換された細胞が、インビボでの腫瘍の発生および増殖を促進することを示す。
【０３２２】
（実施例１２．線維芽細胞増殖因子−２０様タンパク質をコードする核酸を同定する方法）
本発明のさらなる線維芽細胞増殖因子−２０様タンパク質配列（ＦＧＦ２０Ｘ）（本明細書においてクローンＣＧ５３１３５−０２とも呼ぶ）を、この配列のインシリコ予想によって、ｃＤＮＡフラグメントの実験的クローニングによって誘導した。このＤＮＡ配列の全長またはこの配列の一部のいずれか、または両方をカバーするｃＤＮＡフラグメントをクローニングした。インシリコ予想は、ＣｕｒａＧｅｎの自社の配列データベースにおいて、または公的なヒト配列データベースにおいて利用可能な配列に基づき、そして全長ＤＮＡ配列またはそのいくつかの部分のいずれかを提供された。
【０３２３】
実験的クローニングは、以下にまとめられる方法の１つ以上を用いて実施された：
ＳｅｑＣａｌｌｉｎｇ^ＴＭＴｅｃｈｎｏｌｏｇｙ：ｃＤＮＡは、複数の組織型、正常状態および疾患状態、病的状態、ならびに異なるドナーからの発生状態を示す種々のヒトサンプルに由来する。サンプルは組織全体、初代細胞または組織、培養された初代細胞または細胞株として得た。細胞および細胞株は、遺伝子発現を調節する生化学的因子または化学的因子（化合物）（例えば、増殖因子、ケモカインまたはステロイド）で処理されていてもよい。従って誘導されたｃＤＮＡを、次に、ＣｕｒａＧｅｎの自社のＳｅｑＣａｌｌｉｎｇ技術を用いて配列決定した。配列トレースを、手動で評価し、そして必要に応じて補正のために編集した。各アセンブリについてコンセンサス配列を生成するためのバイオインフォマティクスのプログラムを用いて、時には公的なヒト配列を含む、全てのサンプル由来のｃＤＮＡ配列を一緒にアセンブルした。各アセンブリは、ＣｕｒａＧｅｎ　Ｃｏｒｐｏｒａｔｉｏｎのデータベースに含まれる。別の成分との同一性の程度が５０ｂｐにわたって少なくとも９５％であった場合、配列をアセンブリのための成分として含んだ。各アセンブリは、遺伝子またはその部分を示し、そして改変体についての情報（例えば、スプライシング形態、一ヌクレオチド多形態（ＳＮＰ）、挿入、欠失、および他の配列バリエーション）を含む。
【０３２４】
エキソン連結（Ｌｉｎｋｉｎｇ）：ＣＧ５３１３５−０２配列についてのｃＤＮＡコードを以下のプライマーを用いて、ポリメラーゼ連鎖反応（ＰＣＲ）によってクローニングした：５’−ＡＧＧＴＣＡＣＣＡＴＧＧＣＴＧＴＴＡＴＴＧＧＣ−３’（配列番号２４）、および５’−ＣＴＧＴＣＴＧＴＣＣＴＣＡＧＡＡＧＡＡＧＴＴＣＴＴＧＡＴＣ−３’（配列番号２５）。本発明のｃＤＮＡ／タンパク質配列の全長部分またはいくつかの部分（１つ以上のエキソン）のインシリコ予測に基づいてプライマーを設計した。これらのプライマーを用いて、以下の組織由来の、発現されたヒト配列を含むプールからｃＤＮＡを増幅した：副腎、骨髄、脳−扁桃、脳−小脳、脳−海馬、脳−黒質、脳−視床、脳−全脳、胎児脳、胎児腎臓、胎児肝臓、胎児肺、心臓、腎臓、リンパ腫−ラージ、乳腺、膵臓、下垂体、胎盤、前立腺、唾液腺、骨格筋、小腸、脊髄、脾臓、胃、精巣、甲状腺、気管、および子宮。
【０３２５】
各アセンブリについてコンセンサス配列を生成するためのバイオインフォマティクスのプログラムを用いて、時には公的なヒト配列を含む、複数のクローンを配列決定して、これらのフラグメントを一緒にアセンブルした。各アセンブリは、ＣｕｒａＧｅｎ　Ｃｏｒｐｏｒａｔｉｏｎのデータベースに含まれる。別の成分との同一性の程度が５０ｂｐにわたって少なくとも９５％であった場合、配列をアセンブリのための成分として含んだ。各アセンブリは、遺伝子またはその部分を示し、そして改変体についての情報（例えば、スプライシング形態、一ヌクレオチド多形態（ＳＮＰ）、挿入、欠失、および他の配列バリエーション）を含む。
【０３２６】
物理的クローン：エキソン連結によって誘導されたＰＣＲ産物（オープンリーディングフレーム全体をカバーする）を、Ｉｎｖｉｔｒｏｇｅｎ由来のｐＣＲ２．１ベクターにクローニングして、１３７６２７：：１６００８３８７４．１０４３０１０．Ａ９を得た。
【０３２７】
改変体配列はまた、本出願に含まれる。改変体配列は、一ヌクレオチド多形態（ＳＮＰ）を含み得る。ＳＮＰは、ＳＮＰを含有するヌクレオチド配列がｃＤＮＡに由来することを示すために、ある場合には、「ｃＳＮＰ」と呼ばれ得る。ＳＮＰは、いくつかの方法で生じ得る。例えば、ＳＮＰは、多様な部位における１ヌクレオチドの別のヌクレオチドでの置換に起因し得る。このような置換は、トランジションまたはトランスバージョンのいずれかであり得る。ＳＮＰはまた、基準の対立遺伝子に対する、ヌクレオチドの欠失またはヌクレオチドの挿入から生じ得る。この場合、１つの対立遺伝子が、別の対立遺伝子において特定のヌクレオチドに関してギャップを保有する部位は、多様な部位である。遺伝子内に生じるＳＮＰは、このＳＮＰの位置でこの遺伝子によってコードされるアミノ酸の変更を生じ得る。遺伝子内のＳＮＰはまた、ＳＮＰを含むコドンが、この遺伝子コードの冗長性の結果として同じアミノ酸をコードする場合、サイレントであり得る。遺伝子の領域の外側に生じるＳＮＰ、または遺伝子内のイントロンに生じるＳＮＰは、タンパク質のいずれのアミノ酸配列における変化も生じないが、発現パターンの調節の変化を生じ得る。例としては、一時的発現、生理学的反応調節、細胞型発現調節、発現の強度、および転写されたメッセージの安定性における変化が挙げられる。
【０３２８】
新規な線維芽細胞増殖因子−２０様遺伝子のＤＮＡ配列およびタンパク質配列は、エキソン連結によって得られ、そして本明細書においてクローンＣＧ５３１３５−０２として報告され、そして以下の表４および表５に示される。
【０３２９】
（表４．本発明の線維芽細胞増殖因子−２０様タンパク質をコードするヌクレオチド配列（配列番号２６））
【０３３０】
【化２】

（表５．配列番号２６によってコードされるタンパク質配列（配列番号２７））
【０３３１】
【化３】

新規な線維芽細胞増殖因子−２０様タンパク質をコードする５４０ヌクレオチドの新規な核酸（クローンＣＧ５３１３５−０２）を、表４に示す。ヌクレオチド１〜３で始まりそしてヌクレオチド５３８〜５４０で終わるオープンリーディングフレームが同定された。このポリペプチドは、新規の機能的な線維芽細胞増殖因子−２０様タンパク質を示す。オープンリーディングフレームの開始コドンおよび終止コドンを、太字で強調する。推定非翻訳領域（下線部）（存在する場合）は、開始コドンの上流および終止コドンの下流に見出される。１７９アミノ酸残基を有するコードされたタンパク質を、表５に一文字コードを使用して示す。
【０３３２】
（類似性）
配列データベースの検索において、例えば、本発明の核酸が、５０６塩基のうちＨｏｍｏ　ｓａｐｉｅｎｓ由来のｇｂ：ＧＥＮＢＡＮＫ−ＩＤ：ＡＢ０４４２７７｜ａｃｃ：ＡＢ０４４２７７．１　ｍＲＮＡ（ＦＧＦ−２０についてのＨｏｍｏ　ｓａｐｉｅｎｓ　ｍＲＮＡ：完全なｃｄｓ）に同一な４９５塩基（９７％）を有することが見出された。本発明のタンパク質の全長アミノ酸配列は、１６２アミノ酸残基のうちＨｏｍｏ　ｓａｐｉｅｎｓ（ヒト）（線維芽細胞増殖因子−２０（ＦＧＦ−２０））由来の２１１アミノ酸残基のｐｔｎｒ：ＳＷＩＳＳＮＥＷ−ＡＣＣ：Ｑ９ＮＰ９５タンパク質に同一な１６０残基（９８％）、そして１６２アミノ酸残基のうちこれに類似した１６０残基（９８％）を有することが見出された。さらなる相同性を以下の表６に提供する。
【０３３３】
【表１】

複数配列整列を表７に与え、本発明のタンパク質を、本発明のタンパク質と関連タンパク質配列とを比較するＣｌｕｓｔａｌＷ分析において第１列に示す。この配列は、２０〜５１位に示されるように、線維芽細胞増殖因子−２０のスプライス形態を示すことに注意すること。上に示される整列において、黒い輪郭のアミノ酸残基は、配列間で同様に保存された残基（すなわち、構造的または機能的な特性を保存することが必要とされ得る残基）を示す；灰色の背景を有するアミノ酸残基（タンパク質の構造または機能を変更することなく、同等の物理的および／または化学的特性を保有する）（例えば、群Ｌ、Ｖ、Ｉ、およびＭは類似であると考えられ得る）は、配列間で互いに類似している；そして、白色の背景を有するアミノ酸残基は、配列間で保存されていないし類似もしていない。
【０３３４】
（表７．ＣＧ５３１３５−０２タンパク質と関連タンパク質とのＣｌｕｓｔａｌＷ整列）
【０３３５】
【表２】

本明細書中に開示されたタンパク質における同定可能なドメインの存在は、Ｐｆａｍ、ＰＲＯＳＩＴＥ、ＰｒｏＤｏｍ、ＢｌｏｃｋまたはＰｒｉｎｔのようなドメインデータベースに対する検索によって決定され、次いでＩｎｔｅｒｐｒｏドメイン登録番号によって同定される。重要なドメインを表８に要約する。
【０３３６】
【表３】

ペパリン結合増殖因子ＩおよびＩＩ（ＨＢＧＦ）［１，２］（酸性および塩基性の線維芽細胞増殖因子（ＦＧＦ）としてもまた公知である）は、広範な種々の中胚葉起源または神経外胚葉起源の細胞の増殖または分化を刺激する構造的に関連した分裂促進因子である。これらの２つのタンパク質は、インターロイキン−１タンパク質、Ｋｕｎｉｔｚ型ダイズトリプシンインヒビター（ＳＴＩ）（参照）およびヒスタクトフィリン（ｈｉｓｔａｃｔｏｐｈｉｌｉｎ）もまた含むスーパーファミリーのメンバーである増殖因子およびオンコジーンのファミリーに属する。全てが非常に類似した構造を有し、ＨＢＧＦおよびインターロイキン−１ファミリーは、いくらかの配列類似性（約２５％［１］）を共有するが、これらは、ＳＴＩに対して全く類似性を示さない。
【０３３７】
ＨＢＧＦは、細胞の分化および増殖の制御に関連する多数の異なるプロセスに関与する［１］。ＨＢＧＦ１およびＨＢＦＧ２は、同様の効果を有する：これらは、胚発生において中胚葉の形成を誘導し、そして創傷修復、血管形成および神経増殖を媒介する；これらはまた、線維芽細胞、内皮細胞および大グリア細胞の増殖および移動を誘導する。ＨＢＧＦ３（ｉｎｔ−２）およびＨＢＧＦ４（ｈｓｔ／ｋｓ）は、それぞれ胃の腫瘍およびカポージ肉腫由来の公知のオンコジーンである。ＨＢＧＦ５およびＨＢＧＦ６はまた、オンコジーン産物である。ＨＢＧＦ７（ケラチノサイト増殖因子）は、おそらく、正常な上皮細胞増殖の主要なパラクリンエフェクターである。
【０３３８】
これらの増殖因子は、細胞内シグナル伝達を生じるこれらのチロシンキナーゼレセプターの二量体化を引き起こす。現在、線維芽細胞増殖因子について４つの既知のチロシンキナーゼレセプターが存在する。これらのレセプターは各々、このファミリー［３］のいくつかの異なるメンバーに結合し得る。
【０３３９】
ＨＢＧＦ１およびＨＢＧＦ２の結晶構造［４］が解析され、これらがインターロイキン−１およびＫｕｎｉｔｚ型ダイズトリプシンインヒビターの両方と同一の１２鎖のβシート構造を有することが示された［５］；ＨＢＧＦ１およびインターロイキン−１は、類似していることが見出され、そしてこれらは、類似の構造を有することが推定された［６］。βシートは、中央軸の周りに３つの類似の葉において配置され、６つの鎖が逆平行のβバレルを形成する。ＨＢＧＦ１のいくつかの領域（主に、β鎖１〜３および鎖８と９との間のループ）は、レセプター結合に関与する。鎖１０と１１との間のループは、結合ペパリンに関与すると考えられる。
【０３４０】
（表９：ＣｕｒａＧｅｎ登録番号ＣＧ５３１３５−０２についてのＰＳＯＲＴ、ＳｉｇｎａｌＰおよびヒドロパシー結果）
【０３４１】
【表４】

本発明のＦＧＦ２０Ｘタンパク質をコードする新規なＦＧＦ２０Ｘ核酸またはそのフラグメントは、診断用途においてさらに有用であり得、ここで、この核酸またはタンパク質の存在または量が評価される。これらの物質は、治療方法または診断方法における使用のための本発明の新規な物質に免疫特異的に結合する抗体の産生においてさらに有用である。これらの抗体は、以下の「抗ＦＧＦ２０Ｘ抗体」の節において記載されるように、疎水性チャートからの推定を使用して、当該分野で公知の方法に従って産生され得る。開示されたＦＧＦ２０Ｘタンパク質は、複数の親水性領域を有し、その各々が免疫原として使用され得る。１つの実施形態において、意図されるＦＧＦ２０Ｘエピトープは、およそアミノ酸２０〜６５に由来する。別の特異的な実施形態において、ＦＧＦ２０Ｘエピトープは、およそアミノ酸８０〜１７５に由来する。
【０３４２】
このことは、本発明のＦＧＦ２０Ｘ配列が、この／これらのドメインを含むことが知られている他のタンパク質の特性と類似の特性およびこれらのドメインの特性と類似の特性を有することを示す。
【０３４３】
（染色体情報）
本発明において開示されたＦＧＦ２０Ｘ遺伝子は、染色体８ｐ２１．３−ｐ２２にマッピングされる。この割り当ては、開示された配列と配列同一性を共有する、ゲノムクローン、公の遺伝子およびＥＳＴと関連する情報、およびＣｕｒａＧｅｎ　Ｃｏｒｐｏｒａｔｉｏｎ’ｓ　Ｅｌｅｃｔｒｏｎｉｃ　Ｎｏｒｔｈｅｒｎ生命情報工学ツールを使用して行なった。
【０３４４】
（組織発現）
本発明において開示される線維芽細胞増殖因子−２０様遺伝子は、少なくとも以下の組織において発現される：哺乳動物組織、結腸、肺、脳、肝臓、腎臓、および胃。発現情報は、クローンＣＧ５３１３５−０２の配列の誘導物に含まれる配列の組織供給源に由来する。
【０３４５】
【数１】

（等価物）
本発明の特定の実施形態に関する前述の詳細な説明から、核酸、ポリペプチド、抗体を含む特定の新規の組成物ならびに方法、検出そして処置が記載されていることは明らかである。これらの特定の実施形態が、本明細書中で詳細に開示されたが、これは例示目的のためのみに例としてなされ、そして上記の添付した特許請求の範囲に関して制限することを意図されない。特に、種々の置換、変更、および改変が、特許請求の範囲によって規定される本発明の精神および範囲から逸脱することなく、当業者に慣用的事象であるとして、本発明に対してなされ得ることは、本発明者らによって意図される。実際に、本明細書中に記載されるものに加えて、本発明の種々の改変は、前述の説明および添付の図面により当業者に明らかとなる。このような改変は、添付の特許請求の範囲内であることが意図される。
【図面の簡単な説明】
【図１】
図１は、本発明の新規のＦＧＦ−ＣＸポリヌクレオチドのヌクレオチド配列（配列番号１）および本発明の新規のＦＧＦ−ＣＸタンパク質の翻訳されたアミノ酸配列（配列番号２）の説明である。
【図２】
図２は、配列番号１の核酸配列とＦＧＦ−９様グリア活性因子（ＧＡＦ）配列（配列番号５）とのＢＬＡＳＴＮアライメントである。
【図３】
図３は、配列番号１の核酸配列の相補鎖とヒト第８染色体の長いゲノムフラグメント（ＧｅｎＢａｎｋ登録番号ＡＢ０２０８５８）の３つの不連続なセグメント（それぞれＡ〜Ｃにおける、配列番号６〜配列番号８）のＢＬＡＳＴＮアライメントである。
【図４】
図４は、４つの脊椎動物ＦＧＦ様タンパク質（配列番号９〜１２）と、本発明のＦＧＦ−ＣＸタンパク質（配列番号２）とのＣｌｕｓｔａｌＷアライメントである。黒、灰、および白の表現は、それぞれ、アライメント中での、同一の残基、保存された残基、非保存残基である。
【図５】
図５は、ＦＧＦ−ＣＸと、３つの他のＦＧＦファミリーメンバーとのＣｌｕｓｔａｌＷである。ＦＧＦ−ＣＸは、ヒトＦＧＦ−９、ヒトＦＧＦ−１６、およびＸｅｎｏｐｕｓ　ＦＧＦ−ＣＸ（それぞれ、登録番号は、Ｄ１４８３８、ＡＢ００９３９１およびＡＢ０１２６１５である）とアライメントされた。
【図６】
図６は、ＦＧＦ−ＣＸポリペプチド配列（配列番号２）とヒトＦＧＦ−９（配列番号９）とのＢＬＡＳＴＰアライメントであり、同一（「｜」）およびポジティブ（「＋」）である残基を示す。
【図７】
図７は、ＦＧＦ−ＣＸポリペプチド配列（配列番号２）と、マウスＦＧＦ−９（配列番号１０）とのＢＬＡＳＴＸアライメントであり、同一（「｜」）およびポジティブ（「＋」）である残基を示す。
【図８】
図８は、ＦＧＦ−ＣＸポリペプチド配列（配列番号２）と、ラットＦＧＦ−９（配列番号１１）とのＢＬＡＳＴＸアライメントであり、同一（「｜」）およびポジティブ（「＋」）である残基を示す。
【図９】
図９は、ＦＧＦ−ＣＸポリペプチド配列（配列番号２）と、Ｘｅｎｏｐｕｓ　ＸＦＧＦ−ＣＸ（配列番号１２）とのＢＬＡＳＴＸアライメントであり、同一（「｜」）およびポジティブ（「＋」）である残基を示す。
【図１０】
図１０は、１９残基のウインドウで生成された、配列番号２のＦＧＦ−ＣＸポリペプチドのハイドロパシープロット（ｈｙｄｒｏｐａｔｈｙ）の表現である。
【図１１】
図１１は、ＦＧＦ−ＣＸのウエスタン分析を示す。指定された構築物で、一過的にトランスフェクトされた、２９３細胞（パネルＡ）またはＮＩＨ　３Ｔ３細胞（パネルＢ）からのサンプルを、抗Ｖ５抗体を使用するウエスタン分析によって調べた。ＣＭ＝訓化培地、ＳＥ＝スラミン抽出訓化培地。分子量マーカを、左に示す。
【図１２】
図１２は、２９３細胞によって分泌されたＦＧＦ−ＣＸタンパク質のウエスタン分析を示す。
【図１３】
図１３は、ＦＧＦ−ＣＸ遺伝子の分析を表し、これは、ＦＧＦ−ＣＸのヌクレオチドおよび推定アミノ酸配列（配列番号２）を含む。この開始コドンおよび終止コドンは、太字であり、そして５’ＵＴＲ中に存在するインフレーム（ｉｎ−ｆｌａｍｅ）終止コドンが下線で示される。
【図１４】
図１４は、Ｅ．ｃｏｌｉで発現されるＦＧＦ−ＣＸ（配列番号２）タンパク質のウエスタン分析を示す。
【図１５】
図１５は、ＦＧＦ−ＣＸ特異的ＴａｑＭａｎ試薬を使用するリアルタイム定量ＰＣＲによって得られるＦＧＦ−ＣＸの発現の分析を表す。正常なヒト組織サンプル由来の規格化されたＲＮＡについての結果を、パネルＡに示し、そして腫瘍細胞株由来の規格化されたＲＮＡについての結果を、パネルＢに示す。外科手術の間に直接的に得られた腫瘍組織を使用して得られた結果を、パネルＣおよびパネルＤに示す。
【図１６】
図１６は、組換えＦＧＦ−ＣＸのＤＮＡ合成に与える影響によって表される、組換えＦＧＦ−ＣＸの生物学的活性を提示する。細胞は、血清飢餓にし、指定された因子と共に１８時間インキュベートし、ＢｒｕＵ取り込みアッセイによって分析した。サンプルは、三連で実施された。パネルＡは、ＮＩＨ　３Ｔ３マウス線維芽細胞である。パネルＢは、ＣＣＤ−１０７０ヒト線維芽細胞である。パネルＣは、ＣＣＤ−１１０６ヒトケラチノサイトである。
【図１７】
図１７は、細胞増殖に与える組換えＦＧＦ−ＣＸの影響によって表される、組換えＦＧＦ−ＣＸの生物学的活性を示す。ＮＩＨ　３Ｔ３細胞を、指定される因子を補充した無血清培地と共にインキュベートし、４８時間後に計数した。サンプルは、二連で実施された。
【図１８】
図１８は、細胞形態に与える組換えＦＧＦ−ＣＸの影響によって表される組換えＦＧＦ−ＣＸの生物学的活性を表す。ＮＩＨ　３Ｔ３細胞を、ＦＧＦ−ＣＸタンパク質またはコントロールタンパク質と共に４８時間インキュベートし、そして倍率×２５で写真撮影した。
【図１９】
図１９は、ＦＧＦ−ＣＸの腫瘍形成活性を表すグラフを表す。指定された構築物で安定にトランスフェクトされたＮＩＨ　３１３細胞を、胸腺欠損ヌードマウスに皮下組織に注入し、そして２週間の期間にわたって腫瘍形成について調べた。４動物の最小値を、各データポイントとして使用した。[0001]
(Field of the Invention)
The present invention generally relates to nucleic acids and polypeptides. The invention more particularly relates to nucleic acids encoding polypeptides related to members of the fibroblast growth factor family.
[0002]
(Background of the Invention)
The group of cytokines, fibroblast growth factors (FGFs), includes at least 21 members that regulate a wide variety of cellular functions, such as proliferation, survival, apoptosis, motility, and differentiation. These molecules transduce signals through high affinity interactions with the cell surface tyrosine kinase FGF receptor (FGFR). These FGF receptors are expressed on most cell types in tissue culture. It has been reported that dimerization of FGF receptor monomer upon ligand binding is essential for activation of the kinase domain, which leads to receptor phosphorylation. FGF receptor-1 (FGFR-1) exhibits the broadest expression pattern of the four FGF receptors and comprises at least seven tyrosine phosphorylation sites. Many signaling molecules are affected by binding to these phosphorylation sites with different affinities.
[0003]
In addition to being involved in normal growth and development, known FGFs are also involved in the development of pathological conditions, including cancer. FGFs can contribute to malignancy by directly increasing the growth of tumor cells. For example, autocrine growth stimulation via co-expression of FGF and FGFR in the same cell has been reported to lead to cell transformation.
[0004]
(Summary of the Invention)
The present invention is based, in part, on the discovery of nucleic acids encoding novel polypeptides having homology to members of the fibroblast growth factor (FGF) family of proteins. A polynucleotide sequence designated fibroblast growth factor-CX (FGF-CX) is encompassed by the present invention, and FGF-CX polypeptide encoded by its nucleic acid sequence, and fragments, homologs, analogs and derivatives thereof Are claimed in the present invention. An example of an FGF-CX nucleic acid is SEQ ID NO: 1, and an example of an FGF-CX polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. This amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO: 1.
[0005]
In one aspect, the invention includes an isolated FGF-CX polypeptide. In some embodiments, the isolated polypeptide comprises the amino acids of SEQ ID NO: 2 or SEQ ID NO: 27. In other embodiments, the invention includes variants of SEQ ID NO: 2, wherein the variant comprises any number of amino acid residues (eg, no more than 1% of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 27, % Or less, 3% or less, 5% or less, 10% or less, or 15% or less). In some embodiments, the isolated FGF-CX polypeptide is an amino acid sequence in a mature form of the amino acid sequence provided in SEQ ID NO: 2 or SEQ ID NO: 27, or provided in SEQ ID NO: 2 or SEQ ID NO: 27. Variants of the mature form of the amino acid sequence are included. Preferably, 1% or less, 2% or less, 3% or less, 5% or less, 10% or less, or 15% or less of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 27 is a modification of the mature form of this amino acid sequence. Changed in the body.
[0006]
Fragments of the FGF-CX polypeptides are encompassed by the present invention, and include fragments of the modified FGF-CX polypeptide, the mature FGF-CX polypeptide and variants of the mature FGF-CX polypeptide, and the FGF-CX nucleic acid. Includes fragments of FGF-CX polypeptides encoded by allelic variants and single nucleotide polymorphisms (SNPs).
[0007]
In another aspect, the invention includes an isolated FGF-CX nucleic acid molecule. An FGF-CX nucleic acid molecule can include a sequence encoding any of the FGF-CX polypeptides, FGF-CX variants, or FGF-CX fragments described above, or the complement to such a nucleic acid sequence. In one embodiment, the sequence comprises the sequence disclosed in SEQ ID NO: 1. In another embodiment, the sequence comprises the sequence disclosed in SEQ ID NO: 26. In yet other embodiments, the FGF-CX nucleic acid comprises a sequence into which nucleotides different from the sequence given in SEQ ID NO: 1 or SEQ ID NO: 26 can be incorporated. Preferably, no more than 1%, no more than 2%, no more than 3%, no more than 5%, no more than 10%, no more than 15% or no more than 20% of these nucleotides are so modified. In other embodiments, the invention includes fragments or complements of these nucleic acid sequences. Vectors and cells that incorporate an FGF-CX nucleic acid are also encompassed by the present invention.
[0008]
The present invention also includes antibodies that immunospecifically bind to any of the FGF-CX polypeptides described herein. FGF-CX antibodies in various embodiments include, for example, polyclonal, monoclonal, humanized, and / or human antibodies.
[0009]
The present invention further provides a pharmaceutical composition comprising an FGF-CX polypeptide, FGF-CX nucleic acid, or FGF-CX antibody of the present invention. Also included in the invention are kits that include, for example, an FGF-CX polypeptide, an FGF-CX nucleic acid, or an FGF-CX antibody.
[0010]
Several methods are included in the present invention. For example, disclosed are methods for determining the presence or amount of a FGF-CX polypeptide of the invention in a sample. The method comprises contacting the sample with an FGF-CX antibody that immunospecifically binds to the polypeptide; and determining the presence or amount of antibody bound to the polypeptide, such that the antibody Indicating the presence or amount of the polypeptide in the sample.
[0011]
Similarly, the present invention discloses a method for determining the presence or amount of a FGF-CX nucleic acid molecule in a sample. The method comprises the steps of contacting the sample with a probe that binds to the nucleic acid molecule; and determining the presence or amount of antibody bound to the nucleic acid molecule, such that the probe binds FGF-CX in the sample. Indicating the presence or amount of the nucleic acid molecule.
[0012]
A method for identifying a drug that binds to an FGF-CX polypeptide is also provided by the present invention. The method includes determining whether the candidate substance binds to the FGF-CX polypeptide. Binding to the candidate indicates that the drug is an FGF-CX polypeptide binding agent.
[0013]
The invention also includes a method for identifying potential therapeutics for use in treating a medical condition. The condition is associated, for example, with abnormal expression, abnormal processing, or abnormal physiological interaction of an FGF-CX polypeptide of the invention. The method comprises the steps of: expressing a FGF-CX polypeptide and providing a cell having a property or function attributable to the polypeptide; a composition comprising the provided cell and a candidate substance. And determining whether the substance alters a property or function attributable to the polypeptide as compared to control cells. Any such substance is identified as a potential therapeutic. In addition, therapeutics can be identified by subjecting any potential therapeutics so identified to further testing and identifying therapeutics for use in treating this condition.
[0014]
In some embodiments, the property or function is associated with cell growth or cell proliferation, and the substance binds to the polypeptide, thereby causing the activity of the polypeptide to increase. Adjust In some embodiments, the candidate has a molecular weight no greater than about 1500 Da. In some embodiments, the candidate substance is an antibody. The invention further provides any therapeutic agent identified using a method as described herein.
[0015]
A further important aspect of the present invention relates to a method of treating or preventing a disorder associated with an FGF-CX polypeptide. This disorder is characterized by insufficient or inefficient proliferation of cells or tissues, or hyperplasia or neoplasia of cells or tissues. The method comprises administering an FGF-CX polypeptide of the invention or an FGF-CX nucleic acid of the invention or any other therapeutic agent of the invention in an amount and for a duration sufficient to treat or prevent the disorder in the subject. Administering to the subject. In important embodiments, the subject is a human.
[0016]
The invention also encompasses methods of screening FGF-CX polypeptides for modulators of latency or predisposition to disorders associated with abnormal expression, abnormal processing, or abnormal physiological interactions. The method comprises the steps of: recombinantly expressing a FGF-CX polypeptide of the present invention and providing a test animal having an increased risk for a disorder; administering a test compound to the test animal. Measuring the activity of the polypeptide in a test animal after administering the compound; and the activity of the FGF-CX polypeptide in the test animal and the FGF-CX polypeptide in a control animal not receiving the compound. Comparing with the activity of If there is a change in the activity of the polypeptide in the test animal relative to the control animal, the test compound is a modulator of the latency or predisposition to the disorder.
[0017]
The invention also provides a method for determining the presence or predisposition to a disease associated with altered levels of an FGF-CX polypeptide or FGF-CX nucleic acid of the invention in a first mammalian subject. The method comprises the steps of: determining the expression level of the polypeptide or the amount of the nucleic acid in a sample from the first mammalian subject; and determining the amount in the sample and the disease. Comparing to an amount present in a control sample from a second mammalian subject known not to be affected or having a predisposition to suffer from the disease. An alteration in the level of expression of the polypeptide or the level of the amount of the nucleic acid in the first subject, as compared to a control sample, is indicative of the presence or predisposition to the disease.
[0018]
A method for treating a condition in a mammal is also provided by the present invention, wherein the condition is an abnormal expression, abnormal processing, or abnormal physiological interaction of an FGF-CX polypeptide of the invention. Related. The method comprises administering to the mammal an amount of a polypeptide of the invention sufficient to alleviate the condition, wherein the FGF-CX polypeptide comprises SEQ ID NO: 2 or SEQ ID NO: 2. An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or even 99% identical to the polypeptide comprising the amino acid sequence of No. 27, or a biologically active fragment thereof. Polypeptide. In another related method, the antibodies of the invention are administered to a mammal.
[0019]
In another aspect, the invention includes a method of promoting cell proliferation in a subject. The method comprises administering to a subject an FGF-CX polypeptide of the invention in an amount and for a duration effective to promote cell proliferation. In some embodiments, the subject is a human and the cells promoted for proliferation are cells around the wound, cells of the vasculature, cells involved in hematopoiesis, cells involved in erythropoiesis, gastrointestinal tract Cells in the epithelium, and cells in the hair follicle.
[0020]
In a further aspect, the invention provides a method of inhibiting cell proliferation in a subject, wherein the proliferation is associated with expression of a FGF-CX polypeptide of the invention. The method includes administering to the subject a composition that inhibits cell proliferation. In a very important embodiment, the composition contains an antibody of the invention or another therapeutic agent. Of note, the subject is a human and the cells whose growth is inhibited are selected from transformed cells, hyperplastic cells, tumor cells, and neoplastic cells.
[0021]
In a still further aspect, the invention provides a method of treating or preventing or delaying a tissue growth-related disorder. The method comprises administering to the subject an FGF-CX nucleic acid, FGF-CX polypeptide, or FGF-CX antibody in an amount sufficient to treat, prevent, or delay a tissue growth-related disorder. Administering to a subject in whom prevention or delay is desired.
[0022]
A tissue growth-related disorder, diagnosed, treated, prevented, or delayed using an FGF-CX nucleic acid molecule, FGF-CX polypeptide, or FGF-CX antibody, is a disease associated with epithelial cells (e.g., eye disease following surgery). Fibroblasts and keratinocytes in the anterior eye). Other tissue growth-related disorders include, for example, complications of tumor, restenosis, psoriasis, Dupuytren's contracture, diabetes, Kaposi's sarcoma, and rheumatoid arthritis.
[0023]
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0024]
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
[0025]
(Detailed description of the invention)
The present invention is based, in part, on the discovery of a novel FGF-CX nucleic acid sequence, which encodes a polypeptide that is a member of the fibroblast growth factor (FGF) family. As used herein, the designation "FGF-CX" refers to nucleic acids, polynucleotides, proteins, and any of their variants, derivatives, and fragments, and to compounds of these classes. Refers to an antibody that specifically binds.
[0026]
Previously described members of the FGF family regulate a variety of cellular functions, such as proliferation, survival, apoptosis, motility and differentiation (Szebenyi, G. and Fallon, JF (1999) Int. Rev. Cytol.185, 45-106). These molecules transduce signals into cells via high affinity interactions with the cell surface tyrosine kinase FGF receptor (FGFR), four of which have been identified to date ( Xu, X., Weinstein, M., Li, C. and Deng, C. (1999) Cell Tissue Res. 296, 33-43; Klint, P. and Claesson-Welsh, L. (1999) Front. 4, 165-177). These FGF receptors are expressed on most cell types in tissue culture. It has been reported that dimerization of FGF receptor monomer upon ligand binding is necessary for activation of the kinase domain and results in receptor transphosphorylation. FGF receptor-1 (FGFR-1), which exhibits the broadest expression pattern among the four FGF receptors, contains at least seven tyrosine phosphorylation sites. Many signaling molecules are affected by binding with different affinities to these phosphorylation sites.
[0027]
FGF also binds to heparin sulfate proteoglycan (HSPG), which is present on most cell surfaces and extracellular matrix (ECM), despite low affinity. The interaction between FGF and HSPG stabilizes the FGF / FGFR interaction and serves to sequester FGF and protect them from degradation (Szebenyi, G. and Fallon, JF (1999)). ). Due to its growth promoting ability, FGF-7, a member of the FGF family, is currently in clinical trials for the treatment of chemotherapy-induced mucositis (Danilenko, DM). (1999) Toxicol. Pathol. 27, 64-71).
[0028]
In addition to being involved in normal growth and development, known FGFs have also been implicated in the development of pathological conditions, including cancer (Basilica, C and Moscatelli, D. (1992) Adv. Cancer Res. 59, 115-165). FGFs can contribute to malignancy by directly enhancing the growth of tumor cells. For example, stimulation of autocrine growth via co-expression of FGF and FGFR in the same cell results in transformation of the cell (Matsumoto-Yoshitomi, S., Habasita, J., Nomura, C., Kuroshima, K. and Kurokawa). , T. (1997) Int. J. Cancer 71, 442-450). Similarly, constitutive activation of FGFR through mutagenesis or rearrangement results in uncontrolled proliferation (Lorenzi, M., Horii, Y., Yamanaka, R., Sakaguchi, K. and Miki, T. ( Natl. Acad. Sci. USA. 93, 8956-8961; Li, Y., Mangasarian, K., Mansukhani, A. and Basilico, C. (1997) Oncogene 14, 1397-1406). In addition, some FGFs are angiogenic (Gerwins, P., Skoldenberg, E. and Claesson-Welsh, L. (2000) Crit. Rev. Oncol. Hematol. 34, 185-194). Such FGFs can contribute to the tumorigenic process by facilitating the development of the blood supply required for maintenance of tumor growth. Not surprisingly, at least one FGF is currently under investigation as a potential target for cancer treatment (Gasparini, G. (1999) Drugs 58, 17-38).
[0029]
Expression of FGF and their receptors in the birth and adult mouse brain has been tested. Messenger RNA for all FGF genes except FGF-4 is detected in these tissues. The FGF-3, FGF-6, FGF-7 and FGF-8 genes demonstrate higher expression in late embryonic stages than in postnatal stages, indicating that these members are involved in late stages of brain development. Suggest. In contrast, expression of FGF-1 and FGF-5 increased after birth. In particular, FGF-6 expression in mice at birth is restricted to the central nervous system and skeletal muscle, with strong signals in the cerebrum during embryonic development and with strong signals in the cerebellum of 5 day old neonates. Have been reported. FGF receptor (FGFR) -4, a cognate receptor for FGF-6, demonstrates similar spatiotemporal expression, with FGF-6 and FGFR-4 being important ligand-receptor systems in nervous system maturation. Suggest to play a role. According to Ozawa et al., These results indicate that various FGFs and their receptors are involved in various developmental processes of the brain, such as neural progenitor cell proliferation and migration, neural and glial differentiation, neurite outgrowth and synapse formation. It strongly suggests that it is involved in regulation.
[0030]
Another FGF family member, glial activator (GAF), is a heparin-binding growth factor purified from culture supernatants of human glioma cell lines. See Miyamoto et al., 1993, Mol Cell Biol 13 (7): 4251-4259. GAF shows a slightly different range of activity than other known growth factors and is called FGF-9. Human FGF-9 cDNA encodes a polypeptide of 208 amino acids. Sequence similarity with other members of the FGF family was estimated to be about 30%. The two cysteine residues found in other family members and other consensus sequences were also well conserved in the FGF-9 sequence. FGF-9 was found to have no representative signal sequence at the N-terminus as found in acidic and basic FGFs.
[0031]
It is known that acidic and basic FGFs are not secreted from cells in a conventional manner. However, FGF-9 was found to be efficiently secreted from transfected COS cells, despite the lack of a typical signal sequence. This could be detected exclusively in the culture medium of the cells. The secreted protein did not lack the N-terminal amino acid residue relative to the residue predicted by the cDNA sequence, except for the starting methionine. Rat FGF-9 cDNA was also cloned, and structural analysis indicated that the FGF-9 gene was highly conserved.
[0032]
The present invention provides a novel human FGF and its corresponding cDNA. The protein product of this gene has been shown to exhibit growth stimulating and tumorigenic properties. In addition, overexpression of FGF mRNA has been noted in certain cancer cell lines. These observations suggest that novel FGFs can be used by serving as excellent targets in treating human malignancies.
[0033]
The invention also relates to mature FGF-CX polypeptides, variants of mature FGF-CX polypeptides, fragments of mature FGF-CX polypeptides and variants of mature FGF-CX polypeptides, and encoding these polypeptides and fragments. Contains nucleic acids. As used herein, a "mature" form of an FGF-CX polypeptide or protein disclosed in the present invention is a naturally occurring polypeptide or a precursor form or the product of a proprotein. A naturally occurring polypeptide, precursor or proprotein includes, by way of non-limiting example, the full-length gene product encoded by the corresponding gene. In some embodiments, the mature form comprises an FGF-CX polypeptide, precursor or proprotein encoded by an open reading frame described herein. A product "mature" form may occur as a result of one or more naturally occurring processing steps, if it can occur in the cell in which the gene product is produced, or in a host cell.
[0034]
Examples of such processing steps that result in a "mature" form of the polypeptide or protein include cleavage of the N-terminal methionine residue encoded by the initiation codon of the open reading frame, or proteolytic cleavage of the signal peptide or leader sequence. No. Thus, the mature form resulting from the FGF-CX precursor polypeptide or precursor protein, having residues 1 to N, where residue 1 is the N-terminal methionine, remains after removal of the N-terminal methionine. Residues 2 to N. Alternatively, the mature form resulting from the precursor polypeptide or protein, having residues 1 to N, where the N-terminal signal sequence of residues 1 to M is cleaved, It has residues from M + 1 to residue N. Further, a "mature" protein or fragment may result from a cleavage event other than removal of the starting methionine or signal peptide. Further, as used herein, a "mature" form of an FGF-CX polypeptide or protein may result from post-translational modification steps other than proteolytic cleavage events. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. Generally, a mature polypeptide or protein can result from the manipulation of only one of these processes, or a combination of any of these.
[0035]
As used herein, “identical” residues are those residues in a comparison between two sequences where the equivalent nucleotide bases or amino acid residues in the alignment of the two sequences are the same residue. Corresponding to the group. Alternatively, the residue is when the comparison between the two sequences in the alignment indicates that the residue at the equivalent position in the comparison is either the same amino acid or a conserved amino acid as defined below. Described as “similar” or “positive”.
[0036]
An FGF-CX nucleic acid, an isolated nucleic acid encoding an FGF-CX polypeptide or a portion thereof, an FGF-CX polypeptide, a vector containing these nucleic acids, a host cell transformed with the FGF-CX nucleic acid, Anti-FGF-CX antibodies and pharmaceutical compositions are included within the scope of the present invention. Also disclosed are methods of making FGF-CX polypeptides, and methods of screening, diagnosing, treating conditions using these compounds, and screening for compounds that modulate FGF-CX polypeptide activity. Table 1 below depicts the sequence descriptors used throughout the present invention.
[0037]
Table 1
Sequence number Array descriptor
1 Human FGF-CX nucleotide sequence
2 Human FGF-CX polypeptide sequence
3 FGF-CX forward primer
4 FGF-CX reverse primer
5 Glial activator (GAF)
6 human genomic fragment-bp 15927-16214
7 human genomic fragment-bp 7257-7511
8 human genomic fragment-bp 9837-9942
9 Human FGF-9
10 Mouse FGF-9
11 Rat FGF-9
12 Xenopus FGF-CX
13 Human FGF-CX hydrophobic domain (aa90-115)
14 PSec-V5-His forward direction
15 PSec-V5-His reverse direction
16 PSETA linker
17 PSETA linker
18 TaqMan expression analysis forward primer
19 TaqMan expression analysis reverse primer
20 TaqMan expression analysis probe
21 TaqMan expression analysis forward primer
22 TaqMan expression analysis reverse primer
23 TaqMan expression analysis probe
24 PCR oligonucleotides
25 PCR oligonucleotides
26 Fibroblast growth factor-20-like polynucleotide sequence
27 Fibroblast growth factor-20-like polypeptide sequence.
[0038]
The FGF-CX nucleic acids and polypeptides discussed herein, as well as FGF-CX antibodies, therapeutics and pharmaceutical compositions are particularly useful in treating tissue growth-related disorders. These tissue growth-related disorders may include disorders affecting epithelial cells, such as fibroblasts and keratinocytes in front of the eye after surgery. Other tissue growth-related disorders include, for example, tumors, restenosis, psoriasis, Dupuytren's contracture, diabetic complications, Kaposi's sarcoma and rheumatoid arthritis.
[0039]
A nucleotide sequence encoding a novel fibroblast growth factor designated as fibroblast growth factor-20X (FGF-CX) (SEQ ID NO: 1 and SEQ ID NO: 26) is included in the present invention. FIG. 1 shows FGF-CX described by SEQ ID NO: 1. This coding sequence was identified in the human genomic DNA sequence. The disclosed DNA sequence has 633 bases, encoding a polypeptide predicted to have 211 amino acid residues (SEQ ID NO: 2). The predicted molecular weight of FGF-CX based on the sequence shown in FIG. 1 and SEQ ID NO: 2 is 23498.4 Da.
[0040]
This FGF-CX nucleic acid sequence was used as a query nucleotide sequence in a BLASTN search to identify relevant nucleic acid sequences. This FGF-CX nucleotide sequence is identical to mouse fibroblast growth factor 9 (FGF-9) (329 bases out of 543 bases, or 72%; GenBank accession number S82023) and human encoding glial activator (GAF). It has high similarity to DNA (385 bases identical to 554 bases, ie 69%; GenBank accession number E05822, also referred to as FGF-9). In addition, FGF-CX has a comparable degree of identity (424 bases) to the GAF sequence (SEQ ID NO: 5) disclosed by Naroo et al., Japanese Patent: JP 19933301893, entitled "Glia-Activating Factor And Its Production". 311 bases identical, ie, 73%) (see FIG. 2).
[0041]
To confirm that the open reading frame (ORF) identified by genomic mining was correct, a cDNA corresponding to the predicted genomic clone was obtained using PCR amplification. The nucleotide sequence of the obtained product exactly matches the sequence of the predicted gene (see Example 1).
[0042]
The protein encoded by this cDNA is most closely related to Xenopus FGF-20X (referred to herein as XFGF-CX or XFGF-20X) and human FGF-9 and human FGF-16 (respectively). , 80%, 70% and 64% amino acid identity; see FIGS. 4 and 5). Based on the strong homology with XFGF-CX, the gene identified in the present disclosure is believed to represent its human ortholog and is referred to herein as FGF-CX.
[0043]
A BLASTP alignment of the first 208 amino acids of the FGF-CX polypeptide sequence (SEQ ID NO: 2) with human FGF-9 (SEQ ID NO: 9) is shown in FIG. For glial activator precursor (GAF) (fibroblast growth factor-9), see SWISSPROT accession number P31371; Miyamoto et al., 1993 Mol. Cell. Biol. 13: 4251-4259; and Naroo et al., 1993 J. Am. Biol. Chem. 268: 2857-2864. A BLASTX alignment of the first 208 amino acids of the FGF-CX polypeptide (SEQ ID NO: 2 translated from SEQ ID NO: 1) with the mouse FGF-9 sequence (SEQ ID NO: 10) and the rat FGF-9 sequence (SEQ ID NO: 11). 7 and 8 respectively. For glial activator precursor (GAF) (fibroblast growth factor-9), SWISSPROT accession number P54130; for mouse FGF-9, Santos-Ocampo et al., 1996 J. Am. Biol. Chem. 271: 1726-1731; and for rat FGF-9, SWISSPROT accession number P36364, glial activator precursor (GAF) (fibroblast growth factor-9) (FGF-9), Miyamoto, 1993 Mol. Cell. Biol. 13: 4251-4259. As shown by the bars ("|") in FIGS. 5-7, the FGF-9 sequences of all three species are identical to FGF-CX (SEQ ID NO: 2) for 147 out of 208 residues, It has 70% identity over the entire sequence. In addition, 170 of the 208 residues are positive for the sequence of FGF-CX (SEQ ID NO: 2), with an overall percentage of positive residues of 81%. Positive residues are those residues that are either identical (“|”) or have conservative amino acid substitutions (“+”) at the same relative position in the compared sequences when aligned. Including. See below.
[0044]
The full length FGF-CX polypeptide (SEQ ID NO: 2) was also aligned with Blentox with Xenopus XFGF-CX (SEQ ID NO: 12). As shown in FIG. 9, FGF-CX has 170 of 211 identical residues (80%) and 189 of 211 positive residues (89%) compared to Xenopus XFGF-CX. . Xenopus XFGF-CX was recently obtained from a cDNA library prepared at the tailbud stage, using the product of a denatured PCR performed using primers based on mammalian FGF-9 as a probe. See Koga et al., 1999 Biochem Biophys Res Commun 261 (3): 756-765. The predicted 208 amino acid sequence of the XFGF-CX open reading frame contains a motif characteristic of the FGF family. XFGF-CX has an overall similarity of 73.1% to XFGF-9, but differs from XFGF-9 in its amino-terminal region (33.3% similarity). This is similar to the similarity currently seen for the disclosed SEQ ID NO: 2 for the sequences of various mammalian FGF-9 and FGF-16, including humans (see above). See Figures 4, 5, and 7-9.
[0045]
The polypeptide sequence of FIG. 1 (SEQ ID NO: 2) is predicted to have a high potential for sorting through the membrane of the endoplasmic reticulum and microsomes (peroxisomes) by the program PSORT. Furthermore, although it does not have the predicted cleavable signal sequence at its N-terminus, the hydropathy plot of FIG. 10 shows that FGF-CX shows that amino acid positions from about 90 to about 115 (SEQ ID NO: 13) Indicates that it has a marked hydrophobic segment. This single hydrophobic region is known to be a classification signal in other members of the FGF family. Thus, a polypeptide comprising the amino acid sequence of SEQ ID NO: 13 is useful as a classification signal and allows secretion through the membranes of various cells (eg, endoplasmic reticulum, Golgi membrane or plasma membrane).
[0046]
FGF-CX is a PSORT computer algorithm (Nakai, K. and Kanehisa, M. (1992) Genomics, just as found for some of the most closely related human family members (eg, FGF-9 and FGF-16). 14, 897-911) and the SIGNALP computer algorithm (Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997) Protein Eng. 10, 1-6). Lacks the typical amino-terminal signal sequence. Nevertheless, both FGF-9 and FGF-16 are secreted (Matsumoto-Yoshitomi, S., Habasita, J., Nomura, C., Kuroshita, K. and Kurokawa, T. (1997) Int. Miyake, A., Konishi, M., Martin, F.H., Hernday, N.A., Ozaki, K., Yamamoto, S. Mikami, T., Arakawa, T. J. Cancer 71, 442-450; And Itoh, N. (1998) Biochem.Biophys.Res.Comm.243, 148-152; Miyakawa, K., Hatsawawa, K., Kurokawa, T., Asada, M., Kuroiwa, T. and Inamura, T. (1999) J. Biol. Chem. 274, 29352-29357; Revest, J.-M., DeMoerloose, L. and Dickson, C. (2000) J Biol. Chem. 275,8083-8090). . To determine whether FGF-CX was also secreted, the cDNA encoding the full-length FGF-CX protein was subcloned into a mammalian expression vector called pFGF-CX. The protein expressed when human embryonic kidney 293 cells are transfected with this vector is found in conditioned media and has an apparent molecular weight of about 27 kDa in a Western blot, and is directed against the C-terminal V5 epitope. The band detected by the antibody is shown (FIG. 11, Example 7). An additional portion of the expressed protein is released from sequestration on 293 cells by treatment with a substrate that inhibits interaction with heparin sulfate proteoglycan (HSPG). The proteins thus released also show a similar Western blot pattern (FIG. 11). Similarly, when this protein is expressed in HEK293 cells from a recombinant plasmid that incorporates an Igκ signal sequence, a band is detected by Western blot, with an appropriate molecular weight of about 34 kDa (FIG. 12, Example 5). ).
[0047]
ClustalW multiple protein alignments for some vertebrate FGF-like proteins (including FGF-CX described by SEQ ID NO: 2) (Thompson, JD, Higgins, DG, and Gibson, TJ. 1994) Nucleic Acids Res. 22, 4673-4680) is shown in FIGS. The three mammalian proteins (SEQ ID NOs: 9-11) are closely similar to each other, but significantly different from the FGF-CX protein of the present invention (SEQ ID NO: 2). Also, the sequences of Xenopus XFGF-CX (SEQ ID NO: 12) and SEQ ID NO: 2 are more closely similar to each other than the sequence of FGF-9. Internal hydrophobic domains involved in FGF-9 secretion (Miyakawa, K., Hatsawa, K., Kurokawa, T., Asada, M., Kuroiwa, T. and Inamura, T. (1999) J. Biol. Chem. 274, 29352-29357) span residues 95-120 of the FGF-9 sequence. (See FIG. 10 for the hydropathy plot of FGF-CX described by SEQ ID NO: 2).
[0048]
The expression of XFGF-20 and Xenopus FGF-9 are different from each other. XFGF-20 mRNA is specifically expressed in diploid cells, in blastula and post-blastula embryos, and in adult stomach and testis; while XFGF-9 mRNA is expressed maternally in eggs. And is expressed in many adult tissues. Koga et al., Supra. Correct expression of XFGF-20 during gastrulation appears to be required for the formation of normal head structures in Xenopus larvae. When XFGF-20 mRNA was overexpressed in early embryos, gastrulation was abnormal and development of anterior structures was suppressed. See Koga et al., Supra. In such embryos, among other things, the expression of Xbra transcripts was suppressed during gastrulation, indicating that expression of the Xbra gene mediates the effects of XFGF-CX. See Koga et al., Supra.
[0049]
The pattern of expression of the relevant FGF-9 polypeptides in proliferating tissues (including, for example, tissues in eggs, testes, stomach and mother frog) is dependent on the expression of tissues normally undergoing regeneration in functioning organs. Suggests a role for XFGF-20 in maintenance.
[0050]
In Example 8, it is shown that FGF-CX mRNA is expressed in normal cerebellum and some human tumor cell lines, including lung, stomach and colon carcinomas, but not in corresponding normal tissues. It is. The lack of FGF-CX expression in normal lung, stomach and colon, and the presence of FGF-CX expression in tumor lines derived from these tissues, clearly indicates that these cancer cell lines over-express FGF-CX in an inappropriate manner It is expressed. The chromosomal region to which FGF-CX is mapped is commonly altered in colorectal, lung and gastric carcinomas (Emi, M., Fujiwara, Y., Nakajima, T., Tsuchiya, E., Tsuda, H., et al. Hirashi, S., Maeda, Y., Tsuruta, K., Miyaki, M. and Nakamura, Y. (1992) Cancer Res, 52, 5368-5372; Baffa, R., Santoro, R., Bullrich, F. , Mandes, B., Ishii, H. and Croce, CM (2000) Clin. Cancer Res. 6, 1372-1377). The establishment of an FGF-CX driven autocrine growth loop in these cells can contribute to their initial oncogenic transformation and / or subsequent expansion. This scenario is supported by the finding that the generation of FGF-CX driven autocrine loops in NIH 3T3 cells activates their tumorigenic potential (see Example 11). It is also possible that FGF-CX secreted by tumor cells stimulates their proliferation in vivo via a paracrine effect on stromal cells.
[0051]
Expression of heterologous FGF-CX in NIH 3T3 cells has been found to induce their transformation and tumorigenic potential (see Example 11). These effects are mediated by both native FGF-CX (construct pFGF-CX) and FGF-CX expressed at the amino terminus with a heterologous Igκ signal sequence (construct pIgκ-FGF-CX). However, pIgκ-FGF-CX is more oncogenically active than pFGF-CX, as evidenced by higher in vitro transformability (data not shown) and in vivo tumorigenicity (FIG. 19). It should be noted that The superior tumorigenic potential of pIgκ-FGF-CX on pFGF-CX indicates that pIgκ-FGF-CX significantly increases more secreted FGF-CX protein than does pFGF-CX in NIH 3T3 cells. (Fig. 11B).
[0052]
Other FGFs, such as FGF-CX, have been shown to transform cells after ectopic expression, and in some cases, blocking FGF signaling inhibits cell transformation. (Matsumoto-Yoshitomi, S., Habasita, J., Nomura, C., Kuroshima, K. and Kurokawa, T. (1997) Int. J. Cancer 71, 442-450; Li, Y., Basilico, C. and Mansukhani, A. (1994) Mol. Cell. Biol. 14, 7660-7669).
[0053]
Based on the properties of FGF-CX described herein and their similarity to the effects found for the relevant FGF proteins, FGF-CX is believed to play an important role in human malignancies. Can be For these reasons, the FGF-CX polypeptides, nucleic acids and antibodies disclosed herein are useful in methods of diagnosing the presence or amount of these compositions, as therapeutic agents for FGF-CX related pathologies. Useful in screening and identification, and in methods of treating various types of malignancies.
[0054]
(FGF20X clone, functional variant and homolog)
The novel nucleic acids of the invention that encode a fibroblast growth factor-20-like protein include the nucleic acids whose sequences are set forth by SEQ ID NO: 1 or SEQ ID NO: 26, or fragments thereof. The invention also encodes a protein wherein any of its bases can be changed from the corresponding base shown in Tables 4 and 5, but still retains its fibroblast growth factor-20-like activity and physiological function. , Mutant or variant nucleic acids or fragments of such nucleic acids. The invention further includes nucleic acids whose sequences are complementary to the sequences of CuraGen Accession Nos. CG53153-02 and CG53135-01, and includes nucleic acid fragments that are complementary to any of the just described nucleic acids. The invention further includes a nucleic acid or nucleic acid fragment or its complement, the structure of which comprises a chemical modification. Such modifications include, by way of non-limiting example, modified bases and nucleic acids whose sugar phosphate backbone has been modified or derivatized. These modifications are performed, at least in part, to enhance the chemical stability of the modified nucleic acids, so that they can be used as antisense-binding nucleic acids, for example, in therapeutic applications in a subject. obtain. In mutant or variant nucleic acids and their complements, up to about 3% bases can be so altered.
[0055]
The novel proteins of the present invention include a fibroblast growth factor-20-like protein of the sequence set forth by SEQ ID NO: 2 and SEQ ID NO: 27. The present invention also relates to the fibroblast growth factor-20-like protein fibroblast growth factor-20, wherein any of its bases can be changed from the corresponding residue shown in the sequence of SEQ ID NO: 2 and SEQ ID NO: 27. Muteins or modified proteins, or fragments thereof, that still encode proteins that maintain similar activity and physiological function. In muteins or modified proteins, up to about 2% of the amino residues can be changed.
[0056]
(Chimeric and fusion proteins)
The invention includes a chimeric or fusion protein of a fibroblast growth factor-20-like protein, wherein the fibroblast growth factor-20-like protein of the invention is a second polypeptide or a novel polypeptide of the invention. Linked to a protein that is not substantially homologous to the protein. The second polypeptide can be fused to either the amino or carboxyl terminus of the CG53135-02 or CG53135-01 polypeptide of the invention. In certain embodiments, a third heterologous polypeptide or protein can also be fused to a novel fibroblast growth factor-20-like protein, such that the second heterologous polypeptide or protein is amino terminal And a third heterologous polypeptide or protein is linked at the carboxyl terminus of the CG53135-02 or CG53135-01 polypeptide. Examples of heterologous sequences into which either the second or third polypeptide or protein can be incorporated include glutathione S-transferase, a heterologous signal sequence fused at the amino terminus of a fibroblast growth factor-20-like protein, Immunoglobulin sequences or domains, serum proteins or domains thereof (such as serum albumin), antigenic epitopes, and (His) ₆ And a specific portion such as
[0057]
The invention further includes nucleic acids encoding any of the chimeric or fusion proteins described in the above paragraph.
[0058]
(antibody)
The invention further provides antibodies and antibody fragments (Fab, (Fab)) that immunospecifically bind to any of the proteins of the invention. ₂ Or a single chain FV construct). The present invention also includes peptides and polypeptides comprising sequences having high binding affinity for any of the proteins of the present invention. Such peptides and polypeptides include peptides and polypeptides that are fused (or biologically expressed on the surface of a carrier) to any carrier particles, such as bacteriophage particles.
[0059]
(Use of the composition of the present invention)
The protein similarity information, expression patterns, cellular localization, and map locations for proteins and nucleic acids disclosed herein indicate that this fibroblast growth factor-20-like protein has important structures and And / or suggest that they may have physiological functions unique to the fibroblast growth factor family. Thus, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications, and as research tools. These include acting as specific or selective nucleic acid or protein diagnostic and / or prognostic markers, where the presence or amount of the nucleic acid or protein is assessed. These also include the following potential therapeutic applications: (i) protein therapeutics, (ii) small molecule drug targets, (iii) antibody targets (therapeutic antibodies, diagnostic antibodies, drug targeting / cells) (Virulent antibodies), (iv) nucleic acids useful in gene therapy (gene delivery / gene ablation), (v) promoters of tissue regeneration in vitro and in vivo, and (vi) biological defense weapons.
[0060]
The nucleic acids and proteins of the invention have applications in the diagnosis and / or treatment of various diseases and disorders. For example, compositions of the invention have efficacy for treating patients suffering from: Hirschsprung's disease, Crohn's disease, appendicitis, inflammatory bowel disease, diverticulosis, systemic lupus erythematosus, autoimmune disease, asthma, Emphysema, scleroderma, allergy, ARDS, von Hippel-Lindau (VHL) syndrome, liver cirrhosis, transplantation, hypercalcemia, ulcer, cardiomyopathy, atherosclerosis, hypertension, congenital heart defect ( heart defect), aortic stenosis, atrial septal defect (ASD), atrioventricular (AV) tube defect, arterial tract, pulmonary stenosis, lower aortic stenosis, ventricular septal defect (VSD), valve disease , Tuberous sclerosis, scleroderma, obesity, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, tubular acidosis, IgA nephropathy, hypercalcemia, Alzheimer's disease, seizures, tuberous sclerosis, hypercalcemia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, telangiectasia Ataxia, leukodystrophy, behavioral disorders, addictions, anxiety, pain, neurodegeneration, and other diseases, disorders and conditions.
[0061]
These materials are further useful in generating antibodies that immunospecifically bind to the novel substances of the present invention for use in diagnostic and / or therapeutic methods.
[0062]
(FGF-CX nucleic acid)
The nucleic acid of the present invention includes a nucleic acid encoding an FGF-CX protein or an FGF-CX-like protein. In particular, these nucleic acids are nucleic acids of the sequence provided in FIG. 1 (SEQ ID NO: 1, SEQ ID NO: 26) or fragments thereof. An FGF-CX nucleic acid can have a genomic FGF-CX nucleic acid or cDNA nucleotide sequence. Furthermore, the present invention includes a mutant nucleic acid or a modified nucleic acid of the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 26, or a fragment thereof, and any of these bases is shown in the sequence of SEQ ID NO: 1 or SEQ ID NO: 26. Still encodes a protein that can be changed from the corresponding base but retains FGF-CX-like activity and physiological function. The invention further includes the complement of the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 26, including fragments, derivatives, analogs and homologs thereof. An example of a portion of the complementary strand of FGF-CX is shown in FIG. The present invention further includes nucleic acids or nucleic acid fragments or their complements, the structure of which comprises a chemical modification.
[0063]
One aspect of the present invention relates to an isolated nucleic acid molecule encoding a FGF-CX protein or a biologically active portion thereof. Also, polymerase chain reaction (PCR) for amplification or mutation of a nucleic acid fragment and FGF-CX nucleic acid molecule sufficient to be used as a hybridization probe to identify FGF-CX encoding nucleic acids (eg, FGF-CX mRNA). Also included are fragments used as primers. As used herein, the term "nucleic acid molecule" refers to a DNA molecule (e.g., cDNA or genomic DNA), an RNA molecule (e.g., mRNA), of DNA or RNA produced using nucleotide analogs. It is intended to include analogs and their derivatives, fragments and homologs. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
[0064]
"Probe" refers to nucleic acid sequences of various lengths, preferably, depending on the use, at least about 10 nucleotides (nt), 100 nt, or for example, between as large as about 6,000 nt. . Probes are used in the detection of identical, similar or complementary nucleic acid sequences. Longer length probes are usually obtained from natural or recombinant sources, are very specific, and hybridize much slower than oligomers. Probes can be single-stranded or double-stranded and designed to have specificity in techniques such as PCR, membrane-based hybridization techniques or ELISA.
[0065]
An "isolated" nucleic acid molecule is one that has been separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Examples of isolated nucleic acid molecules include recombinant DNA molecules contained in vectors, recombinant DNA molecules maintained in heterologous host cells, partially or substantially purified nucleic acid molecules, and synthetic DNA molecules. Or, but not limited to, synthetic RNA molecules. Preferably, an “isolated” nucleic acid is a sequence that is naturally adjacent to the nucleic acid molecule in the genomic DNA of the organism from which the nucleic acid is derived (ie, the sequences located at the 5 ′ and 3 ′ ends of the nucleic acid). Not included. For example, in various embodiments, an isolated FGF-CX nucleic acid molecule is less than about 50 kb, less than 25 kb, less than 5 kb, 4 kb that is naturally adjacent to the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid was derived. Less than 3 kb, less than 2 kb, less than 1 kb, less than 0.5 kb, or less than 0.1 kb. Further, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, when produced by recombinant technology, is substantially free of other cellular material or culture media, or is chemically synthesized. It may be substantially free of chemical precursors or other chemicals.
[0066]
Nucleic acid molecules of the invention, for example, nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 26, or any complement of these nucleotide sequences, are provided by standard molecular biology techniques and herein. Can be isolated using sequence information. Using all or part of the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 26 as a hybridization probe, FGF-CX nucleic acid sequences can be synthesized using standard hybridization and cloning techniques (eg, Sambrook et al., Eds., MOLECULAR CLONING: ALABORATORY MANUAL 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel et al., Eds., CURRENT PROTOCOLS INMOILEBOILON, YOULYON ).
[0067]
The nucleic acids of the invention can be amplified using cDNA, mRNA or genomic DNA as a template, and appropriate oligonucleotide primers, according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into a suitable vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to FGF-CX nucleotide sequences can be prepared by standard synthetic techniques, eg, using an automated DNA synthesizer.
[0068]
As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues, wherein the oligonucleotide has a sufficient number of nucleotide bases that can be used in a PCR reaction. Short oligonucleotide sequences can be based on or designed from genomic or cDNA sequences, and amplify, confirm, or their presence in a particular cell or tissue the same, similar or complementary DNA or RNA. Used to indicate Oligonucleotides comprise a portion of a nucleic acid sequence having a length of about 10 nt, 50 nt, or 100 nt, preferably about 15 nt to 30 nt. In one embodiment, the oligonucleotide comprising a nucleic acid molecule of less than 100 nt in length further comprises at least six consecutive nucleotides of SEQ ID NO: 1 or SEQ ID NO: 26, or the complement thereof. Oligonucleotides can be chemically synthesized and used as probes.
[0069]
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is the complement of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 26. In another embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleic acid molecule that is the complement of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 26, or a portion of the nucleotide sequence. A nucleic acid molecule that is complementary to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 26 is a nucleic acid molecule that is sufficiently complementary to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 26, It can hydrogen bond with little or no mismatch to the nucleotide sequence shown at 26, thereby forming a stable duplex.
[0070]
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule, and the term "binding" refers to two polypeptides or compounds or related polypeptides or compounds or It means a physical or chemical interaction between those combinations. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. Physical interactions can be either direct or indirect. An indirect interaction may be through or due to another polypeptide or compound. Direct binding refers to an interaction that is neither through another polypeptide or compound, nor occurs due to its effects, and is free of other substantial chemical intermediates.
[0071]
Further, the nucleic acid molecules of the invention may encode only a portion of the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 26 (e.g., a fragment that can be used as a probe or primer, or a biologically active portion of FGF-CX Fragment). The fragments provided herein provide for specific hybridization of at least six (contiguous) nucleic acid sequences or at least four (contiguous) amino acid sequences (each in the case of nucleic acids, Or, in the case of amino acids, a length sufficient to allow specific recognition of the epitope) and is at most some shorter than the full length sequence. Fragments can be derived from any contiguous portion of a selected nucleic acid or amino acid sequence. A derivative is a nucleic acid or amino acid sequence formed from a native compound, either directly or by modification or partial substitution. An analog is a nucleic acid or amino acid sequence that has a structure similar to (but not identical to) a native compound, but that differs from the native compound with respect to particular components or side chains. Analogs may be synthetic or derived from an evolutionarily different source and may have a similar or opposite metabolic activity as compared to the wild type.
[0072]
Derivatives and analogs can be full length or other than full length if the derivative or analog comprises a modified nucleic acid or amino acid, as described below. In various embodiments, a derivative or analog of a nucleic acid or protein of the present invention, when compared to a nucleic acid or protein of the present invention over an identically sized nucleic acid or amino acid sequence, or with an aligned sequence, wherein the alignment is Performed by computer homology programs known in the art), at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (a preferred identity is 80%). ~ 99%) or the nucleic acid encoding it hybridizes under stringent, moderately stringent, or low stringent conditions to the complement of the sequence encoding the protein. Includes, but is not limited to, molecules containing regions that can soy. See, e.g., Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below. Exemplary programs include the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX (registered trademark), Genetics Computer Group, University Research Park, Madison, Wisconsin, Wisconsin, Wisconsin, Wisconsin; Appl. Math., 1981, 2: 482-489, which are incorporated herein by reference in their entirety).
[0073]
"Homologous nucleic acid sequence" or "homologous amino acid sequence", or a variant thereof, refers to a sequence characterized by homology at the nucleotide or amino acid level, as discussed above. A homologous nucleotide sequence encodes a sequence that encodes an isoform of the FGF-CX polypeptide. Isoforms can be expressed in different tissues of the same organism, for example, as a result of alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the present invention, homologous nucleotide sequences include non-human species, including but not limited to mammals, and thus include, for example, mice, rats, rabbits, dogs, cats, cows, horses, and other organisms. And FGF-CX polypeptides that may be mentioned. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variants and variants of the nucleotide sequences described herein. However, a homologous nucleotide sequence does not include the nucleotide sequence encoding the human FGF-CX protein. Homologous nucleic acid sequences include conservative amino acid substitutions of SEQ ID NO: 2 or SEQ ID NO: 27 (see below), and nucleic acid sequences encoding polypeptides having FGF-CX activity. The biological activity of the FGF-CX protein is described below. A homologous amino acid sequence does not encode the amino acid sequence of a human FGF-CX polypeptide.
[0074]
The nucleotide sequence determined from the cloning of the human FGF-CX gene identifies and / or clones FGF-CX homologs in other cell types (eg, from other tissues), as well as FGF-CX homologs from other mammals. Allows the production of probes and primers designed for use in The probe / primer typically comprises a substantially purified oligonucleotide. The oligonucleotide typically comprises at least about 12, about 25, about 50, about 100, about 150, about 200, about 250, about 300 of SEQ ID NO: 1 or SEQ ID NO: 26. About 350 or more than about 400 contiguous sense strand nucleotide sequences; or the antisense strand nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 26; or a naturally occurring variant of SEQ ID NO: 1 or SEQ ID NO: 26, Region of the nucleotide sequence that hybridizes under mild conditions.
[0075]
Probes based on the human FGF-CX nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached to the probe, for example, the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used, for example, to reduce the level of FGF-CX-encoding nucleic acid in a sample of cells from a subject as part of a diagnostic test kit for identifying cells or tissues that mis-express the FGF-CX protein. It can be used by measuring (eg, detecting FGF-CX mRNA levels or determining whether a genomic FGF-CX gene is mutated or deleted).
[0076]
“Polypeptide having a biologically active portion of FGF-CX” refers to a polypeptide that exhibits activity similar to, but not necessarily identical to, the activity of a polypeptide of the present invention, whether or not dose-dependent. , Including the mature form as measured in a particular biological assay. A nucleic acid fragment encoding a “biologically active portion of FGF-CX” is a polypeptide having the biological activity of FGF-CX (the biological activity of the FGF-CX protein is described below). Is isolated, expressing the encoded portion of the FGF-CX protein (e.g., by recombinant expression in vitro), and encoding the FGF-CX encoded It can be prepared by assessing the activity of the moiety. For example, a nucleic acid fragment encoding a biologically active portion of FGF-CX can optionally include an ATP binding domain. In another embodiment, a nucleic acid fragment encoding a biologically active portion of FGF-CX comprises one or more regions.
[0077]
(Variant of FGF-CX)
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 26 due to the degeneracy of the genetic code. Thereby, these nucleic acids encode the same FGF-CX protein as the protein encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 26. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence that encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 27.
[0078]
In addition to the human FGF-CX nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 26, DNA sequence polymorphisms that lead to changes in the amino acid sequence of FGF-CX may be present in a population (eg, a human population). It will be understood by those skilled in the art. Such genetic polymorphisms in the FGF-CX gene may exist between individuals in a population due to natural allelic variations. As used herein, the terms "gene" and "recombinant gene" refer to a nucleic acid molecule comprising an open reading frame encoding a FGF-CX protein, preferably a mammalian FGF-CX protein. Such natural allelic variations can typically result in 1-5% variability in the nucleotide sequence of the FGF-CX gene. Any and all such nucleotide variations within FGF-CX and the resulting amino acid polymorphisms that are the result of natural allelic variation and do not alter the functional activity of FGF-CX are within the scope of the invention. It is intended to be
[0079]
In addition, nucleic acid molecules encoding FGF-CX proteins from other species, and thus having a nucleotide sequence that differs from the human sequence of SEQ ID NO: 1 or SEQ ID NO: 26, are intended to be within the scope of the invention. . Nucleic acid molecules corresponding to natural allelic variants and homologs of the FGF-CX cDNAs of the present invention may be used under stringent hybridization conditions based on their homology to the human FGF-CX nucleic acids disclosed herein. Can be isolated using human cDNA or a portion thereof as a hybridization probe according to standard hybridization techniques. For example, soluble human FGF-CX cDNA can be isolated based on its homology to human membrane-bound FGF-CX. Similarly, membrane-bound human FGF-CX cDNA can be isolated based on its homology to soluble human FGF-CX.
[0080]
Thus, in another embodiment, an isolated nucleic acid molecule of the present invention is at least 6 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 26. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length. In another embodiment, an isolated nucleic acid molecule of the invention hybridizes to a coding region. As used herein, the term "hybridizes under stringent conditions" refers to conditions under which, for hybridization and washing, nucleotide sequences that are at least 60% homologous to each other typically remain hybridized to each other. It is intended to be described.
[0081]
Homologs (i.e., nucleic acids encoding FGF-CX proteins from non-human species) or other related sequences (e.g., paralogs) can be probed with all or a portion of a particular human sequence, using nucleic acid hybridization. And may be obtained by low, moderate or high stringency hybridization using methods well known in the art for cloning.
[0082]
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide hybridizes to its target sequence but does not hybridize to other sequences. Say. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 ° C. below the thermal melting point (Tm) of the particular sequence at a defined ionic strength and pH. This Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequence is generally present in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions are pH 7.0-8.3 and salt concentrations less than about 1.0 M sodium ion, typically about 0.01-1.0 M sodium ion (or other Salt), and conditions such that the temperature is at least about 30 ° C. for short probes, primers or oligonucleotides (eg, 10 nt to 50 nt) and at least about 60 ° C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
[0083]
Stringent conditions as described above are known to those of skill in the art, and are described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NJ. Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences that are at least about 65%, about 70%, about 75%, about 85%, about 90%, about 95%, about 98% or about 99% homologous to each other, typically to each other. Conditions that remain hybridized. Non-limiting examples of stringent hybridization conditions include 6 × SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg Hybridization at 65 ° C. in a high salt buffer containing / ml denatured salmon sperm DNA. After this hybridization, the plate is washed at least once in 0.2 × SSC, 0.01% BSA at 50 ° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule having a naturally occurring nucleotide sequence (eg, encoding a naturally occurring protein).
[0084]
Homologs (i.e., nucleic acids encoding FGF-CX proteins from non-human species) or other related sequences (e.g., paralogs) can be probed with all or a portion of a particular human sequence and used for nucleic acid hybridization and cloning. It may be obtained by low, moderate or high stringency hybridization using methods well known in the art.
[0085]
In a second embodiment, there is provided a nucleic acid sequence capable of hybridizing under moderate stringency conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 26, or a fragment, analog or derivative thereof. Non-limiting examples of moderate stringency hybridization conditions include hybridization in 6 × SSC, 5 × Denhardt's solution, 0.5% SDS and 100 mg / ml denatured salmon sperm DNA at 55 ° C., followed by Wash one or more times at 37 ° C. in 1 × SSC, 0.1% SDS. Other conditions of moderate stringency that can be used are well known in the art. See, for example, Ausubel et al.
[0086]
In a third embodiment, there is provided a nucleic acid capable of hybridizing under low stringency conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 26, or a fragment, analog or derivative thereof. Non-limiting examples of low stringency hybridization conditions include 35% formamide, 5 × SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0. Hybridization at 40 ° C. in 2% BSA, 100 mg / ml denatured salmon sperm DNA, 10% (weight / volume) dextran sulfate, followed by 2 × SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0% Wash at least once at 50 ° C. in 1% SDS. Other conditions of low stringency that can be used are well known in the art (eg, as used for species cross-hybridization). See, for example, Ausubel et al. (Eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY and Kriegler, 1990, GENE TRANSFER AND EXARBION, EXPRESS, ALBAS ACCESS, AT,,,,,,,,,,,,,,,,,,,,,,,,,, See Sci USA 78: 6789-6792.
[0087]
(Conservative mutation)
In addition to naturally occurring allelic variants of the FGF-CX sequence that may be present in the population, those skilled in the art will appreciate that alterations can be introduced by mutations to the nucleotide sequence of SEQ ID NO: 1 or 26, whereby FGF -It is further understood that changes are made in the amino acid sequence of the encoded FGF-CX protein without altering the functional capacity of the CX protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1 or 26. "Nonessential" amino acid residues are those residues that can be altered from the wild-type sequence of FGF-CX without altering the biological activity, while "essential" amino acid residues are those that Is required for For example, amino acid residues conserved among the FGF-CX proteins of the present invention are expected to be particularly unacceptable for changes.
[0088]
In addition, amino acid residues conserved between FGF family members are not expected to be sensitive to changes, as indicated by the alignment represented as FIG. For example, an FGF-CX protein of the invention can include at least one domain that is a region typically conserved in members of the FGF family (ie, FGF-9 and XFGF-CX proteins, and FGF-CX homologs). . As such, these conserved domains are unlikely to be sensitive to mutations. However, other amino acid residues (eg, amino acid residues that are not conserved or are only semi-conserved among members of the FGF protein) may not be essential for activity and are therefore more susceptible to changes. Seems to respond to
[0089]
Another aspect of the invention pertains to nucleic acid molecules encoding FGF-CX proteins that include changes in amino acid residues that are not essential for activity. Such FGF-CX proteins differ in amino acid sequence from SEQ ID NO: 2 or 27, but still retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein, wherein the protein comprises an amino acid sequence that is at least about 75% homologous to the amino acid sequence of SEQ ID NO: 2 or 27. . Preferably, the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO: 2 or 27, more preferably at least about 90%, about 95%, about 98% homologous, and most preferably the sequence At least about 99% homologous to number 2.
[0090]
An isolated nucleic acid molecule encoding an FGF-CX protein homologous to the protein of SEQ ID NO: 2 is made by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS: 1 or 26. As a result, one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
[0091]
Mutations can be introduced into SEQ ID NO: 1 or 26 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more putative non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. A particular amino acid has reference to a side chain that has more than one categorizable characteristic. These families include: amino acids having a basic side chain (eg, lysine, arginine, histidine), amino acids having an acidic side chain (eg, aspartic acid, glutamic acid), having an uncharged polar side chain Amino acids (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), amino acids having non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), Amino acids with β-branched side chains (eg, threonine, valine, isoleucine) and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, a putative non-essential amino acid residue in a growth factor is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, the mutation can be introduced randomly (eg, by saturation mutagenesis) along all or part of the growth factor coding sequence, and the resulting mutant Can be screened for biological activity to identify those variants that retain activity. Following mutagenesis of SEQ ID NOs: 1, 3, and 26, the encoded protein can be expressed by any recombinant technique known in the art, and the activity of the protein can be determined.
[0092]
In one important embodiment, the mutant FGF-CX protein may be assayed for: (1) other FGF-CX proteins, other cell surface proteins, or biologically active portions thereof, and the protein : Ability to form protein interactions, (2) complex formation between mutant FGF-CX protein and FGF-CX receptor; (3) mutant FGF-CX protein is an intracellular target protein or its biological (E.g., avidin protein); or (4) the ability to specifically bind to an anti-FGF-CX protein antibody.
[0093]
(Antisense)
Another aspect of the invention relates to an isolated antisense nucleic acid molecule that is capable of hybridizing to or complementary to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 26 or a fragment, analog or derivative thereof. . An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid that encodes a protein (eg, complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). . In certain aspects, an antisense nucleic acid comprising a sequence complementary to at least about 10, about 25, about 50, about 100, about 250 or about 500 nucleotides or the entire FGF-CX coding strand, or only a portion thereof. A molecule is provided. Further provided are nucleic acid molecules encoding fragments, homologs, derivatives and analogs of the FGF-CX protein of SEQ ID NO: 2 or 27, or antisense nucleic acids complementary to the nucleic acid sequence of FGF-CX of SEQ ID NO: 1 or 26.
[0094]
In one embodiment, the antisense nucleic acid molecule is antisense to the "coding region" of the coding strand of a nucleotide sequence encoding FGF-CX. The term "coding region" refers to a region of the nucleotide sequence that includes codons translated into amino acid residues (e.g., the protein coding region of human FGF-CX corresponding to SEQ ID NO: 2 or 27). In another embodiment, the antisense nucleic acid molecule is antisense to a "non-coding region" of the coding strand of a nucleotide sequence encoding FGF-CX. The term "non-coding region" refers to 5 'and 3' sequences that are adjacent to the coding region and are not translated into amino acids (ie, also referred to as 5 'and 3' untranslated regions).
[0095]
In view of the coding strand sequence encoding FGF-CX disclosed herein (e.g., SEQ ID NOS: 1 or 26), the antisense nucleic acids of the present invention are compatible with Watson and Crick or Hoogsteen base pairing rules. Can be designed according to The antisense nucleic acid molecule can be complementary to the entire coding region of FGF-CX mRNA, but more preferably is an oligonucleotide that is antisense only to a portion of the coding or non-coding region of FGF-CX mRNA. . For example, an antisense oligonucleotide can be complementary to the region surrounding the translation start site of FGF-CX mRNA. An antisense oligonucleotide can be, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 nucleotides in length. Antisense nucleic acids of the invention can be constructed using chemical synthesis or enzymatic ligation using procedures known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) are formed to increase the biological stability of naturally occurring nucleotides, or the molecule, or between the antisense and sense nucleic acids. It can be chemically synthesized using various modified nucleotides designed to increase the physical stability of the duplex (eg, phosphorothioate derivatives and acridine substituted nucleotides can be used).
[0096]
Examples of modified nucleotides that can be used to make antisense nucleic acids include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- Acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcuosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylamino Methylura 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylcuosin, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid ( v), wife toxosin, pseudouracil, cuocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (V), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector in which the nucleic acid has been subcloned in the antisense orientation (ie, RNA transcribed from the inserted nucleic acid is further described in the following subsections). In the antisense direction to the target nucleic acid of interest).
[0097]
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ, such that they hybridize to cellular mRNA and / or genomic DNA encoding an FGF-CX protein. Or binds thereto, thereby inhibiting the expression of the protein, for example, by inhibiting transcription and / or translation. Hybridization can be by conventional nucleotide complementarity forming a stable duplex or, for example, in the case of an antisense nucleic acid molecule binding to a DNA duplex, specific in the major groove of the duplex. Through a statistical interaction. Examples of routes of administration of the antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to a receptor or antigen expressed on the selected cell surface. This is, for example, by linking the antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecule, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
[0098]
In yet another embodiment, an antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms a specific double-stranded hybrid with the complementary RNA. Here, in contrast to the normal β-unit, the chains run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The antisense nucleic acid molecule can also include 2'-o-methyl ribonucleotides (Inoue et al., (1987) Nucleic Acids Res 15: 6131- 6148) or chimeric RNA-DNA analogs (Inoue et al. (1987) FEBS Lett 215: 327-). 330).
[0099]
(Ribozyme and PNA part)
Such modifications include, by way of non-limiting example, modified bases and nucleic acids with modified or derivatized sugar phosphate backbones. These modifications are made, at least in part, to enhance the chemical stability of the modified nucleic acid, such that they are used, for example, as antisense-binding nucleic acids in therapeutic applications in a subject. obtain.
[0100]
In yet another embodiment, the antisense nucleic acids of the invention are ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that can cleave single-stranded nucleic acids (eg, mRNA), which have regions complementary to the single-stranded nucleic acids. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) are used to catalytically cleave FGF-CX mRNA transcripts and thereby FGF-CX mRNA transcripts. -It can inhibit the translation of CX mRNA. A ribozyme having specificity for a nucleic acid encoding FGF-CX can be designed based on the nucleotide sequence of FGF-CX DNA disclosed herein (ie, SEQ ID NO: 1). For example, a derivative of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to a nucleotide sequence that is cleaved in mRNA encoding FGF-CX. See, for example, Cech et al., US Pat. No. 4,987,071; and Cech et al., US Pat. No. 5,116,742. Alternatively, FGF-CX mRNA can be used to select a catalytic RNA with specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel et al. (1993) Science 261: 1411-1418.
[0101]
Alternatively, FGF-CX gene expression targets a nucleotide sequence that is complementary to a regulatory region of FGF-CX (eg, an FGF-CX promoter and / or enhancer) and prevents transcription of the FGF-CX gene in target cells. By forming a triple helical structure. Generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene et al. (1992) Ann. N. Y. Acad. Sci. 660: 27-36; and Maher (1992) Bioassays 14: 807-15.
[0102]
In various embodiments, FGF-CX nucleic acids can be modified with a base moiety, a sugar moiety, or a phosphate backbone, for example, to improve the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of a nucleic acid can be modified to produce a peptide nucleic acid (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the term “peptide nucleic acid” or “PNA” refers to a nucleic acid mimetic in which the deoxyribose phosphate backbone has been replaced by a pseudopeptide backbone and only four natural nucleobases have been retained. (Eg, a DNA mimic). The neutral backbone of PNA has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996); Perry-O'Keefe et al. (1996) PNAS 93: 14670-675, supra.
[0103]
FGF-CX PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigenetic agents for sequence-specific regulation of gene expression, for example, by inducing transcription or translation arrest or inhibiting replication. FGF-CX PNAs can also be used to analyze single base pair mutations in genes, for example, by PNA-directed PCR clamping; as an artificial restriction enzyme when used in combination with other enzymes (eg, S1 nuclease) (Hyrup B). (1996) supra); or as a DNA sequence and hybridization probe or primer (Hyrup et al. (1996) supra; Perry-O'Keefe (1996) supra).
[0104]
In another embodiment, the PNAs of FGF-CX can be linked to a PNA-DNA chimera by attaching a lipophilic group or other helper group to the PNAs, for example, to enhance their stability or cellular uptake. It can be modified by formation or by use of liposomes or other techniques of drug delivery known in the art. For example, a PNA-DNA chimera of FGF-CX can be generated that can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (eg, RNase H and DNA polymerase) to interact with the DNA moiety, while the PNA moiety provides high binding affinity and specificity. PNA-DNA chimeras can be ligated using linkers of appropriate length, selected taking into account the stacking of bases, the number and orientation of nucleobase linkages (Hyrup (1996) supra). Synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) supra and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA strand can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs (eg, 5 '-(4-methoxytrityl) amino-5'- Deoxy-thymidine phosphoramidite) can be used between PNA and the 5 'end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). The PNA monomers are then coupled in a stepwise fashion to produce a chimeric molecule having a 5 'PNA segment and a 3' DNA segment (Finn et al. (1996) supra). Alternatively, chimeric molecules can be synthesized using a 5 'DNA segment and a 3' PNA segment. See Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
[0105]
In other embodiments, the oligonucleotide may include other accessory groups, such as: a peptide (eg, for targeting a host cell receptor in vivo), or an agent that facilitates transport across the cell membrane ( For example, Letsinger et al., 1989, Proc.Natl.Acad.Sci.USA 86: 6553-6556; Lemaitre et al., 1987, Proc.Natl.Acad.Sci.84: 648-652; PCT Publication No. WO88. / 09810), or the blood-brain barrier (see, eg, PCT Publication No. WO 89/10134). In addition, the oligonucleotide can be a hybridization-triggered cleavage agent (see, eg, Krol et al., 1988, BioTechniques 6: 958-976), or an intercalating agent (eg, Zon, 1988, Pharm. Res. 5: 539). 549). For this purpose, the oligonucleotide can be conjugated to another molecule, such as a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
[0106]
(FGF-CX polypeptide)
The novel proteins of the present invention include the FGF-CX-like proteins whose sequence is provided in FIG. 1 (SEQ ID NO: 2) or SEQ ID NO: 27. The present invention also provides a protein, or a functional fragment thereof, wherein any of its residues has been changed from the corresponding residue shown in FIG. 1, but retains its FGF-CX-like activity and physiological function. Or mutant or variant proteins. In this mutant or variant protein, up to 20% or more of the residues can be so altered.
[0107]
Generally, an FGF-CX-like variant that retains an FGF-CX-like function is one in which the residue at a particular position in the sequence is replaced by another amino acid, and furthermore, additional residues between the two residues of the original protein. And any variants that include the possibility of deleting one or more residues from the original sequence. Any amino acid substitutions, insertions or deletions are encompassed by the present invention. In a preferred situation, the substitution is a conservative substitution as defined above. Further, without limiting the scope of the invention, the following positions in Table 2 (using the numbering defined in SEQ ID NO: 2) may be substituted as indicated, so that the mutant or A variant protein may include one or more of the indicated substitutions. The suggested substitutions do not limit the scope of possible substitutions that may be made at a given position.
[0108]
(Table 2)
Position Possible substitution
6: Glu to Asp
9: Gly to Ser, Thr, or Asn
10: Phe to Tyr
11: Leu to Phe or Ile
15: Glu to Asp
16: Gly to Ala
17: Leu to Ile or Val
19: Gln may be deleted
21: Val to Phe or Ile
31: Gly to Lys, Arg, Ser, or Ala
33: Arg to Lys or Ser
35: Pro to Leu or Val
38: Gly to Asn or Ser
39: Glu to Asp
40: Arg to Lys, His, or Pro
42: Ser to Thr, Ala, or Gly
43: Ala to Gln, Asn, or Ser
48: Ser or Gly from Ala
51: Gly to Ala
53: Ala or deletion from Gly
54: Gly, Val, or deletion from Ala
55: Ser or Thr from Ala
56: Gln to Asp, Glu, or Asn
58: Ala to Ser, Thr, Asn, Gln, Asp, or Glu
61: His to Gln, Asn, Lys, or Arg
78: Gln to Asn, Glu, or Asp
80: Leu to Phe or Ile
82: Asp to Glu, Asn, or Gln
84: Ser to Asn, Thr, or Gln
85: Val to Ile
90: Gln to Asn or Lys
103: Val to Ile
115: Ser to Thr
123: Asp to Glu
128: Tyr to Phe
135: Ser to Thr, Gln, or Asn
138: Ile to Val or Leu
155: Ile to Leu
159: Gly to Val or Ala
161: Thr to Ser
166: Phe to Tyr
177: Asp to Glu
181: Ser to Ala or Thr
198: Glu to Asp
199: Arg to Lys
207: Leu to Ile or Val
209: any residue from Met
211: Thr to Ser
One aspect of the present invention relates to isolated FGF-CX proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-FGF-CX antibodies. In one embodiment, native FGF-CX protein can be isolated from a cell or tissue source by a suitable purification scheme using standard protein purification techniques. In another embodiment, the FGF-CX protein is produced by recombinant DNA technology. Instead of recombinant expression, an FGF-CX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
[0109]
An “isolated” or “purified” protein, or biologically active portion thereof, is substantially free of cellular material or other contaminating proteins from cells or tissue sources from which the FGF-CX protein is derived. It is free or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of FGF-CX protein in which the FGF-CX protein has been separated from the cellular components of the isolated or recombinantly produced cell. In one embodiment, the term “substantially free of cellular material” refers to less than about 30% (by dry weight) of non-FGF-CX protein (also referred to herein as “contaminating protein”); More preferably, the FGF-CX protein has less than about 20% non-FGF-CX protein, even more preferably less than about 10% non-FGF-CX protein, and most preferably less than about 5% non-FGF-CX protein. Including preparations. If the FGF-CX protein or a biologically active portion thereof is produced recombinantly, preferably the preparation is also substantially free of culture medium. That is, the culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
[0110]
The term "substantially free of chemical precursors or other chemicals" refers to a preparation of an FGF-CX protein in which the protein is separated from the chemical precursors or other chemicals involved in the synthesis of the protein. Including. In one embodiment, the term "substantially free of chemical precursors or other chemicals" means less than about 30% (by dry weight) of chemical precursors or non-FGF-CX chemicals, more preferably Has less than about 20% of a chemical precursor or non-FGF-CX chemical, even more preferably less than about 10% of a chemical precursor or non-FGF-CX chemical, and most preferably a chemical precursor or non-FGF-CX chemical. Include preparations of FGF-CX protein that have less than about 5% material.
[0111]
The biologically active portion of the FGF-CX protein comprises fewer amino acids than the full-length FGF-CX protein and exhibits at least one activity of the FGF-CX protein (eg, SEQ ID NO: 2) or a peptide containing an amino acid sequence derived from the amino acid sequence of the FGF-CX protein. Typically, a biologically active portion comprises a domain or motif having at least one activity of an FGF-CX protein. A biologically active portion of an FGF-CX protein can be, for example, a polypeptide that is 10, 25, 50, 100, or more amino acid residues in length.
[0112]
A biologically active portion of an FGF-CX protein of the present invention can include at least one of the above-identified domains that is substantially conserved between FGF family proteins. In addition, other biologically active portions lacking other regions can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native FGF-CX protein. obtain.
[0113]
In one embodiment, the FGF-CX protein has the amino acid sequence shown in SEQ ID NO: 2 or 27. In other embodiments, the FGF-CX protein is substantially homologous to SEQ ID NO: 2 or 27, and as described in more detail below, contains amino acids due to natural allelic variants or mutagenesis. The sequences differ, but retain the functional activity of the protein of SEQ ID NO: 2 or 27. Thus, in another embodiment, the FGF-CX protein is a protein comprising amino acids having at least about 45% homology to the amino acid sequence of SEQ ID NO: 2 or 27, and the FGF-CX protein of SEQ ID NO: 2 or 27. A protein that retains the functional activity of the protein. The FGF-CX protein may, in another embodiment, be at least about 45%, and more preferably 55%, 65%, 70%, 75%, 80% of the amino acid sequence of SEQ ID NO: 2 or 27. , 85%, 90%, 95%, 98% or even 99% homologous amino acid sequence, and the corresponding polypeptide having the sequence of SEQ ID NO: 2 or 27, the functional activity of the FGF-CX protein Is a protein that retains
[0114]
(Determination of homology between two or more sequences)
To determine the percentage of homology between two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (eg, gaps are used for optimal alignment between sequences). Can be introduced into any sequence to be compared). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (ie, as used herein). , Amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
[0115]
Nucleic acid sequence homology can be determined as the degree of identity between two sequences. Homology can be determined using computer programs known in the art, such as the GAP software provided in the GCG program package. See Needleman and Wunsch, 1970 J Mol Biol 48: 443-453. Using the GCG GAP software with the following settings for nucleic acid sequence comparison (GAP production penalty, 5.0, and GAP extension penalty, 0.3), the coding region for similar nucleic acid sequences referred to above , Preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to the CDS (coding) portion of the DNA sequence shown in SEQ ID NO: 1 or 26. Is shown.
[0116]
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by: comparing two sequences optimally aligned over this comparison region, nucleobases identical in both sequences (eg, A in the case of nucleic acids). , T, C, G, U, or I) to determine the number of locations where there is, and derive the number of matched positions, and determine the number of matched positions by the total number of locations in the comparison area (ie, (Window size) and multiplying the result by 100 to derive a percentage of sequence identity. The term "substantial identity," as used herein, refers to a characteristic of a polynucleotide sequence, wherein the polynucleotide has at least 80% sequence over a comparison region compared to a reference sequence. Include sequences that have identity, preferably at least 85% sequence identity, and often 90-95% sequence identity, more usually at least 99% sequence identity. The term “percentage of positive residues” is calculated by: comparing two sequences optimally aligned over their comparison region, wherein identical amino acid and conservative amino acid substitutions Determining the number of positions present in both sequences and deriving the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison region (ie, window size), and Multiply the result by 100 to derive the percentage of positive residues.
[0117]
(Chimeric and fusion proteins)
The present invention also provides FGF-CX chimeric or fusion proteins. As used herein, an FGF-CX “chimeric protein” or FGF-CX “fusion protein” comprises an FGF-CX polypeptide operably linked to a non-FGF-CX polypeptide. “FGF-CX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to FGF-CX, and “non-FGF-CX polypeptide” is a protein that is not substantially homologous to an FGF-CX protein (eg, , A protein different from the FGF-CX protein and corresponding to the same or different organism). In an FGF-CX fusion protein, the FGF-CX polypeptide may correspond to all or a portion of an FGF-CX protein. In one embodiment, an FGF-CX fusion protein comprises at least one biologically active portion of an FGF-CX protein. In another embodiment, an FGF-CX fusion protein comprises at least two biologically active portions of an FGF-CX protein. In another embodiment, the FGF-CX fusion protein comprises at least two biologically active portions. In a fusion protein, the term "operably linked" is intended to indicate that the FGF-CX polypeptide and the non-FGF-CX polypeptide are fused together in frame. Non-FGF-CX polypeptides can be fused to the N-terminus or C-terminus of the FGF-CX polypeptide.
[0118]
For example, in one embodiment, an FGF-CX fusion protein comprises an FGF-CX polypeptide operably linked to the extracellular domain of a second protein. Such fusion proteins can further be used in screening assays for compounds that modulate FGF-CX activity (such assays are described in detail below).
[0119]
In another embodiment, the fusion protein is a GST-FGF-CX fusion protein, wherein the FGF-CX sequence is fused to the C-terminus of a GST (ie, glutathione S-transferase) sequence. Such a fusion protein may facilitate purification of the recombinant FGF-CX.
[0120]
In yet another embodiment, the fusion protein is an FGF-CX protein that includes a heterologous signal sequence at the N-terminus. For example, the native FGF-CX signal sequence (ie, amino acids 1-20 of SEQ ID NO: 2 or 27) can be removed and replaced with a signal sequence from another protein. In certain host cells (eg, mammalian host cells), expression and / or secretion of FGF-CX can be increased through the use of a heterologous signal sequence.
[0121]
In another embodiment, the fusion protein is an FGF-CX-immunoglobulin fusion protein, wherein the FGF-CX sequence is fused to one or more domains from a sequence derived from a member of the immunoglobulin protein family. including. This FGF-CX-immunoglobulin fusion protein of the invention is incorporated into a pharmaceutical composition and administered to a subject to allow interaction between the FGF-CX ligand and the FGF-CX protein on the surface of cells. It may inhibit action, thereby suppressing FGF-CX-mediated signaling in vivo. In one non-limiting example, a possible FGF-CX ligand is an FGF-CX receptor. This FGF-CX-immunoglobulin fusion protein can be used to affect the bioavailability of a FGF-CX cognate ligand. Inhibition of the FGF-CX ligand / FGF-CX interaction may be therapeutically useful for treating both proliferative and differentiation disorders, and for modulating (eg, promoting or inhibiting) cell survival. Further, this FGF-CX-immunoglobulin fusion protein of the present invention can be used as an immunogen to raise anti-FGF-CX antibodies in a subject, purifying FGF-CX ligand, and Can be used in screening assays to identify molecules that inhibit the interaction of FGF-CX with.
[0122]
An FGF-CX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences may be ligated in accordance with conventional techniques, eg, blunt or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, Cohesive ends are ligated in-frame by fill-in, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In another embodiment, the fusion gene may be synthesized by conventional techniques including an automated DNA synthesizer. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that create a complementary overhang between two consecutive gene fragments, and these fragments can then be annealed and reamplified to produce a chimeric gene sequence (eg, Ausbel et al. (Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). In addition, many expression vectors are commercially available that already encode a fusion component (eg, a GST polypeptide). The nucleic acid encoding FGF-CX can be cloned into such an expression vector such that the fusion component is linked in-frame to the FGF-CX protein.
[0123]
(FGF-CX agonist and antagonist)
The present invention also relates to variants of the FGF-CX protein that function as FGF-CX agonists (mimetics) or as FGF-CX antagonists. Variants of the FGF-CX protein can be generated by mutagenesis (eg, discrete point mutations or truncations of the FGF-CX protein). An agonist of an FGF-CX protein may retain substantially the same biological activity, or a subset of that biological activity, as the naturally occurring form of the FGF-CX protein. An antagonist of an FGF-CX protein can competitively bind one or more activities of a naturally occurring form of the FGF-CX protein, eg, to a downstream or upstream member of a cell signaling cascade that includes the FGF-CX protein. Can be inhibited by Thus, specific biological effects can be induced by treatment with variants of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activity of the naturally occurring form of the protein is less than treatment with a naturally occurring form of the FGF-CX protein. Has fewer side effects in the subject.
[0124]
Variants of the FGF-CX protein that function as either FGF-CX agonists (mimetics) or as FGF-CX antagonists are variants of the FGF-CX protein (eg, truncations) for FGF-CX protein agonist or antagonist activity. (Type variants) by screening combinatorial libraries. In one embodiment, a versatile library of FGF-CX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a versatile gene library. A versatile library of FGF-CX variants can be used, for example, to mix mixtures of synthetic oligonucleotides, degenerate sets of potential FGF-CX sequences, as individual polypeptides, or within FGF-CX sequences. It can be produced by enzymatically linking to a gene sequence so that it can be expressed as a larger set of fusion proteins comprising the set (eg, for phage display). There are various methods that can be used to generate libraries of potential FGF-CX variants from degenerate oligonucleotide sequences. Chemical synthesis of the degenerate gene sequence can be performed in an automated DNA synthesizer, and the synthetic gene is then ligated into an appropriate expression vector. The use of a degenerate set of genes allows the provision of all of the sequences encoding the desired set of potential FGF-CX sequences in one mixture. Methods for synthesizing degenerate oligonucleotides are well known in the art (eg, Narang (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucl. Acid Res. 11: 477).
[0125]
(Polypeptide library)
In addition, a library of fragments of the FGF-CX protein coding sequence can be used to generate a diverse population of FGF-CX fragments for screening and subsequent selection of variants of the FGF-CX protein. In one embodiment, the library of coding sequence fragments comprises treating a double-stranded PCR fragment of an FGF-CX coding sequence with a nuclease under conditions that produce only about one nick per molecule. Denaturing the DNA, regenerating the DNA to form a double-stranded DNA that may contain sense / antisense pairs from different nick products, from re-forming the duplex by treatment with S1 nuclease It can be produced by removing the single-stranded portion and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be obtained that encode N-terminal and internal fragments of various sizes of the FGF-CX protein.
[0126]
Various techniques are known in the art for screening gene products of combinatorial libraries created by point mutation or truncation, and for screening cDNA libraries of gene products having selected properties. Such techniques are applicable to rapid screening of gene libraries generated by combinatorial mutagenesis of FGF-CX proteins. The most widely used techniques suitable for high-throughput analysis for screening large gene libraries are typically cloning gene libraries into replicable expression vectors, and libraries of resulting vectors. Transforming the appropriate cells, and expressing the combinatorial gene under conditions that detecting the desired activity include conditions that facilitate isolation of a vector encoding the gene whose product was detected. A novel technique for increasing the frequency of functional variants in the library, Recruitable ensemble mutagenesis (REM), can be used in conjunction with screening assays to identify FGF-CX variants ( See, e.g., Arkin and Youvan (1992) PNAS 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6: 327-331).
[0127]
(Anti-FGF-CX antibody)
As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules (ie, specifically binds (immunoreacts with) an antigen. Molecule containing an antigen binding site). Such antibodies include polyclonal, monoclonal, chimeric, single chain, Fab, Fab 'and F (ab') 2 fragments, and F (ab ') 2 fragments. _ab An expression library includes, but is not limited to. In general, human-derived antibody molecules relate to any class of IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chains present in the molecule. Certain classes have subclasses as well (eg, IgG1, IgG2, etc.). Further, in humans, the light chain can be a kappa or lambda chain. References herein to antibodies include references to all such classes, subclasses, and types of human antibody species.
[0128]
Using the isolated protein of the present invention, which is intended to serve as an antigen, or a portion or fragment thereof, as an immunogen, using standard techniques for the preparation of polyclonal and monoclonal antibodies, Antibodies can be generated that immunospecifically bind to. The full length protein can be used, or the invention provides an antigenic peptide fragment of an antigen for use as an immunogen. The antigenic peptide fragment comprises at least six amino acid residues of the amino acid sequence of the full-length protein (eg, the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 27) and includes their epitopes, such that the peptide Antibodies form specific immune complexes with the full-length protein or any fragment containing this epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes comprised by this antigenic peptide are the regions of the protein located on its surface; usually these are hydrophilic regions.
[0129]
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of FGF-CX located on the surface of the protein, for example, a hydrophilic region. Hydrophobic analysis of the human FGF-CX protein sequence will reveal which regions of the FGF-CX polypeptide are particularly hydrophilic, and thereby which regions encode surface residues useful for targeting antibody production. Indicates that it seems to Means for targeting antibody production include hydropathy plots showing hydrophilic and hydrophobic regions, including, for example, the Kyte Doolittle or Hopp Woods method, either with or without Fourier transforms, It can be produced by any method known in the art. See, for example, Hopp and Woods, 1981, Proc. Nat. Acad. Sci. ScL USA 78: 3824-3828; Kyte and Doolittle 1982, J. Am. Mol. Biol. 157: 105-142. Antibodies specific to one or more domains within the antigenic protein or derivative, fragment, analog or homolog thereof are also provided herein.
[0130]
The proteins of the invention, or derivatives, fragments, analogs, homologs or orthologs thereof, can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components.
[0131]
Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the proteins of the invention, or derivatives, fragments, analogs, homologs or orthologs thereof (eg, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference. Some of these antibodies are discussed below.
[0132]
(1. polyclonal antibody)
For the production of polyclonal antibodies, various suitable host animals (eg, rabbits, goats, mice or other animals) may be raised once with a native protein of FGF-CX, a synthetic variant thereof, or a derivative thereof as described above. It can be immunized by the above injection. Suitable immunogenic preparations include, for example, a naturally occurring immunogenic protein, a chemically synthesized polypeptide displaying the immunogenic protein, or a recombinantly expressed immunogenic protein. obtain. Further, the FGF-CX protein can be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation may further include an adjuvant. Various adjuvants have been used to increase the immunological response, such as Freund (complete and incomplete), mineral gels (eg, aluminum hydroxide), surfactants (eg, lysolecithin, pluronic) (Pluronic) polyols, polyanions, peptides, oil emulsions, dinitrophenols, and the like, adjuvants that can be used in humans (eg, Bacille Calmette-Guerin and Corynebacterium parvum) or similar immune stimulants Not done. Further examples of adjuvants that can be used include MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).
[0133]
Polyclonal antibody molecules directed against the immunogenic FGF-CX protein can be isolated from mammals (eg, from blood) and can be isolated by well-known techniques (eg, affinity chromatography using Protein A or Protein G (eg, This can be further purified by mainly providing the IgG fraction of the immune serum))). Subsequently, or alternatively, the specific antigen or epitope of that antigen that is the target of the immunoglobulin sought is immobilized on a column, and the immunospecific antibody can be purified by immunoaffinity chromatography. Purification of immunoglobulins is described, for example, in Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA), Vol. 14, No. 8 (April 17, 2000), pages 25-28.
[0134]
(2. Monoclonal antibody)
As used herein, the term "monoclonal antibody" (MAb) or "monoclonal antibody composition" includes only one species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. Refers to a population of antibody molecules. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all populations of molecules. Thus, MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen, characterized by a unique binding affinity for the antigen.
[0135]
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to produce antibodies that specifically bind to the immunizing agent, or to produce these antibodies. Induces lymphocytes to be obtained. Alternatively, the lymphocytes can be immunized in vitro.
[0136]
The immunizing agent typically includes an FGF-CX protein antigen, a fragment thereof, or a fusion protein thereof. Generally, peripheral blood lymphocytes are used when cells of human origin are desired, or spleen cells or lymph node cells are used when non-human mammalian sources are desired. Either. The lymphocytes are then fused to the immortalized cell line using an appropriate fusion factor (eg, polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59- 103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable culture medium, preferably containing one or more substances, which inhibits the growth and survival of the unfused immortalized cells. For example, if the parent cell lacks the enzyme (hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT)), the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine ("HAT medium"). ), Those substances inhibit the growth of HGPRT deficient cells.
[0137]
Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virgin. Human myeloma and mouse-human myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor: J. Immunol., 133: 3001 (1984); Brodeur et al .: Monoclonal Antibody Production Techniques Applications, Demographics, Applications). , Inc., New York (1987) 51-63).
[0138]
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of a monoclonal antibody directed against the antigen. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay (eg, a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA)). Is done. Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by the method of Munson and Polard, Anal. Biochem. , 107: 220 (1980). It is an object to identify antibodies with a high degree of specificity and high binding affinity for a target antigen, and is particularly important in therapeutic applications of monoclonal antibodies.
[0139]
After the desired hybridoma cells have been identified, clones can be subcloned by limiting dilution procedures and expanded by standard methods (Goding, 1986). For example, culture media suitable for this purpose include Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
[0140]
Monoclonal antibodies secreted by the subclone are isolated from the culture medium or ascites by conventional immunoglobulin purification procedures (eg, protein A sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, etc.). Or it can be purified.
[0141]
Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in US Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the present invention can be readily prepared using conventional procedures (eg, by using oligonucleotide probes capable of binding specifically to the genes encoding the heavy and light chains of the mouse antibody). Can be isolated and sequenced. The hybridoma cells of the present invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then converted to host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that would not otherwise produce immunoglobulin proteins. ) To obtain monoclonal antibody synthesis in recombinant host cells. DNA can also be obtained, for example, by replacing the encoding sequence with human heavy and light chain constant domains in place of the homologous mouse sequence (U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13). (1994)) or by covalent linkage to an immunoglobulin encoding all or part of the coding sequence for a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies of the invention, or for the variable regions of one of the antigen binding sites of the antibodies of the invention, to produce chimeric bivalent antibodies. .
[0142]
(3. Humanized antibody)
Antibodies directed against the FGF-CX protein antigens of the present invention can further include humanized or human antibodies. These antibodies are suitable for administration to humans that do not provoke a human immune response to the administered immunoglobulin. Humanized forms of antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′, F (ab ′)). ₂ Or other antigen-binding sequences of an antibody), which are composed primarily of human immunoglobulin sequences and include minimal sequences derived from non-human immunoglobulins. Humanization is by replacement of the rodent CDR (s) sequence with the corresponding sequence of a human antibody by the method of Winter and coworkers (Jones et al., Nature, 31: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1544-1536 (1988)) (see also US Patent No. 5,225,539). In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. A humanized antibody may also contain residues that are not found in the recipient's antibody, nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions. Within these regions, all or substantially all CDR regions correspond to regions of a non-human immunoglobulin, and all or substantially all framework regions correspond to regions of a human immunoglobulin consensus sequence. The humanized antibody also optionally contains at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)).
[0143]
(4. Human antibody)
Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light and heavy chains, including the CDRs, arise from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies directed against the FGF-CX protein can be prepared by trioma technology; human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72) and human monoclonal antibodies. EBV hybridoma technology for production (see Cole et al., 1985: Monoclonal Antibodies and Cancer Therapy, Alan R. License, Inc., pp. 77-96). Human monoclonal antibodies can be used in the practice of the present invention and can be used by the use of human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80: 2026-1030), or in vitro Epstein Barr Virus. (Cole et al., 1985: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0144]
In addition, human antibodies have also been developed using additional techniques (Phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)). ). Similarly, human antibodies can be made by introducing a human immunoglobulin locus into a transgenic animal (eg, a mouse in which the endogenous immunoglobulin gene has been partially or completely inactivated). Upon challenge, human antibody production was observed, which closely resembles in all respects what was observed in humans (including gene rearrangement, assembly, and antibody repertoire). This approach is described, for example, in: US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; Nos. 5,633,425; 5,661,016, and Marks et al. (Bio / Technology 10,779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison (Nature) 368, 812-13 (1994)); Fishwild et al. (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. .Immunol.13 65~93 (1995)).
[0145]
Human antibodies that specifically bind to the FGF-CX protein can be obtained using transgenic non-human animals that have been modified to produce fully human antibodies rather than the animal's endogenous antibodies in response to antigen challenge. It can be produced further (see PCT publication WO 94/02602). Endogenous genes encoding heavy and light chain immunoglobulins in non-human hosts have become intolerable, and active loci encoding human heavy and light chain immunoglobulins have been inserted into the host genome. Is done. For example, human genes are integrated using yeast artificial chromosomes containing the required human DNA segments. An animal that provides all the desired modifications is then obtained as a progeny by hybridizing an intermediate transgenic animal that contains less than the complement of the fully modified modification. A preferred embodiment of such a non-human animal is a mouse, Xenomoous as disclosed in PCT publications WO 96/33735 and WO 96/34096. ^TM Called. This animal produces B cells that secrete human immunoglobulin well. Obtaining antibodies directly from animals after immunization with the desired FGF-CX immunogen (eg, preparation of polyclonal antibodies) or from immortalized B cells derived from animals (eg, hybridomas producing monoclonal antibodies) Is possible. In addition, genes encoding immunoglobulins having human variable regions can be restored and directly expressed to obtain the antibody, or further modified to obtain an analog of the antibody (eg, a single chain Fv molecule). Can be done.
[0146]
An example of a method of making a non-human host lacking expression of an endogenous immunoglobulin heavy chain (exemplified as a mouse) is described in US Pat. No. 5,939,598. It can be obtained by a method comprising the steps of: preventing rearrangement of the locus and forming a transcript of the rearranged immunoglobulin heavy chain locus, and a gene encoding a selectable marker Deleting a J segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent a deletion affected by a targeting vector comprising: and a transgenic mouse, whose somatic and germ cells are , Including a gene encoding a selectable marker) from embryonic stem cells.
[0147]
Methods for producing an antibody of interest (eg, a human antibody) are disclosed in US Pat. No. 5,916,771. This involves the steps of: introducing an expression vector comprising a nucleotide sequence encoding the heavy chain into one mammalian host cell in culture, providing an expression vector comprising the nucleotide sequence encoding the light chain, Introducing into another mammalian host cell, and fusing the two cells to form a hybrid cell. Hybrid cells express antibodies that include heavy and light chains.
[0148]
In a further refinement of this procedure, methods for identifying clinically relevant epitopes on immunogens and correlating methods for selecting antibodies that immunospecifically bind with high affinity to relevant epitopes include: It is disclosed in PCT publication WO 99/53049.
[0149]
(5. Fab fragments and single chain antibodies)
In accordance with the present invention, techniques can be applied to the production of single chain antibodies specific for the antigenic FGF-CX proteins of the present invention (see, eg, US Patent No. 4,946,778). In addition, the method can be applied to the construction of a Fab expression library (see, eg, Huse et al., 1989 Science 246: 1275-1281), with the desired specificity for the protein or derivative, fragment, analog or homolog thereof. Enables rapid and effective identification of monoclonal Fab fragments having Antibody fragments containing the idiotype to the protein antigen include (i) F (ab ') 2 fragments produced by pepsin digestion of the antibody molecule; (ii) F (ab') ₂ Fab fragments produced by reducing the disulfide bridges of the fragments; (iii) Fab fragments produced by treatment of the antibody molecule with papain and a reducing agent; and (iv) Fv fragments. Can be produced by techniques known in the art.
[0150]
(6. Bispecific antibody)
The bispecific antibody is a monoclonal antibody, preferably a human or humanized monoclonal antibody, that has binding specificities for at least two different antigens. In this case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and is advantageously a cell surface protein or receptor or receptor subunit.
[0151]
Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein And Cuello, Nature, 305: 537-539 (1983)). Because of the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one is the correct bispecific Having a structure. Purification of this precise molecule is usually achieved by an affinity chromatography step. Similar procedures are described in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J. Clin. , 10: 3655-3659 (1991).
[0152]
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably has an immunoglobulin heavy chain constant domain, comprising at least a portion of the hinge region, a CH2 region, and a CH3 region. It is preferred to have the first heavy chain constant region (CH1) containing the necessary site for light chain binding present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details of generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).
[0153]
According to another approach described in WO 96/27011, the interface between pairs of antibody molecules can be manipulated to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces include at least a portion of the CH3 region 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). Compensatory "cavities" of the same or similar size to a large side chain are created by replacing a large amino acid side chain with a smaller amino acid side chain (eg, alanine or threonine) to form a second antibody molecule. Generated on the interface. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.
[0154]
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F (ab ') 2 bispecific antibodies). Techniques for making bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describe a procedure for proteolytic cleavage of intact antibodies to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiol and prevent intermolecular disulfide formation. The generated 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 this is mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. To form The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
[0155]
In addition, Fab 'fragments have been used for E. coli. E. coli and can be chemically coupled to form bispecific antibodies. Shalaby et al. Exp. Med. 175: 217-225 (1992) describes the generation of two fully humanized bispecific antibody F (ab ') molecules. Each Fab ′ fragment was prepared from E. coli. E. coli are separately secreted and subjected to direct chemical coupling in vitro to form bispecific antibodies. The bispecific antibody thus formed can bind to cells overexpressing the ErbB2 receptor and normal human T cells and trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
[0156]
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have been described. For example, bispecific antibodies are produced using leucine zippers. Kostelny et al. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins are linked to the Fab 'portions of two different antibodies by gene fusion. The homodimeric antibody is reduced at the hinge region to form monomers and then re-oxidized to form a heterodimeric antibody. This method can also be used for the production of homodimeric antibodies. See Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 64444-6448 (1993), this "diabody" technology provided an alternative mechanism for making bispecific antibody fragments. This fragment contains a heavy chain variable region (VH) connected to a light chain variable region (VL) by a linker that is too short to pair between the two domains on the same chain. Thus, the VH and VL domains of one fragment pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. Gruber et al. Immunol. 152: 5368 (1994).
[0157]
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. Immunol. 147: 60 (1991).
[0158]
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates from a protein antigen of the invention. Alternatively, the anti-antigenic arm of the immunoglobulin molecule focuses on the defense mechanism of the cell against cells that express a particular antigen, such as leukocytes (eg, T cell receptor molecules (eg, CD2, CD3, CD28, or B7)). Or an arm that binds to a triggering molecule on an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Bispecific antibodies can also be used to direct cytotoxic factors to cells that express a particular antigen. These antibodies possess an antigen-binding arm and an arm that binds a cytotoxic factor or a radionuclide chelator (eg, EOTUBE, DPTA, DOTA, or TETA). Another bispecific antibody of interest binds a protein antigen described herein, and further binds tissue factor (TF).
[0159]
(7. Heteroconjugate antibody)
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been 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; EP03089) has been proposed. It is contemplated that the antibodies, including those containing cross-linking agents, can be prepared in vitro using known methods in synthetic protein chemistry. For example, immunotoxins can be constructed by 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 the reagents disclosed, for example, in U.S. Patent No. 4,676,980.
[0160]
(8. Operation of effector function)
It may be desirable to modify the FGF-CX antibodies of the invention for effector function, for example, to increase the efficacy of the antibody in treating cancer. For example, a cysteine residue can be introduced into the Fc region, thereby allowing for the formation of interchain disulfide bonds within this region. The homodimeric antibody thus generated may have improved potential for internalization and / or increased complement-mediated cell killing, and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al. Exp. Med. 176: 1191-1195 (1192) and Shopes, J. Mol. Immunol. 148: 2918-2922 (1992). Homodimer antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff et al., Cancer Research, 53: 2560-2565 (1993). Alternatively, antibodies can be engineered that have two Fc regions, which may have the potential for enhanced complement lysis and ADCC. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
[0161]
(9. Immunoconjugate)
The invention also provides a cytotoxic agent (eg, a chemotherapeutic agent, a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof)) or a radioisotope (ie, Radioconjugates) comprising an FGF-CX antibody conjugated to the immunoconjugate.
[0162]
Chemotherapeutic agents useful in making such immunoconjugates are described above. Examples of enzymatically active toxins and their fragments that can be used include diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, and modeccin. A) A-chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, fincin, ficin, crocin Gelonin, mitogellin, restri Examples include restrictocin, phenomycin, enomycin, and tricothecen. A variety of radionuclides are available for making radioactively linked antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.
[0163]
Conjugates of antibodies and cytotoxic factors include various bifunctional protein binding factors (eg, N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), imidoesters). Functional derivatives (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis (p-azidobenzoyl)) Hexanediamine), bis-diazonium derivatives (eg, bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg, triene 2,6-diisocyanate), and bis-active fluorine compounds (For example, 1,5-difluoro-2,4-dinitrobenzene). 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 exemplary chelator for conjugation of radionucleotides to antibodies. See WO94 / 11026.
[0164]
In another embodiment, for use in pre-targeting tumors, the antibody can be conjugated to a “receptor” (eg, streptavidin), wherein the antibody-receptor conjugate is administered to a patient, Unbound conjugate is removed from the circulation using a clearing agent, and then a "ligand" (eg, avidin) that binds sequentially to the cytotoxic factor is administered.
[0165]
(10. Immunoliposome)
The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art (eg, Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and the methods described in U.S. Patent Nos. 4,485,045 and 4,544,545). Liposomes with enhanced circulation time are described in US Pat. No. 5,013,556.
[0166]
Particularly useful liposomes can be produced by a reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a predetermined pore size to obtain liposomes having a desired diameter. Fab 'fragments of the antibodies of the present invention are described in Martin et al. Biol. Chem. , 257: 286-288 (1982), and can be conjugated to liposomes via a disulfide exchange reaction. A chemotherapeutic agent (eg, doxorubicin) is optionally contained within the liposome. Gabizon et al. National Cancer Inst. , 81 (19): 1484 (1989).
[0167]
(11. Diagnostic application of antibody against protein of the present invention)
Antibodies to the FGF-CX proteins of the present invention can be obtained by methods known in the art relating to protein localization and / or quantification (eg, use to measure protein levels in appropriate physiological samples, diagnostics, etc.). Use in methods, in imaging proteins, and the like). In certain embodiments, antibodies to a protein or a derivative, fragment or homolog thereof comprising an antigen binding domain are utilized as pharmacologically active compounds (see below).
[0168]
Antibodies specific for an FGF-CX protein of the invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Such antibodies may facilitate the purification of natural protein antigens from cells, and of recombinantly produced antibodies expressed in host cells. In addition, such antibodies can be used to detect antigenic proteins (eg, in cell lysates or cell supernatants) to assess the production and expression pattern of the antigenic proteins. Antibodies to the FGF-CX protein can be used diagnostically to monitor protein levels in tissues as part of a clinical trial procedure, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; Examples of materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.
[0169]
(12. Antibody therapeutic agent)
The FGF-CX antibodies of the present invention (including polyclonal, monoclonal, humanized and fully human antibodies) can be used as therapeutics. Such agents are commonly used to treat or prevent a disease or pathology in a subject. An antibody preparation (preferably an antibody with high specificity and high affinity for the target antigen) is administered to the subject and generally has an effect due to binding to the target. Such effects can be of two types, depending on the nature of the specific interaction between the given antibody molecule and the target antigen of interest. In the first case, administration of the antibody may abrogate or inhibit the binding of the naturally binding endogenous ligand to the target. In this case, the antibody binds to the target and masks the binding site of the naturally occurring ligand, where the ligand serves as an effector molecule. Thus, the receptor mediates the signaling pathway to which the ligand responds.
[0170]
Alternatively, the effect may be one that causes a physiological consequence by binding of the antibody to an effector binding site on the target molecule. In this case, this target (a receptor with an endogenous ligand that may be absent or defective in the disease or pathology) binds to the antibody as a surrogate effector ligand and initiates a receptor-based signaling event by the receptor.
[0171]
A therapeutically effective amount of an antibody of the invention will generally be related to the amount required to achieve a therapeutic purpose. As mentioned above, this may be a binding interaction between the antibody and its target antibody, interfering with the function of this target in certain cases and promoting a physiological response in other cases. The required amount to be administered further depends on the binding affinity of the antibody for the specific antigen and the rate at which the administered antibody is removed from the free volume of the other subject to which it is administered. A general range for a therapeutically effective dose of an antibody or antibody fragment of the present invention can be, by way of non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dose frequencies may range, for example, from twice a day to once a week.
[0172]
(13. Pharmaceutical composition of antibody)
Antibodies that specifically bind to the FGF-CX proteins of the present invention and other molecules identified by the screening assays disclosed herein are administered in the form of pharmaceutical compositions for the treatment of various disorders. Can be done. Principles and considerations involved in preparing such compositions, and guidance in the selection of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th Edition (Alfonso R. Gennaro et al.) Mack Pub. . Co. , Easton, Pa .; 1995: Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Howood Acessic Publishers, Langhorne, Pa. And Peptide And Protein Drug Delivery (Advances In Parental Science, Vol. 4, 1991); Dekker, New York.
[0173]
Where the antigen protein is intracellular and whole antibodies are used as inhibitors, it is preferred to internalize the antibodies. However, liposomes can also be used to deliver antibodies or antibody fragments to cells. When using antibody fragments, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulations herein may also, where necessary, have more than one active compound required to treat a particular indication, preferably those with complementary activities that do not adversely affect each other. It may contain more active compounds. Alternatively, or in addition, the composition may include an agent that enhances its function, such as a cytotoxic, cytokine, chemotherapeutic or growth inhibitor. Such molecules are suitably present in an effective amount combination for the intended purpose.
[0174]
The active ingredient can be produced, for example, in colloid drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, respectively, by conventional techniques or interfacial polymerization (eg, hydroxymethylcellulose, respectively) Or it can be encapsulated in microcapsules prepared by gelatin-microcapsules and poly- (methyl methacrylate) microcapsules.
[0175]
Formulations to be used for in vivo administration must be sterile. This is easily achieved by filtration through a sterile filtration membrane.
[0176]
(FGF-CX recombinant expression vector and host cell)
Another aspect of the present invention relates to a vector, preferably an expression vector, comprising a nucleic acid encoding an FGF-CX protein, or a derivative, fragment, analog or homolog thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors may direct the expression of an operably linked gene. Such vectors are referred to herein as "expression vectors". Generally, expression vectors useful in recombinant DNA technology are often in the form of a plasmid. As used herein, "plasmid" and "vector" may be used interchangeably as this plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
[0177]
The recombinant expression vector of the present invention contains the nucleic acid of the present invention in a form suitable for expressing the nucleic acid in a host cell. This means that the recombinant expression vector contains one or more regulatory sequences, which are selected based on the host cell used for expression, and is operably linked to the nucleic acid sequence to be expressed. I do. In a recombinant expression vector, "operably linked" refers to a form in which the nucleotide sequence of interest is capable of expressing the nucleotide sequence (eg, in an in vitro transcription / translation system or when the vector is introduced into a host cell). In a host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of the nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). It is understood by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired protein, and the like. An expression vector of the invention is introduced into a host cell, whereby a protein or peptide (including a fusion protein or fusion peptide) encoded by a nucleic acid as described herein (eg, an FGF-CX protein, Mutant forms of FGF-CX proteins, fusion proteins, etc.).
[0178]
The recombinant expression vectors of the invention can be designed for expression of FGF-CX in prokaryotic or eukaryotic cells. For example, FGF-CX can be expressed in bacterial cells (eg, E. coli), insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are described in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, a recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.
[0179]
Expression of proteins in prokaryotes is most often performed in E. coli with vectors containing constitutive or inducible promoters that direct the expression of either fusion or non-fusion proteins. E. coli. Fusion vectors add a number of amino acids to an encoded protein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase the expression of the recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) 3.) To assist in purifying the recombinant protein by acting as a ligand in affinity purification. Frequently, in fusion expression vectors, a proteolytic cleavage site is introduced into the fusion moiety and the binding moiety of the recombinant protein such that the recombinant protein can be separated from the fusion moiety after purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. As a typical fusion expression vector, glutathione S-transferase (GST), maltose E binding protein, or protein A is fused to the target recombinant protein, respectively, pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene, respectively). 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ).
[0180]
Suitable inducible non-fusion E. Examples of E. coli expression vectors include pTrc (Amran et al. (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California, 90-90, Canada). 89).
[0181]
E. FIG. One strategy for maximizing recombinant protein expression in E. coli is to express the protein in a host bacterium that has an impaired ability to proteolytically cleave the recombinant protein. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is that the individual codons for each amino acid are E. coli. altering the nucleic acid sequence of a nucleic acid to be inserted into an expression vector so as to be preferentially used in E. coli (see, for example, Wada et al. (1992) Nucl. Acids Res. 20: 2111-2118). . Such alterations of the nucleic acid sequences of the present invention can be performed by standard DNA synthesis techniques.
[0182]
In another embodiment, the FGF-CX expression vector is a yeast expression vector. Yeast S. Examples of vectors for expression in cerevisae include pYepSec1 (Baldari et al., (1987) EMBO J 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 et al. , (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), And picZ (InVitrogen Corp., San Diego, Calif.).
[0183]
Alternatively, FGF-CX can be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (eg, SF9 cells) include the pAc series (Smith et al. (1983) Mol Cell Biol 3: 2156-2165) and the pVL series (Lucklow). And Summers (1989) Virology 170: 31-39).
[0184]
In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NJ. Y. , 1989, Chapters 16 and 17.
[0185]
In another embodiment, a recombinant mammalian expression vector can direct the expression of a nucleic acid preferentially in a particular cell type (eg, using a tissue-specific regulatory element to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev 1: 268-277), the lymph-specific promoter (Calame and Eaton (1988) Adv Immunol 43). : 235-275), especially the T cell receptor promoter (Winoto and Baltimore (1989) EMBO J 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell. 33: 741-748), neuron-specific promoters (eg, the neurofilament promoter; Byrne and Ruddle (1989). PNAS 86: 5473-5777), pancreas-specific promoter (Edlund et al. (1985) Science 230: 912-916), and mammary gland-specific promoter (eg, milk whey promoter; US Pat. No. 4,873,316 and Europe). Application Publication No. 264,166). Developmentally regulated promoters are also included (eg, the mouse hox promoter (Kessel and Gruss (1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3: 537-546).
[0186]
The present invention further provides a recombinant expression vector comprising a DNA molecule of the present invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to regulatory sequences in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to FGF-CX mRNA. Regulatory sequences can be selected that are operably linked to the cloned nucleic acid in the antisense orientation, directing the continuous expression of the antisense RNA molecule in various cell types. For example, viral promoters and / or enhancers or regulatory sequences that direct constitutive, tissue-specific, or cell-type-specific expression of the antisense RNA can be selected. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus, wherein the antisense nucleic acid is produced under the control of a high efficiency regulatory region, the activity of which is determined by the vector into which the vector is introduced. Cell type. For a discussion of the regulation of gene expression using antisense genes, see, for example, Weintraub et al., "Antisense RNA as a molecular tool for genetic analysis", Reviews-Trends in Genetics, Vol. 1 (1) 1986.
[0187]
Another aspect of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the cell of a particular subject, but also to the progeny or potential progeny of such a cell. Although certain progeny may be present in the next generation, due to either mutations or environmental effects, such progeny may in fact not be identical to the parental cells, but are still described herein. Included within the scope of the terms as used in.
[0188]
A host cell can be any prokaryotic or eukaryotic cell. For example, FGF-CX protein can be expressed in bacterial cells (eg, E. coli), insect cells, yeast or mammalian cells (eg, human Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
[0189]
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to various art-recognized techniques for introducing foreign nucleic acids (eg, DNA) into host cells. And include calcium phosphate or calcium chloride co-precipitation, DEAE dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are described in Sambrook et al. (MOLECULAR CLONGING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory, NY, Cold Spring Harbor Laboratory. 1989) and other laboratory manuals.
[0190]
For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small portion of the cell can integrate foreign DNA into its genome. To identify and select for these elements, a gene encoding a selectable marker (eg, resistance to an antibiotic) is generally introduced into the host cell along with the gene of interest. Various selectable markers include markers that confer resistance to drugs, such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the vector encoding the growth promoter, or can be introduced on a separate vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have taken up the selectable marker gene will survive, but other cells will die).
[0191]
The host cells of the invention (eg, prokaryotic and eukaryotic host cells in culture) can be used to produce (ie, express) FGF-CX protein. Accordingly, the present invention further provides a method for producing an FGF-CX protein using the host cell of the present invention. In one embodiment, the method comprises introducing the host cell of the invention (wherein the recombinant expression vector encoding FGF-CX is introduced into a suitable medium such that the FGF-CX protein is produced. ). In another embodiment, the method further comprises isolating FGF-CX from the medium or the host cell.
[0192]
(Transgenic animals)
The host cells of the present invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or embryonic stem cell into which an FGF-CX coding sequence has been introduced. Such host cells can then be used to create non-human transgenic animals, where the exogenous FGF-CX sequences are replaced by their genomic or homologous recombinant animals, where the endogenous FGF- CX sequence has been changed). Such animals are useful for studying FGF-CX function and / or activity, and for identifying and / or evaluating modulators of FGF-CX activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or a mouse, wherein these animals One or more of the cells contains the transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. The transgene is exogenous DNA that is integrated into the genome of the cell (from which the transgenic animal develops) and remains in the genome of the mature animal, thereby producing one or more cells of the transgenic animal. Directs expression of the encoded gene product in the type or tissue. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, wherein the endogenous FGF-CX gene is It has been altered by homologous recombination between the sex gene and an exogenous DNA molecule introduced into the animal's cells (eg, animal embryonic cells) prior to animal development.
[0193]
The transgenic animals of the invention are capable of transfecting a nucleic acid encoding FGF-CX (eg, by microinjection, retroviral infection, by introducing into the male pronucleus of a fertilized oocyte) and transforming the oocyte into a pseudo-nucleus. It can be made by allowing it to occur in pregnant female foster animals. The human FGF-CX DNA sequence of SEQ ID NO: 1 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homolog of the human FGF-CX gene (eg, the mouse FGF-CX gene) can be isolated based on hybridization to the human FGF-CX cDNA (further described above) and used as a transgene. Can be done. Intron sequences and polyadenylation signals may also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence is operably linked to the FGF-CX transgene to direct FGF-CX protein expression to a particular cell. Methods for generating transgenic animals (especially animals such as mice) via embryo manipulation and microinjection have become conventional in the art and are described, for example, in US Pat. No. 4,736,866. No. 4,870,009; and No. 4,873,191; and Hogan 1986, MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NJ; Y. It is described in. Similar methods are used for the production of other transgenic animals. Transgenic primary animals can be identified based on the presence of the FGF-CX transgene in their genome and / or expression of FGF-CX mRNA in the tissues or cells of the animal. The transgenic primary animal can then be used to breed additional animals with the transgene. In addition, transgenic animals having a transgene encoding FGF-CX can further be bred to other transgenic animals having other transgenes.
[0194]
To create a homologous recombinant animal, at least one of the FGF-CX genes wherein a deletion, addition, or substitution has been introduced, thereby altering (eg, functionally disrupting) the FGF-CX gene. Prepare a vector containing the parts. The FGF-CX gene can be a human gene (eg, SEQ ID NO: 1), but is more preferably a non-human homolog of the human FGF-CX gene. For example, the mouse homolog of the human FGF-CX gene of SEQ ID NO: 1 can be used to construct a suitable homologous recombination vector to alter the endogenous FGF-CX gene in the mouse genome. In one embodiment, the vector is designed such that upon homologous recombination, the endogenous FGF-CX gene is functionally disrupted (ie, no longer encodes a functional protein; also referred to as a "knock-out" vector). Is done.
[0195]
Alternatively, the vector is designed such that upon homologous recombination, the endogenous FGF-CX gene is mutated or otherwise altered, but still encodes a functional protein (eg, The upstream regulatory region may be altered, thereby altering expression of the endogenous FGF-CX protein). In a homologous recombination vector, the altered portion of the FGF-CX gene is flanked at its 5 'and 3' ends by additional nucleic acids of the FGF-CX gene, and homologous recombination is carried out by the exogenous FGF carried by the vector. -Allow what happens between the CX protein and the endogenous FGF-CX protein in embryonic stem cells. Additional flanking FGF-CX protein nucleic acids are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of adjacent DNA (both 5 'and 3' ends) are included in the vector. See, for example, Thomas et al. (1987) Cell 51: 503 for a description of homologous recombination vectors. This vector is introduced into an embryonic stem cell line (eg, by electroporation), and cells in which the introduced FGF-CX gene has been homologously recombined with the endogenous FGF-CX gene are selected (eg, (See Li et al. (1992) Cell 69: 915).
[0196]
The selected cells are then injected into a blastocyst of an animal (eg, a mouse) to form an aggregate chimera. See, for example, Bradley (1987) Teratocarcinomas and Embryonic STEM Cells: A PRACTICAL APPROACH, edited by Robertson, IRL, Oxford, pp. 113-152. The chimeric embryo can then be implanted into a suitable pseudopregnant female Foster animal and the embryo left for a period of time. Progeny that carry the homologous recombination DNA in their germ cells can be used to breed animals, wherein all cells of the animal carry this homologous recombination DNA by germline transmission of the transgene. Including. Homologous recombination vectors and methods for constructing homologous recombination animals are further described below; Bradley (1991) Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication Numbers: WO90 / 11354; WO91 / 01140; WO92 / 0968; and WO93 / 04169.
[0197]
In another embodiment, non-human transgenic animals can be produced that contain a selected system that allows for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, for example, Lakso et al., 1992, PNAS 89: 6232-6236. Another example of a recombinase system is the Saccharomyces cerevisiae FLP recombinase system (see O'Gorman et al. (1991) Science 251: 1351-1355). If the cre / loxP recombinase system is used to regulate transgene expression, animals containing transgenes encoding both Cre recombinase and the selected protein are required. Such animals can be "duplicated" by, for example, crossing two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. "Can be provided by the construction of transgenic animals.
[0198]
Clones of the transgenic non-human animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385: 810-813. Briefly, cells (eg, somatic cells) from a transgenic animal are isolated, removed from the growth cycle, and ₀ May be entered. The quiescent cells can then be fused to an excised oocyte from the same animal from which the quiescent cells are isolated, for example, by use of an electrical pulse. The reconstructed oocyte is then cultured, whereby it develops into a morula or undifferentiated germ cell, which is then transferred to a pseudopregnant female Foster animal. The producing offspring of the female Foster animal is a clone of the animal from which the cells (eg, somatic cells) are isolated.
[0199]
(Pharmaceutical composition)
The FGF-CX nucleic acid molecules, FGF-CX proteins, and anti-FGF-CX antibodies (referred to herein as "active compounds") of the present invention, and derivatives, fragments, analogs, and homologs thereof, are suitable for administration. Can be incorporated into any pharmaceutical composition. Such compositions typically include the nucleic acid molecule, protein, or antibody, and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents, which are compatible with pharmaceutical administration. And delaying agents and the like. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference in the art, which is incorporated herein by reference. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents, which are pharmaceutically active, is well known in the art. Except for the scope of any conventional vehicle or agent that is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[0200]
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (eg, intravenous, intradermal, subcutaneous), oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following ingredients: sterile excipients (water for injection, saline solution, fixed oils, Polyethylene glycol, glycerin, propylene glycol or other synthetic solvents); antibacterial agents (eg, benzyl alcohol or methyl paraben); antioxidants (eg, ascorbic acid or sodium bisulfite); chelating agents (eg, ethylenediaminetetraacetic acid); Buffers (eg, acetate, citrate or phosphate); and agents for tonicity adjustment (eg, sodium chloride or dextrose). pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0201]
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ^TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of a coating such as lecithin, by dispersion, by maintaining the required particle size, and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0202]
A sterile injectable solution incorporates the required amount of the active compound (e.g., an FGF-CX protein or anti-FGF-CX antibody) in a suitable solvent, together with one or a combination of the above-listed components. If so, it can be prepared by subsequent filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of a sterile injectable solution, the preparation methods are vacuum drying and lyophilization, whereby the powder of the active ingredient and any further desired ingredients from the previously sterile filtered solution Get.
[0203]
Oral compositions generally include an inert diluent or an edible carrier. These can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and rapidly moved ( Swish, and are exhaled or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds having similar properties: binders (eg, microcrystalline cellulose, gum tragacanth or gelatin); excipients (eg, Starches or lactose), disintegrants (eg, alginic acid), Primogel, or corn starch; lubricants (eg, magnesium stearate or Sterates); glidants (eg, colloidal silicon dioxide); sweeteners (eg, sucrose) Or saccharin); or a flavoring agent (eg, peppermint, methyl salicylate, or orange flavor).
[0204]
For administration by inhalation, the compound is delivered in the form of an aerosol spray from compressed container or dispenser, which contains a suitable propellant (eg, a gas such as carbon dioxide), or a nebulizer.
[0205]
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
[0206]
The compounds can also be prepared in the form of suppositories for rectal delivery (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.
[0207]
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body (eg, a controlled release formulation), including implants and microencapsulations. Delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations will be apparent to those skilled in the art. These materials are also available from Alza Corporation and Nova Pharmaceuticals, Inc. It is commercially available from. Liposomal suspensions (including liposomes targeted to infected cells, including monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.
[0208]
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit is associated with a required pharmaceutical carrier. A predetermined amount of the active compound calculated to produce the desired therapeutic effect. The details of the dosage unit forms of the present invention are or are directly dependent on the unique properties of the active compound and the particular therapeutic effect to be achieved.
[0209]
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by any of a number of routes, such as those described in US Pat. No. 5,703,055. Thus, delivery can be, for example, intravenous injection, topical administration (see US Pat. No. 5,328,470) or stereotactic injection (see, eg, Chen et al. (1994) PNAS 91: 3054-3057). Is mentioned. Pharmaceutical preparations of a gene therapy vector may include the gene therapy vector in an acceptable diluent, or may include a slow release matrix incorporating the gene delivery vehicle. Alternatively, a complete gene delivery vector can be produced intact from recombinant cells (eg, a retroviral vector), and the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
[0210]
The pharmaceutical compositions can be included in a kit (eg, container, package, or dispenser) with instructions for administration.
[0211]
(Uses and Methods of the Invention)
The nucleic acid molecules, proteins, protein homologs, and antibodies described herein can be used in one or more of the following methods: (a) screening assays; (b) detection assays (eg, chromosome mapping, tissue (C) predictive medicine (eg, diagnostic assays, prognostic assays, clinical trial monitoring, and pharmacogenomics); and (d) methods of treatment (eg, treatment and prevention). As described herein, in one embodiment, the FGF-CX proteins of the invention have the ability to bind ATP.
[0212]
The isolated nucleic acid molecules of the invention can express FGF-CX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), as described further below, to produce FGF-CX protein. It can be used to detect genetic damage in CX mRNA (eg, in a biological sample) or the FGF-CX gene, as well as to modulate FGF-CX activity. In addition, FGF-CX proteins may be used to screen for drugs or compounds that modulate the activity or expression of FGF-CX, as well as disorders characterized by insufficient or excessive production of FGF-CX protein, or FGF-CX It can be used to treat a disorder characterized by the production of an FGF-CX protein form that has reduced or abnormal activity compared to a wild-type protein (eg, a proliferative or differentiation disorder). Further, the anti-FGF-CX antibodies of the present invention can be used to detect and isolate FGF-CX proteins and to modulate FGF-CX activity.
[0213]
The present invention further relates to novel agents identified by the above-described screening assays, and their use for treatment as described herein.
[0214]
(Screening assay)
The present invention relates to a candidate or test compound or agent that binds to a modulator, i.e., FGF-CX protein, or has, for example, a stimulatory or inhibitory effect on FGF-CX expression or activity. , Peptides, peptidomimetics, small molecules or other drugs) (also referred to herein as "screening assays").
[0215]
In one embodiment, the invention is directed to screening for candidate or test compounds that bind to or modulate the activity of a protein or polypeptide of FGF-CX, or a biologically active portion thereof. Assay. Test compounds of the present invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including: biological libraries; Parallel solid-phase or solution-phase libraries that can be addressed at room temperature; synthetic library methods requiring deconvolution; "one bead one compound" library methods; and synthetic library methods using affinity chromatography selection. Biological library approaches are limited to peptide libraries, while the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug Design 12: 145).
[0216]
Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al. (1993) Proc Natl Acad Sci U. S. A. 90: 6909; Erb et al. (1994) Proc Natl Acad Sci U. S. A. 91: 11422; Zuckermann et al. (1994) J Med Chem 37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew Chem Int Ed Engl 33: 2059; Carell et al. 33: 2061; and Gallop et al. (1994) J Med Chem 37: 1233.
[0219]
Compound libraries may be in solution (eg, Houghten (1992) Biotechniques 13: 412-421), or on beads (Lam (1991) Nature 354: 82-84), or on chip (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US Pat. No. 5,223,409), spores (Ladner US Pat. No. 5,233,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89: 1865-1869). ) Or on phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al. (1990) Proc Natl). Acad Sci USA 87: 6378-6382; Felici (1991) J Mol Biol 222: 301-310; Ladner (supra).
[0218]
In one embodiment, the assay is a cell-based assay, wherein cells expressing a membrane-bound form of FGF-CX protein, or a biologically active portion thereof, on the cell surface are contacted with a test compound. And the ability of the test compound to bind to the FGF-CX protein is determined. For example, the cells can be of mammalian origin or yeast cells. Determining the ability of the test compound to bind to the FGF-CX protein can be accomplished, for example, by coupling the test compound to a radioisotope label or an enzyme label. As a result, the binding of the test compound to the FGF-CX protein or a biologically active portion thereof can be determined by detecting the labeled compound in the complex. For example, a test compound ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³ H can be labeled either directly or indirectly, and the radioisotope can be detected by direct counting of radiation emission or by scintillation counting. Alternatively, the test compound can be enzymatically labeled, for example, with horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determining the conversion of the appropriate substrate to product. . In one embodiment, the assay involves contacting a cell that expresses a membrane-bound form of the FGF-CX protein, or a biologically active portion thereof, on its cell surface with a known compound that binds to FGF-CX. And forming an assay mixture, contacting the assay compound with a test compound, and determining the ability of the test compound to interact with the FGF-CX protein, wherein the test compound comprises Determining the ability to interact with the FGF-CX protein determines the ability of the test compound to preferentially bind to FGF-CX or a biologically active portion thereof as compared to a known compound. Steps.
[0219]
In another embodiment, the assay is a cell-based assay, which comprises contacting cells expressing a membrane-bound form of FGF-CX protein or a biologically active portion thereof on the cell surface with a test compound. And determining the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of the FGF-CX protein or a biologically active portion thereof. The determination of the ability of the test compound to modulate the activity of FGF-CX or a biologically active portion thereof can be determined, for example, by determining whether the FGF-CX protein binds to or binds to an FGF-CX target molecule. This can be achieved by determining the ability to interact. As used herein, a "target molecule" is a molecule to which an FGF-CX protein naturally binds or interacts, for example, a molecule on a cell surface that expresses an FGF-CX interacting protein. A molecule on the surface of a second cell, a molecule in the extracellular environment, a molecule associated with the inner surface of a cell membrane, or a cytoplasmic molecule. An FGF-CX target molecule can be a non-FGF-CX molecule or FGF-CX protein or polypeptide of the invention. In one embodiment, the FGF-CX target molecule is a component of a signaling pathway that is an extracellular signal into the cell through the cell membrane (eg, where the compound binds to a membrane-bound FGF-CX molecule). Of the signal generated by the above. The target can be, for example, a second intracellular protein with catalytic activity, or a protein that facilitates the association of a downstream signaling molecule with FGF-CX.
[0220]
Determining the ability of an FGF-CX protein to bind to or interact with an FGF-CX target molecule can be accomplished by one of the above methods for determining direct binding. In one embodiment, determining the ability of an FGF-CX protein to bind to or interact with an FGF-CX target molecule can be achieved by determining the activity of the target molecule. For example, the activity of the target molecule is determined by the cellular second messenger (ie, intracellular Ca ²⁺ , Diacylglycerol, IP ₃ Etc.), detecting the catalytic / enzymatic activity of the target on a suitable substrate, detecting a reporter gene (FGF operably linked to a nucleic acid encoding a detectable marker (eg, luciferase)). -Including induction of CX-responsive regulatory elements, or by detecting a cellular response (eg, cell viability, cell differentiation, or cell proliferation).
[0221]
In yet another embodiment, the assay of the invention is a cell-free assay, which comprises contacting a test compound with an FGF-CX protein or a biologically active portion thereof, and wherein the test compound is an FGF-CX protein. -Determining the ability to bind to the CX protein or a biologically active part thereof. The binding of the test compound to the FGF-CX protein can be determined either directly or indirectly, as described above. In one embodiment, the assay comprises contacting the FGF-CX protein or a biologically active portion thereof with a known compound that binds to FGF-CX to form an assay mixture. And determining the ability of the test compound to interact with the FGF-CX protein, wherein the ability of the test compound to interact with the FGF-CX protein is determined. The steps include determining the ability of the test compound to bind preferentially to FGF-CX or a biologically active portion thereof as compared to a known compound.
[0222]
In another embodiment, the assay is a cell-free assay, which comprises contacting an FGF-CX protein or a biologically active portion thereof with a test compound, and wherein the test compound is an FGF-CX protein or Determining the ability to modulate (eg, stimulate or inhibit) the activity of the biologically active moiety. The determination of the ability of the test compound to modulate the activity of FGF-CX can be determined, for example, by determining the ability of the FGF-CX protein to bind to an FGF-CX target molecule by one of the methods described above for determining direct binding. It can be achieved by making a decision. In an alternative embodiment, determining the ability of the test compound to modulate the activity of FGF-CX can be achieved by determining the ability of the FGF-CX protein to further modulate an FGF-CX target molecule. For example, the catalytic activity / enzymatic activity of the target molecule on a suitable substrate can be determined as described above.
[0223]
In yet another embodiment, the cell-free assay comprises contacting an FGF-CX protein or a biologically active portion thereof with a known compound that binds to FGF-CX to form an assay mixture, the assay comprising: Contacting the mixture with a test compound and determining the ability of the test compound to interact with the FGF-CX protein, wherein determining the ability of the test compound to interact with the FGF-CX protein comprises: , Determining the ability of the FGF-CX protein to bind preferentially to the FGF-CX target molecule or to modulate the activity of the target molecule preferentially.
[0224]
The cell-free assays of the present invention accept the use of both soluble or membrane-bound forms of FGF-CX. In the case of cell-free assays that include a membrane-bound form of FGF-CX, it may be desirable to utilize a solubilizing agent so that the membrane-bound form of FGF-CX is maintained in solution. Examples of such solubilizers include nonionic surfactants, such as, for example, n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N -Methylglucamide, Triton (R) X-100, Triton (R) X-114, Thesit (R), isotridecyl poly (ethylene glycol ether) _n (Isotridepoly (ethylene glycol ether)) _n ), N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, 3- (3-cholamidopropyl) dimethylammonio-1-propanesulfonate (3- (3-cholamidopropyl) dimethylamminol-1-) propane sulfonate (CHAPS) or 3- (3-cholamidopropyl) dimethylammonio-2-hydroxy-1-propanesulfonate (3- (3-cholamidopropyl) dimethylaminominyl-2-hydroxy-1-propane sulfonate) (CHAP) ).
[0225]
In one or more embodiments of the above assay methods of the invention, immobilization of either FGF-CX or its target molecule to separate the complexed form from the uncomplexed form of one or both of the proteins. It may be desirable to facilitate and automate the assay. Binding of the test compound to FGF-CX, or the interaction of FGF-CX with the target molecule in the presence and absence of the candidate compound, can be carried out in any vessel suitable for containing these reactants. Can be achieved. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to bind to a matrix. For example, a GST-FGF-CX fusion protein or a GST target fusion protein can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized microtiter plates, which are then combined with the test compound and Either combined or combined with the test compound and either the unadsorbed target protein or the FGF-CX protein, and the mixture is incubated under conditions leading to complex formation (eg, physiological conditions for salt and pH). Is done. Following incubation, the beads or microtiter plate wells are washed to remove any unbound components and, in the case of beads, the matrix is fixed, e.g., directly or indirectly, as described above. Decide on one of the targets. Alternatively, the complex can be dissociated from the matrix and the level of binding or activity of FGF-CX can be determined using standard techniques.
[0226]
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, FGF-CX or any of its target molecules can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated FGF-CX or target molecule is prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, Ill.). Can be obtained and fixed in wells of a 96-well plate (Pierce Chemical) coated with streptavidin. Alternatively, an antibody that is reactive with FGF-CX or a target molecule but does not interfere with the binding of the FGF-CX protein to its target molecule can be derivatized into the wells of the plate and the unbound target or FGF -CX can be trapped in the well by conjugation of the antibody. Methods for detecting such complexes include, in addition to those described above for the GST-immobilized complex, immunodetection of the complex using FGF-CX or an antibody reactive with the target molecule, as well as FGF-CX. Enzyme binding assays that rely on detecting enzymatic activity associated with CX or the target molecule.
[0227]
In another embodiment, modulators of FGF-CX expression are identified in a method in which a cell is contacted with a candidate compound and the expression of FGF-CX mRNA or FGF-CX protein in the cell is determined. The level of expression of FGF-CX mRNA or FGF-CX protein in the presence of the candidate compound is compared to the level of expression of FGF-CX mRNA or FGF-CX protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of FGF-CX expression based on this comparison. For example, if the expression of FGF-CX mRNA or FGF-CX protein is greater (in a statistically significant manner) in the presence of the candidate compound than in the absence of the candidate compound, the candidate compound is expressed as FGF-CX mRNA Alternatively, it is identified as a stimulator of FGF-CX protein expression. Alternatively, if the expression of FGF-CX mRNA or FGF-CX protein is less (statistically significantly less) in the presence of the candidate compound than in the absence of the candidate compound, then the candidate compound is FGF-CX mRNA or Identified as an inhibitor of FGF-CX protein expression. The level of expression of FGF-CX mRNA or FGF-CX protein in a cell can be determined by the methods described herein for detecting FGF-CX mRNA or FGF-CX protein.
[0228]
In yet another aspect of the invention, the FGF-CX protein may be used as a "bait protein" in a two-hybrid assay or a three-hybrid assay (eg, US Patent No. 5,283,317; Zervos et al., (1993) Cell 72: 223-232; Madura et al., (1993) J Biol Chem 268: 12046-12054; Bartel et al., (1993) Biotechniques 14: 920-924; Iwabuchi et al., (1993) Oncogene 8: 1693-1696. And Brent WO94 / 10300), other proteins that bind to or interact with FGF-CX ("FGF-CX binding proteins" or "FGF-CX- bp "), as well as other proteins that regulate the activity of FGF-CX. Such FGF-CX binding proteins also appear to be involved in signal transduction by the FGF-CX protein, for example, as upstream or downstream elements of the FGF-CX pathway.
[0229]
The two-hybrid system is based on the modular nature of most transcription factors, consisting of a separable DNA binding domain and an activation domain. Briefly, this assay uses two different DNA constructs. In one construct, the gene encoding FGF-CX is fused to a gene encoding the DNA binding domain of a known transcription factor (eg, GAL-4). In the other construct, a DNA sequence encoding an unidentified protein ("prey" or "sample") from a library of DNA sequences is fused to a gene encoding the activation domain of a known transcription factor. You. If the "bait" and "prey" proteins can interact in vivo, an FGF-CX-dependent complex is formed, bringing the DNA binding and activation domains of the transcription factor into close proximity. This proximity allows for transcription of a reporter gene (eg, LacZ) operably linked to a transcription regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain a cloned gene encoding a protein that interacts with FGF-CX.
[0230]
The present invention further relates to novel reagents identified by the above screening assays and their use for treatment as described herein.
[0231]
(Detection assay)
Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in many ways as polynucleotide reagents. For example, these sequences may (i) map the respective gene on a chromosome; thus, locate a gene region associated with a genetic disease; (ii) identify an individual from a small number of biological samples (tissue Typing); and (iii) can be used to aid in forensic identification of biological samples.
[0232]
The FGF-CX sequences of the present invention can also be used to identify individuals from a small number of biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed on a Southern blot to obtain unique bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphism" described in US Patent No. 5,272,057).
[0233]
In addition, the sequences of the present invention may be used to provide an alternative technique for determining the actual base-by-base DNA sequence for selected portions of an individual's genome. Thus, using the FGF-CX sequence described herein, two PCR primers can be prepared from the 5 'and 3' ends of the sequence. These primers can then be used to amplify and subsequently sequence the DNA of the individual.
[0234]
A panel of corresponding DNA sequences from individuals prepared in this manner may provide a unique individual identification as each individual has a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identified sequences from individuals and tissues. The FGF-CX sequences of the present invention uniquely represent portions of the human genome. Allelic variation occurs to some extent in the coding regions of these sequences, and to a greater extent in non-coding regions. It is estimated that allelic variation among individual humans occurs at a frequency of about once for each 500 bases. Many allelic variations result from single nucleotide polymorphisms (SNPs), including restriction fragment length polymorphisms (RFLPs).
[0235]
Each of the sequences described herein can to some extent be used as a standard against which DNA from an individual can be compared for identification purposes. Not so many sequences are required to distinguish individuals, as more polymorphisms occur in non-coding regions. The non-coding sequence of SEQ ID NO: 1 or SEQ ID NO: 26, as described above, may provide unambiguous positive individual identification, perhaps using a panel of 10-1,000 primers. These primers each yield an amplified non-coding sequence of 100 bases. If a putative coding sequence is used, a more suitable number of primers for positive individual identification is 500-2,000.
[0236]
(Predictive medicine)
The invention also relates to the field of predictive medicine, wherein diagnostic assays, prognostic assays, pharmacogenomics and monitoring clinical tests are used for prognostic (predictive) purposes, thereby treating an individual prophylactically. Thus, one aspect of the present invention is to determine the expression of FGF-CX protein and / or nucleic acid, and the activity of FGF-CX in the context of a biological sample (eg, blood, serum, cells, tissues), This relates to diagnostic assays for determining whether an individual has, or is at risk for developing, a disease or disorder associated with abnormal FGF-CX expression or activity. The present invention also provides prognostic (or predictive) assays for determining whether an individual is at risk for developing a disorder associated with the expression or activity of FGF-CX proteins, nucleic acids. For example, mutations in the gene for FGF-CX can be assayed in a biological sample. Such assays can be used for prognostic or predictive purposes, whereby an individual is characterized by the expression or activity of an FGF-CX protein, nucleic acid, or prevents an individual prior to the onset of a disorder associated therewith. Treatment.
[0237]
Another aspect of the present invention provides a method for determining the expression of FGF-CX protein, nucleic acid or the activity of FGF-CX in an individual, whereby an appropriate therapeutic or prophylactic agent for that individual ( (Referred to herein as "pharmacogenomics"). Pharmacogenomics is a method for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (eg, the genotype of the individual tested to determine the ability of the individual to respond to a particular drug). Enables selection of drugs (eg, drugs).
[0238]
Yet another aspect of the invention relates to monitoring the effect of an agent (e.g., drug, compound) on FGF-CX expression or activity in a clinical trial.
[0239]
These and other agents are described in further detail in the following sections.
[0240]
(Diagnostic assay)
Fibroblast growth factors FGF-1 to FGF-9 generally promote cell growth in cells having specific growth factor receptors. Examples of FGF growth factors include epithelial cells (eg, fibroblasts and keratinocytes) in the front of the eye after surgery. Other conditions in which cell proliferation plays a role include tumor, restenosis, psoriasis, Dupuytren's contracture, diabetic complications, Kaposi's sarcoma and rheumatoid arthritis.
[0241]
FGF-CX can be used in the methods of the invention to detect its corresponding fibroblast growth factor receptor CX (FGFRX) in a sample or tissue. The method includes contacting the sample or tissue with FGF-CX, forming a receptor-ligand pair, and detecting any FGRCX: FGF-CX pair. Compositions comprising FGF-CX may be used to increase FGRCX activity, for example, to stimulate cartilage or bone repair. Compositions comprising an FGF-CX antagonist or FGF-CX binding agent (e.g., an anti-FGF-CX antibody) may cause a disease caused by excess FGF-CX or excessive activity of FGF-CX (particularly solitary or hereditary). Exostoses, hallux valgus deformity, achondroplasia, synovial chondromatosis and chondroma).
[0242]
Glial activator (GAF) and DNA encoding GAF act to specifically promote glial cell proliferation. Some examples of glial-related disorders that can modulate glial cell activity using GAF are brain injury, cerebral edema, senile dementia, Alzheimer's disease, diabetic neuropathy, and the like. Similarly, FGF-CX can be used in the diagnosis and treatment of glial cell-related disorders. The glial cell modulating activity of FGF-CX can be a neuroprotective-like activity, and FGF-CX can be used as a neuroprotective factor. Due to the close homology of FGF-CX to FGF-9, which was originally identified as a glial activator, the FGF-CX sequence may also be speculated to be a glial activator. . Thus, FGF-CX can be used to stimulate glial cell proliferation and to promote healing of brain injury or treat cerebral edema, senile dementia, Alzheimer's disease or diabetic neuropathy Can be used to
[0243]
FGF-CX also stimulates fibroblasts (to promote healing of burns, wounds, ulcers, etc.), megakaryocytes (to increase platelet count), hematopoietic cells, immune system cells, and vascular smooth muscle cells Can be used for FGF-CX is also predicted to have osteogenic promoting activity and can be used to treat fractures and osteoporosis. Assays of the polypeptide or nucleic acid portion of FGF-CX can be useful in the diagnosis of brain tumors, and antibodies to them can be used to treat such tumors. It can also be used as a reagent to stimulate the growth of cultured cells. Estimated dosages are from 1 ng to 0.1 mg / kg / day, but treatment may vary depending on the type or severity of the disorder being treated. The FGF-CX polypeptide can be used as a platelet-increasing factor, a bone formation promoting factor, or can be used to treat a cranial nerve disease or liver disorder (eg, cirrhosis). They can also be used to treat cancer when used in combination with anti-cancer agents. Antibodies to the FGF-CX polypeptide, or fragments, derivatives or analogs thereof, can be used to detect or determine the biological activity of the FGF-CX polypeptide, or to purify the FGF-CX polypeptide. Can be used for These antibodies, which also neutralize the cell growth activity of FGF-CX, can be used as anti-cancer agents.
[0244]
It is known in the art that many, if not all, homologous proteins have closely related or identical functions. See, for example, Lewin, "Chapter 21: Structural Genes Belg to Families": Gene II, 1985, John Wiley and Sons, Inc. , New York. FGF-CX polypeptide is very similar to Xenopus XFGF-CX protein. The XFGF-CX protein has previously been shown to be specifically expressed in highly proliferative tissues (see, eg, Koga et al., Supra). Therefore, it is speculated that FGF-CX also regulates cellular activity in highly proliferative tissues. Thus, FGF-CX is useful in diagnosing proliferative disorders and in stimulating the growth of such cells and tissues to overcome pathological conditions where cell and tissue growth is suppressed or inhibited. It can be particularly useful. Oligonucleotides corresponding to any part of the FGF-CX nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 26 can be used to detect expression of a FGF-CX-like gene. The proteins of the present invention can be used to stimulate the production of antibodies that specifically bind to these proteins. Such antibodies can be used in immunodiagnostic procedures to detect the presence of a protein in a sample. The proteins of the present invention can be used to stimulate such cell growth in situations where cell growth is desired. One example is to offset the toxic side effects of chemotherapeutic agents, for example, on hematopoiesis and platelet formation, the lining of the gastrointestinal tract, and hair follicles. They can also be used to stimulate new cell proliferation in neurological disorders, including, for example, Alzheimer's disease. Alternatively, antagonist treatment can be administered, wherein antibodies that specifically bind to the FGF-CX-like proteins of the invention abrogate certain growth-inducing effects of these proteins. Such antibodies can be useful, for example, in treating proliferative disorders, including, for example, various tumors and benign hyperplasia.
[0245]
An exemplary method for detecting the presence or absence of FGF-CX in a biological sample comprises obtaining a biological sample from a test subject, and FGF-CX protein or FGF-CX encoding FGF-CX protein. Contacting a biological sample with a compound or agent capable of detecting CX nucleic acids (eg, mRNA, genomic DNA), such that the presence of FGF-CX is detected in the biological sample. . An agent for detecting FGF-CX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to FGF-CX mRNA or genomic DNA. The nucleic acid probe is, for example, a full-length FGF-CX nucleic acid (eg, a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 26) or a portion thereof (eg, at least 15, 30, 50, 100, 250 or 500 nucleotides in length, and As described above, oligonucleotides that are sufficient to specifically hybridize under stringent conditions to mRNA or genomic DNA of FGF-CX). Other suitable probes for use in the diagnostic assays of the invention are described herein.
[0246]
The agent for detecting the FGF-CX protein is an antibody capable of binding to the FGF-CX protein, preferably an antibody having a detectable label. The antibody can be a polyclonal antibody, or more preferably, a monoclonal antibody. Intact antibodies or fragments thereof (eg, Fab or F (ab ')) ₂ ) Can be used. With respect to a probe or antibody, the term “labeled” refers to direct labeling of the probe or antibody (by coupling (ie, physically linking) a detectable substance to the probe or antibody), Also, it is intended to include indirect labeling of the probe or antibody (due to its reactivity with another reagent that is directly labeled). Examples of indirect labeling include detection of the primary antibody using a fluorescently labeled secondary antibody, and end labeling of the DNA probe with biotin (so that it can be detected using fluorescently labeled streptavidin). Get). The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the present invention can be used to detect FGF-CX mRNA, protein or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro techniques for the detection of FGF-CX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of FGF-CX protein include enzyme linked immunosorbent assays (ELISAs), western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detecting FGF-CX genomic DNA include Southern hybridizations. In addition, in vivo techniques for the detection of FGF-CX protein include the introduction of a labeled anti-FGF-CX antibody into a subject. For example, the antibodies can be labeled with a radioactive marker. The presence and location of a radioactive marker in a subject can be detected by standard imaging techniques.
[0247]
In one embodiment, the biological sample contains protein molecules from a test subject. Alternatively, a biological sample can include mRNA molecules from a test subject or genomic DNA molecules from a test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated from a subject by conventional means.
[0248]
In another embodiment, the method comprises obtaining a control biological sample from a control subject, contacting a compound or agent capable of detecting FGF-CX protein, mRNA or genome with the control sample. A process wherein the presence of the FGF-CX protein, mRNA or genomic DNA is detected in the biological sample, and the presence of the FGF-CX protein, mRNA or genomic DNA in the control sample. Comparing with the presence of FGF-CX protein, mRNA or genomic DNA in the test sample.
[0249]
The invention also includes a kit for detecting the presence of FGF-CX in a biological sample. For example, the kit may comprise: a labeled compound or factor capable of detecting a protein or mRNA of FGF-CX in a biological sample; for determining the amount of FGF-CX in the sample Means; and means for comparing the amount of FGF-CX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit may further comprise instructions for using the kit for detecting a protein or nucleic acid of FGF-CX.
[0250]
(Prognostic assay)
The diagnostic methods described herein can be further utilized to identify subjects having or at risk of developing a disease or disorder associated with abnormal FGF-CX expression or activity. For example, utilizing the assays described herein (eg, the diagnostic assays described above or the assays described below), for example, disorders of proliferation or differentiation (eg, hyperplasia, tumor, restenosis, psoriasis, Dupuytren's contracture) Expression or activity of FGF-CX proteins, nucleic acids in diabetic complications, or rheumatoid arthritis, etc., and glial-related disorders (eg, cerebral injury, diabetic neuropathy, cerebral edema, senile dementia, Alzheimer's disease, etc.) A subject having a disorder associated with, or at risk of developing, may be identified. Alternatively, the prognostic assay may be used to identify a subject having or at risk for developing a disease or disorder. Accordingly, the invention provides a method for identifying a disease or disorder associated with abnormal FGF-CX expression or activity. Here, a test sample is obtained from the subject and an FGF-CX protein or nucleic acid (eg, mRNA, genomic DNA) is detected, wherein the presence of the FGF-CX protein or nucleic acid is indicative of an abnormal FGF-CX A diagnostic indicator for a subject having or at risk of developing a disease or disorder associated with expression or activity. As used herein, “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (eg, serum), a cell sample, or a tissue.
[0251]
Further, using the prognostic assays described herein, a subject is administered a drug (eg, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate). To determine if a disease or disorder associated with abnormal FGF-CX expression or activity can be treated. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder (eg, a proliferative disorder, a differentiation disorder, a glial-related disorder, etc.). Accordingly, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abnormal FGF-CX expression or activity. Here, a test sample is obtained and an FGF-CX protein or nucleic acid is detected (eg, where the presence of an FGF-CX protein or nucleic acid indicates a disorder associated with abnormal FGF-CX expression or activity). It is a diagnostic indicator for a subject that can be administered an agent for treatment).
[0252]
The methods of the present invention also detect genetic damage in the FGF-CX gene, whereby a subject having the damaged gene is at risk for or has a disorder such as a proliferative disorder, a disorder of differentiation, a glial-related disorder, or the like. It can also be used to determine if a subject is affected. In various embodiments, the methods of the present invention comprise, in a sample of cells from the subject, a genetic damage characterized by at least one of the alterations affecting the integrity of the gene encoding the FGF-CX protein, Alternatively, the method includes a step of detecting the presence or absence of erroneous expression of the FGF-CX gene. For example, such genetic damage can be detected by confirming the presence of at least one of the following: (1) deletion of one or more nucleotides from the FGF-CX gene; (2) FGF-CX gene (3) substitution of one or more nucleotides of the FGF-CX gene; (4) chromosomal rearrangement of the FGF-CX gene; (5) messenger RNA transcript of the FGF-CX gene. (6) abnormal alteration of the FGF-CX gene (eg, abnormal alteration of the methylation pattern of genomic DNA), (7) non-wild-type splicing pattern of the messenger RNA transcript of the FGF-CX gene. Presence, (8) non-wild type levels of FGF-CX protein, (9) deletion of alleles of the FGF-CX gene, and ( 0) modified post inappropriate translation of FGF-CX protein. As described herein, there are a number of known assay techniques in the art that can be used to detect damage in the gene for FGF-CX. A preferred biological sample is a peripheral blood leukocyte sample isolated from a subject by conventional means. However, any biological sample containing nucleated cells can be used, including, for example, buccal mucosal cells.
[0253]
In certain embodiments, detection of the damage is accomplished by polymerase chain reaction (PCR) (eg, anchor PCR or RACE PCR) or ligation chain reaction (LCR) (eg, US Pat. Nos. 4,683,195 and 4,683). 683, 202) (see, eg, Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) PNAS 91: 360-364). . The latter may be particularly useful for detecting point mutations in the gene for FGF-CX) (see Abravaya et al. (1995) Nucl Acids Res 23: 675-682). The method includes collecting a sample of cells from the patient, isolating nucleic acid (eg, genomic, mRNA, or both) from cells of the sample, one or more specifically hybridizing to the FGF-CX gene. Contacting the primers with the nucleic acid sample under conditions such that hybridization and amplification of the FGF-CX gene (if present) occurs, and detecting the presence or absence of the amplification product, or Detecting the size of the amplification product and comparing its length to a control sample may be included. It is anticipated that PCR and / or LCR may be desirable to be used as a preliminary amplification step with any of the techniques used to detect mutations described herein.
[0254]
Alternative amplification methods include: self-sustaining sequence replication (Guatelli et al., 1990, Proc Natl Acad Sci USA 87: 1874-1878), a transcription amplification system (Kwoh, et al., 1989, Proc Natl Acad Sci USA). 86: 1173-1177), Q-β replicase (Lizardi et al., 1988, BioTechnology 6: 1197), or any other method of nucleic acid amplification, followed by its amplified molecule using techniques well known to those skilled in the art. Detection. These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very small numbers.
[0255]
In an alternative embodiment, a mutation in the FGF-CX gene from a sample cell can be identified by an alteration in the restriction enzyme cleavage pattern. For example, sample and control DNA is isolated, amplified (if necessary), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. . Differences in the size of the fragment length between the sample DNA and the control DNA indicate a mutation in the sample DNA. In addition, the use of sequence specific ribozymes (see, eg, US Pat. No. 5,493,531) can be used to score for the presence of specific mutations by the occurrence or loss of a ribozyme cleavage site. .
[0256]
In other embodiments, the genetic variation in FGF-CX comprises hybridizing a sample nucleic acid and a control nucleic acid (eg, DNA or RNA) to a high-density array containing hundreds or thousands of oligonucleotide probes. (Cronin et al. (1996) Human Mutation 7: 244-255; Kozal et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in FGF-CX can be identified in a two-dimensional array containing light-generating DNA probes as described in Cronin et al. (Supra). Briefly, using a hybridization array of the first probe, scanning over long stretches of DNA in samples and controls, creating a linear array of consecutively overlapping probes, the base changes between that sequence. Can be identified. This step allows the identification of point mutations. Following this step, a second hybridization array that allows the characterization of a particular mutation by using a smaller, specialized probe array that is complementary to all variants or variants detected There is. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
[0257]
In yet another embodiment, the FGF-CX gene can be sequenced directly using any of a variety of sequencing reactions known in the art and the wild-type (control ) Mutations can be detected by comparing to the sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert (1977) PNAS 74: 560 or Sanger (1977) PNAS 74: 5463. It is also contemplated that when performing a diagnostic assay, any of a variety of automated sequencing procedures may be utilized (Naeve et al. (1995) Biotechniques 19: 448). These include sequencing by mass spectrometry (eg, PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv Chromatogr 36: 127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38: 147-159. See).
[0258]
Other methods for detecting mutations in the FGF-CX gene include using mismatch protection to detect mismatched bases in RNA / RNA or RNA / DNA heteroduplexes ( Myers et al. (1985) Science 230: 1242). In general, the art of "mismatch cleavage" involves hybridizing (labeled) RNA or DNA containing wild-type FGF-CX sequences with potential mutant RNA or DNA obtained from a tissue sample. By providing a heteroduplex formed thereby. An agent that cleaves this double-stranded duplex using a single-stranded region of the duplex (eg, one present due to base pair mismatch between the control strand and the sample strand) is used. To process. For example, an RNA / DNA duplex can be treated with RNase, and a DNA / DNA hybrid can be treated with S1 nuclease to enzymatically digest the mismatched region. In other embodiments, the duplex, either DNA / DNA or RNA / DNA, can be treated with hydroxylamine or osmium tetroxide, and piperidine to digest the mismatched region. Then, after digestion of the mismatched region, the resulting material is separated by size on a denaturing polyacrylamide gel to determine the site of mutation. See, for example, Cotton et al. (1988) Proc Natl Acad Sci USA 85: 4397; Saleba et al. (1992) Methods Enzymol 217: 286-295. In one embodiment, control DNA or RNA may be labeled for detection.
[0259]
In yet another embodiment, the mismatch cleavage reaction comprises converting one or more proteins that recognize mismatched base pairs in double-stranded DNA (a so-called “DNA mismatch repair” enzyme) into FGF-CX cDNA obtained from a sample of cells. Used in a defined system to detect and map point mutations in. For example, E. The mutY enzyme of E. coli cleaves A at G / A mismatch and thymidine DNA glycosylase from HeLa cells cleaves T at G / T mismatch (Hsu et al. (1994) Carcinogenesis 15: 1657-1662). According to an exemplary embodiment, a probe based on an FGF-CX sequence (eg, a wild-type FGF-CX sequence) is hybridized to cDNA or other DNA product from a test cell. The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols and the like. See, for example, U.S. Patent No. 5,459,039.
[0260]
In other embodiments, changes in electrophoretic mobility are used to identify mutations in the FGF-CX gene. For example, single-stranded conformational polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc Natl Acad Sci USA). : 86: 2766, and Cotton (1993) Mutat Res 285: 125-144; Hayashi (1992) Genet Anal Tech Appl 9: 73-79). Single stranded DNA fragments of the sample and control FGF-CX nucleic acids are denatured and regenerated. The secondary structure of single-stranded nucleic acids varies according to sequence, and the changes obtained in electrophoretic mobility allow even the detection of single base changes. DNA fragments can be labeled or detected using a labeled probe. The sensitivity of the assay can be enhanced by using RNA (rather than DNA), where the secondary structure is more sensitive to sequence changes. In one embodiment, the methods of the invention utilize heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility (eg, Keen et al. ( 1991) Trends Genet 7: 5).
[0261]
In yet another embodiment, migration of a mutant or wild-type fragment on a polyacrylamide gel containing a constant gradient of denaturing agent is assayed using denaturing gradient gel electrophoresis (DGGE) (eg, Myers et al. ( 1985) Nature 313: 495). If DGGE is used as a method of analysis, the DNA is modified to ensure that it is not completely denatured, for example by adding a GC clamp of high melting point GC-rich DNA of about 40 bp by PCR. In a further embodiment, a temperature gradient is used instead of a denaturing gradient to identify differences in the mobility of control and sample DNA (see, eg, Rosenbaum and Reissner (1987) Biophys Chem 265: 12753). thing).
[0262]
Examples of other techniques for detecting point mutations include, but are not limited to: selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers can be prepared such that the known mutation is centrally located and then hybridized to the target DNA under conditions that permit hybridization only when a perfect match is found ( (Saiki et al. (1986) Nature 324: 163); Saiki et al. (1989) Proc Natl Acad. Sci USA 86: 6230). Such an allele-specific oligonucleotide hybridizes to the PCR-amplified target DNA or many different mutations when the oligonucleotide is attached to a hybridizing membrane and the labeled target DNA is hybridized. Is done.
[0263]
Alternatively, allele-specific amplification techniques that rely on selective PCR amplification can be used in conjunction with the present invention. Oligonucleotides used as primers for specific amplification should be in the center of the molecule (so amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res 17: 2434-2448). Alternatively, the 3'most end of one primer may carry the mutation of interest (Prossner (1993) Tibtech 11: 238), which may prevent mismatches or reduce polymerase extension under appropriate conditions. In addition, it may be desirable to introduce a new restriction site in the region of the mutation to perform cleavage-based detection (see, for example, Gasparini et al. (1992) Mol Cell Probes 6: 1). In certain embodiments, it is anticipated that amplification may also be performed using Taq ligase for amplification (see, for example, Barany (1991) Proc Natl Acad Sci USA 88: 189). In such cases, ligation will only occur if there is a perfect match at the 3 'end of the 5' sequence, and by searching for the presence or absence of amplification, the presence of a known mutation at a particular site will be determined. Enable to detect.
[0264]
The methods described herein can be performed, for example, by utilizing a pre-packaged diagnostic kit that includes at least one probe nucleic acid or antibody reagent described herein, For example, it can be conveniently used in a clinical setting to diagnose patients exhibiting symptoms or a family history of a disease or disorder involving the FGF-CX gene.
[0265]
Further, any cell type or tissue in which FGF-CX is expressed, preferably peripheral blood leukocytes, can be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells (eg, including buccal mucosal cells) can be used.
[0266]
(Pharmacogenomics)
Factors having a stimulatory or inhibitory effect on FGF-CX activity (eg, FGF-CX gene expression), ie, modulators, are identified as abnormal FGF-CXs as identified by the screening assays described herein. An individual can be administered to treat (prophylactically or therapeutically) a disorder associated with CX activity, such as a neurological disorder, a cancer-related disorder or a pregnancy disorder. In conjunction with such treatment, the pharmacogenomics of an individual (ie, a study of the relationship between an individual's genotype and the individual's response to a foreign compound or drug) may be considered. Differences in the metabolism of the therapeutic agent can lead to severe toxicity or treatment failure by altering the relationship between pharmacologically active drug dose and blood concentration. Thus, the pharmacogenomics of an individual allows for the selection of effective agents (eg, drugs) for prophylactic or therapeutic treatment based on considerations of the individual's genotype. Such pharmacogenomics can be further used to determine appropriate dosages and therapeutic regimes. Thus, the activity of the FGF-CX protein, the expression of the FGF-CX nucleic acid, or the mutation content of the FGF-CX gene in the individual is determined, thereby selecting an appropriate drug for therapeutic or prophylactic treatment of the individual. obtain.
[0267]
Pharmacogenomics deals with clinically significant genetic changes in response to drugs due to altered drug properties and abnormal effects in affected individuals. See, for example, Eichelbaum, 1996, Clin Exp Pharmacol Physiol, 23: 983-985 and Linder, 1997, Clin Chem, 43: 254-266. In general, two types of pharmacogenomic conditions can be distinguished. The genetic condition is transmitted as one factor that alters the way the drug acts on the body (altered drug action), or the genetic condition is one factor that alters the way the body acts on the drug. As (modified drug metabolism). These pharmacogenomic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common hereditary enzyme disease, the major clinical complications of which are oxidant drugs (antimalarials, sulfonamides, analgesics, nitrofurans) ) And hemolysis after fava bean ingestion.
[0268]
In an exemplary embodiment, drug metabolizing enzyme activity is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (eg, N-acetyltransferase 2 (NAT2) and the cytochrome P450 enzymes CYP2D6 and CYP2C19) indicates that some patients do not get the expected drug effects or And provided explanations for showing excessive drug response and severe toxicity after taking a safe dose of the drug. These polymorphisms are expressed in the population in two phenotypes: an extensible metabolizer (EM) and a poor metabolizer (PM). The prevalence of PM varies between different populations. For example, the gene encoding CYP2D6 is highly polymorphic, and some mutations have been identified in PM, all of which lead to the absence of functional CYP2D6. People with poor metabolic capacity of CYP2D6 and CYP2C19 fairly frequently experience excessive drug response and side effects when they receive standard doses. When the metabolite is the active therapeutic moiety, PM does not show a therapeutic response, as demonstrated for the analgesic effects of codeine mediated by its CYP2D6-forming metabolite, morphine. At the other extreme are those with a so-called ultra-rapid metabolic capacity that does not respond to standard doses. Recently, the reference molecule for ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
[0269]
Thus, the activity of the protein of FGF-CX, the expression of the nucleic acid of FGF-CX, or the mutation content of the gene for FGF-CX in an individual is determined, thereby making it suitable for therapeutic or prophylactic treatment of that individual. You can choose the right drug In addition, pharmacogenomic studies can be used to apply the genotype of a polymorphic allele encoding a drug metabolizing enzyme to the identification of an individual's drug responsive phenotype. This finding, when applied to dosing or drug selection, may avoid adverse reactions or treatment failures, and thus subject the subject to modulators of FGF-CX, such as the exemplary described herein. The therapeutic or prophylactic efficacy can be enhanced when treated with a modulator identified by one of the various screening assays.
[0270]
(Clinical effect monitoring)
Monitoring the effect of an agent (e.g., drug, compound) on FGF-CX expression or activity (e.g., the ability to modulate abnormal cell proliferation and / or differentiation) can be performed in basic drug screening and clinical trials. It can also be applied. For example, an agent determined by a screening assay as described herein has reduced FGF-CX gene expression, protein levels, or the ability to up-regulate FGF-CX activity. -Can be monitored in clinical trials of subjects that show CX gene expression, protein levels, or down-regulated FGF-CX activity. Alternatively, the agent determined by the screening assay has increased potency in reducing FGF-CX gene expression, protein levels, or down-regulating FGF-CX activity, increasing FGF-CX gene expression, protein levels, Alternatively, it can be monitored in clinical trials of subjects that show up-regulated FGF-CX activity. In such clinical trials, the expression or activity of FGF-CX, and preferably, other genes involved in, for example, proliferative or neurological disorders, may be "read out", ie, specific readouts. It can be used as a marker of cell responsiveness.
[0271]
For example, an agent (eg, a compound, drug or small molecule) that modulates FGF-CX-containing gene, which modulates FGF-CX activity (eg, identified in a screening assay as described herein). Is regulated in the cell by treatment with). Thus, to study the effects of drugs on cellular proliferative disorders, for example, in clinical trials, cells can be isolated and RNA prepared, and FGF-CX and other genes involved in the disorder can be isolated. It can be analyzed for the level of expression. The level of gene expression (ie, gene expression pattern) can be determined by Northern blot analysis or RT-PCR, as described herein, or by measuring the amount of protein produced, or It can be quantified by one of the methods as described herein or by measuring the level of activity of FGF-CX or other genes. In this manner, the gene expression pattern can serve as a marker that is indicative of the physiological response of the cell to the agent. Thus, the response status can be determined before and at various points during treatment of the individual with the agent.
[0272]
In one embodiment, the invention relates to agents (eg, agonists, antagonists, proteins, peptides, nucleic acids, peptidomimetics, small molecules, or other drug candidates identified by the screening assays described herein). A method for monitoring the efficacy of a treatment in a subject, comprising the steps of: (i) obtaining a pre-dose sample from the subject prior to administration of the agent; (Ii) detecting the level of expression of FGF-CX protein, mRNA, or genomic DNA in the pre-dose sample; (iii) obtaining one or more post-dose samples from the subject; (iv) ) In the post-administration sample, the level of expression or activity of FGF-CX protein, mRNA, or genomic DNA is detected. (V) comparing the level of FGF-CX protein, mRNA or genomic DNA expression or activity in the pre-administration sample with the FGF-CX protein, mRNA or genomic DNA in the post-administration sample. And (vi) altering the administration of the drug to the subject according to the comparison. For example, increased administration of the agent may be desirable to increase FGF-CX expression or activity to a higher level than is detected (ie, to increase the efficacy of the agent). Alternatively, reduced administration of the agent may be desirable to reduce FGF-CX expression or activity to a lower level than detected (ie, to reduce the efficacy of the agent).
[0273]
(Method of treatment)
The present invention provides both prophylactic and therapeutic methods of treating a subject at risk for (or susceptibility to) or having a disorder associated with aberrant expression or activity of FGF-CX. .
[0274]
Diseases and disorders characterized by increased levels or biological activity (as compared to a subject not afflicted with the disease or disorder) use therapeutic agents that antagonize (ie, reduce or inhibit) the activity. Can be treated. Therapeutics that antagonize activity can be administered in a therapeutic or prophylactic manner. Therapeutic agents that may be utilized include, but are not limited to: (i) an FGF-CX polypeptide, or an analog, derivative, fragment or homolog thereof; (ii) an antibody against the FGF-CX peptide; (iii) ) A nucleic acid encoding an FGF-CX peptide; (iv) administration of an antisense nucleic acid and a nucleic acid that is “dysfunctional” (ie, due to a heterologous insertion within the coding sequence of the coding sequence for the FGF-CX peptide) Utilized to "knock out" the endogenous function of the FGF-CX peptide by homologous recombination (see, e.g., Capecchi, 1989, Science 244: 1288-1292); or (v) Modifies the interaction with its binding partner Nodal factors (ie, inhibitors, agonists and antagonists, including additional peptidomimetics of the invention or antibodies specific for the peptides of the invention).
[0275]
Diseases and disorders characterized by reduced levels or biological activity (as compared to a subject not afflicted with the disease or disorder) require a therapeutic agent that increases the activity (ie, is an agonist for the activity). And can be treated. Therapeutic agents that upregulate activity can be administered in a therapeutic or prophylactic manner. Therapeutic agents that may be utilized include, but are not limited to: FGF-CX peptide, or an analog, derivative, fragment or homolog thereof; or an agonist that increases bioavailability.
[0276]
Increased or decreased levels can be easily detected by quantifying the peptide and / or RNA. This quantification involves obtaining a patient tissue sample (eg, from a biopsy tissue) and analyzing the sample for RNA or peptide levels, structure and / or activity of the expressed peptide (or FGF-CX peptide mRNA). By assaying in vitro. Methods well known in the art include, but are not limited to, immunoassays such as by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, and the like. ) And / or hybridization assays to detect expression of mRNA (eg, Northern assays, dot blots, in situ hybridization, etc.).
[0277]
In one aspect, the present invention provides a method for treating a disease or condition associated with aberrant expression or activity of FGF-CX in a subject, wherein the agent modulates FGF-CX expression or at least one FGF-CX activity in the subject. And methods for prevention by administering to a subject. A subject at risk for a disease caused or caused by aberrant expression or activity of FGF-CX can be, for example, any of the diagnostic or prognostic assays described herein, or any thereof. By combination, they can be identified. Administration of the prophylactic agent can be prior to the onset of the symptoms characteristic of this FGF-CX abnormality so that the disease or disorder is prevented or slowed. Depending on the type of FGF-CX abnormality, for example, an FGF-CX agonist agent or an FGF-CX antagonist agent can be used to treat the subject. The appropriate agent can be determined based on the screening assays described herein.
[0278]
Another aspect of the invention relates to a method of modulating FGF-CX expression or activity for therapeutic purposes. The modulation method of the invention comprises the step of contacting a cell with an agent that modulates one or more of the activities of FGF-CX protein activity on the cell. Agents that modulate FGF-CX protein activity can be referred to herein as nucleic acids or proteins, naturally occurring cognate ligands of FGF-CX proteins, peptides, FGF-CX peptidomimetics, or other small molecules. It can be an agent as described. In one embodiment, the agent stimulates one or more FGF-CX protein activities. Examples of such stimulating agents include active FGF-CX proteins and nucleic acid molecules encoding FGF-CX introduced into the cell. In another embodiment, the agent inhibits one or more FGF-CX protein activities. Examples of such inhibitory agents include antisense FGF-CX nucleic acid molecules, and anti-FGF-CX antibodies. These modulation methods can be performed in vitro (eg, by culturing the cell with the agent) or in vivo (eg, by administering the agent to a subject). Thus, the present invention provides a method of treating an individual suffering from a disease or disorder characterized by aberrant expression or aberrant activity of a protein or nucleic acid molecule of FGF-CX. In one embodiment, the method comprises an agent that modulates (eg, up-regulates or down-regulates) the expression or activity of FGF-CX (eg, an agent identified by a screening assay described herein). Or administering a combination of such agents. In another embodiment, the method comprises administering an FGF-CX protein or nucleic acid molecule as a treatment to compensate for reduced or abnormal FGF-CX expression or activity.
[0279]
The present invention is further described in the following non-limiting examples.
[0280]
(Example)
(Example 1. Identification of FGF-CX gene)
The FGF-CX gene was synthesized using Xenopus FGF-CX (Koka, C., Adati, N., Nakata, K., Mikoshiba, K., Furuhata, Y., Sata, S., Tei, H., Sakati, Y. , Kurokawa, T., Shiokawa, K. and Yokoyama, KK (1999) Biochem. Biophys. Res. Comm. 261, 756-765; Accession No. AB012615) as queries and TBLAST of Genbank human genomic DNA sequences. (Altschul, SF, Gish, W., Miller, W., Myers, EW and Lipman, DJ (1990) J. Mol. Biol. 215, 403-410). did. This search identified a highly homologous locus on chromosome 8 (Accession number AB020858). Intron / exon boundaries were estimated using standard consensus splicing parameters (Mount, SM (1996) Science 271, 1690-1692) with known homology from FGF. The FGF-CX start codon is located at bp 16214 of the sequence of AB020858, and the remaining 3 'portion of this exon continues to bp 15930. The 5′UTR of FGF-CX was extended an additional 606 bp upstream of this start codon using public ESTs (Accession numbers AA232729, AA236522, AI272876 and AI2727878). The remaining structure of the FGF-CX gene, when related to locus AB020858, is as follows: intron 1 (bp 15929-9942); exon 2 (bp 9941-9838); intron 2 (bp 9837-7500); exon 3 (starting at bp 7499 and continuing as shown in FIG. 13; the structure of the 3 ′ untranslated region is undetermined).
[0281]
The gene found by the procedure of the previous paragraph contains three exons and two introns (FIG. 13). The DNA sequence predicts an ORF of 211 amino acid residues with an in-frame stop codon 117 bp upstream of the initiation methionine. The DNA segment from which this gene was derived is mapped to chromosome 8p21.3-p22 (location confirmed by radiation hybrid analysis) (see Example 2).
[0282]
The FGF signature motif (GX- [LI] -X) located between amino acid residues 125-148, identified by a PROSITE search (Bucher, P. & Bairoch, A. (1994) Ismb. 2, 53-61). -[STAGP] -X (6,7)-[DE] -CX- [FLM] -XEX (6) -Y) is double underlined and the intron / exon boundary is marked with an arrow Indicated by Intron 1 and Intron 2 are 5988 bp and 2338 bp long, respectively. The 5'UTR sequence was derived from a published EST and is not shown in its entirety.
[0283]
(Example 2. Irradiation hybrid mapping of FGF-CX)
Irradiation hybrid mapping with human chromosome markers was performed on FGF-CX. The procedure used was described by Steen, RG et al. (A High-Density Integrated Generic Linkage and Radiation Hybrid Map of the Laboratory Rat, Genome Research 1999, AP, published online on May 21, 1999, AP on May 21, 1999, AP on May 21, 1999). , 1999). A panel of 93 cell clones containing randomized radiation-induced human chromosome fragments was screened in 96-well plates using PCR primers designed to identify the clones sought in a unique manner. The DNA segment for which the nucleotide sequence encoding FGF-CX was identified was marked as a mapping to chromosome 8p21.3-p22. This analysis demonstrates that FGF-CX maps to chromosome 8 at a locus that overlaps with marker AFM177XB10 and at loci that are markers WI-5104 to 1.6 cR and WI-9262 to 3.2 cR. Made this result accurate.
[0284]
Example 3 Molecular Cloning of Sequence Encoding FGF-CX Protein Oligonucleotide primers were designed for amplification by PCR of a DNA segment exhibiting an open reading frame encoding full-length FGF-CX. The forward primer contains a BglII restriction site (AGATCT) and a consensus Kozak sequence (CCACC). The reverse primer contains an in-frame XhoI restriction site for further subcloning purposes. Both the forward and reverse primers contain a 5 'clamp sequence (CTCGTC). The sequences of the primers are as follows:
[0285]
Embedded image

.
[0286]
In a 50 μl volume, a total of 5 ng human prostate cDNA template, 1 μM FGF-CX forward and FGF-CX reverse primers, 5 μM dNTP (Clontech Laboratories, Palo Alto CA) and 1 μl of 50 × Advantage-HF2 polymerase ( PCR reactions were performed using Clontech Laboratories). The following PCR reaction conditions were used:
a) 96 ° C for 3 minutes
b) Denaturation at 96 ° C for 30 seconds
c) Primer annealing at 70 ° C. for 30 seconds (this temperature was gradually reduced at 1 ° C./cycle).
d) Extension at 72 ° C for 1 minute.
[0287]
Steps (b) to (d) are repeated 10 times.
[0288]
e) Denaturation at 96 ° C for 30 seconds
f) Annealing at 60 ° C for 30 seconds
g) Extension at 72 ° C for 1 minute
Steps (e) to (g) are repeated 25 times.
[0289]
h) Final extension at 72 ° C for 5 minutes.
[0290]
A single PCR product with the expected size of about 640 bp was isolated after electrophoresis on agarose gel and ligated into the pCR2.1 vector (Invitrogen, Carlsbad, CA). The cloned insert was sequenced using a vector-specific M13 forward primer (−40) and an M13 reverse primer so that the nucleotide sequence was between the upstream BglII cloning site and the downstream XhoI cloning site. Was confirmed to be 100% identical to the sequence of FIG. The cloned sequence constitutes an open reading frame encoding a putative FGF-CX full length protein. This clone is called TA-AB02085-S274-F19.
[0291]
(Example 4. Preparation of mammalian expression vector pCEP4 / Sec)
Oligonucleotide primers, pSec-V5-His forward (CTCGT CCTCG AGGGT AAGCC TATCC CTAAC (SEQ ID NO: 14)) and pSec-V5-His reverse (CTCGT CGGGC CCCTGATCAG CGGGT TTAAAC (SEQ ID NO: 15 and V), The fragment was designed to amplify a fragment from a pcDNA3.1-V5His (Invitrogen, Carlsbad, CA) expression vector containing His6. The PCR product was digested with XhoI and ApaI and ligated into the pSecTag2 B vector (Invitrogen, Carlsbad CA) carrying the XhoI / ApaI digested Igκ leader sequence. The exact structure of the resulting vector (pSecV5His) containing the Igκ leader and V5-His6 in frame was confirmed by DNA sequence analysis. This vector pSecV5His was digested with PmeI and NheI to provide a fragment that retained the above elements in the correct frame. The Pmel-Nhel fragment was ligated into the vector pCEP4 (Invitrogen, Carlsbad, CA) that had been treated with BamHI / Klenow and Nhel. The resulting vector is named pCEP4 / Sec, and contains the Igκ leader in frame, a site for insertion of the clone of interest, and the V5 epitope and 6 × His under the control of the PCMV and / or PT7 promoter. pCEP4 / Sec is an expression vector that allows the expression and secretion of heterologous proteins by fusing any protein to a multiple cloning site following an Igκ chain signal peptide. The expressed protein is detected and purified with the aid of the presence of a C-terminal V5 epitope tag and a 6 × His tag (Invitrogen, Carlsbad, Calif.).
[0292]
(Example 5. Expression of FGF-CX in human embryonic kidney (HEK) 293 cells)
The BglII-XhoI fragment containing the FGF-CX sequence was isolated from TA-AB02085-S274-F19 (Example 3) and subcloned into BamHI-XhoI digested pCEP4 / Sec to obtain the expression vector pCEP4 / Sec-FGF. -CX was produced. The pCEP4 / Sec-FGF-CX vector was transfected into 293 cells using LipofectaminePlus reagent (Gibco / BRL / Life Technologies, Rockville, MD) according to the manufacturer's instructions. Cell pellets and supernatants were collected 72 hours after transfection and examined for FGF-CX expression by Western blotting (reducing conditions) with anti-V5 antibody. FIG. 12 shows that FGF-CX is expressed as a polypeptide secreted by 293 cells with an apparent molecular weight (Mr) of about 34 kDa. In addition, a minor band is observed at about 31 ka.
[0293]
(Example 6. Expression of FGF-CX in E. coli.)
The vector pRSETA (InVitrogen Inc., Carlsbad, CA) was digested with XhoI and NcoI restriction enzymes. Oligonucleotide linkers of the sequences 5'CATGGTCGCCCTAC 3 '(SEQ ID NO: 16) and 5'TCGAGGTAGCTGAC 3' (SEQ ID NO: 17) were annealed at 37 ° C and ligated to XhoI-NcoI treated pRSETA. The resulting vector was confirmed by restriction analysis and sequencing and was named pETMY. The BglII-XhoI fragment of the sequence encoding FGF-CX (see Example 3) was ligated into the vector pETMY digested with BamHI and XhoI restriction enzymes. The expression vector is named pETMY-FGF-CX. In this vector, hFGF-CX had its N-terminus fused to a 6 × His tag and a T7 epitope. The plasmid pETMY-FGF-CX was then transferred to E. coli. coli expression host BL21 (DE3, pLys) (Novagen, Madison, WI) and the expression of the protein FGF-CX was induced according to the manufacturer's instructions. After induction, total cells were harvested and proteins were analyzed by Western blotting using an anti-HisGly antibody (Invitrogen, Carlsbad, CA). FIG. 14 shows that FGF-CX was expressed as a protein of about 32 kDa.
[0294]
(Example 7. Comparison of expression of recombinant FGF-CX protein with and without cloned signal peptide)
a) Expression without signal peptide
As described in the detailed description of the invention, FGF-CX clearly lacks the classic amino terminal signal sequence. To determine whether FGF-CX was secreted from mammalian cells, cDNA obtained as a BglII-XhoI fragment encoding full-length FGF-CX protein was cloned into TA-AB02085-S274-F19 (Example 3). Was subcloned into BamHI / XhoI digested pcDNA3.1 (Invitrogen). This provides a mammalian expression vector called pFGF-CX. This construct should incorporate a V5 epitope tag and a polyhistidine tag at the carboxy terminus of the protein, assist in the identification and purification of the protein, respectively, and should produce a polypeptide of approximately 27 kDa. After transient transfection of 293 human embryonic kidney cells, conditioned media was collected 48 hours after transfection.
[0295]
In addition to secretion of FGF-CX into conditioned media, it was also found that FGF-CX associates with cell pellet / ECM (data not shown). FGF is known to bind to heparin sulfate proteoglycan (HSPG), which is present on the cell surface and in the extracellular matrix (ECM), and we believe that FGF-CX may be sequestered in this manner. investigated. To this end, cells transfected with FGF-CX were transfected with 100 M suramin (a compound known to disrupt the low affinity interaction between growth factors and HSPGs (La Rocca, R.V. , Stein, CA & Myers, CE (1990) Cancer Cells 2, 106-115)) with 0.5 ml DMEM for 30 minutes at 4 ° C. The suramin-extracted conditioned media was then collected and clarified by centrifugation (5 min; 2000 × g).
[0296]
The conditioned medium and suramin extract were then mixed with an equal volume of 2 × gel loading buffer. Samples were boiled for 10 minutes, resolved by SDS-PAGE on a 4-20% gradient polyacrylamide gel (Novex, Dan Diego, CA) under reducing conditions, and transferred to nitrocellulose filters (Novex). Western analysis was performed using an HRP-conjugated anti-V5 antibody (Invitrogen) and an ECL detection system (Amersham Pannacia Biotech, Piscataway, NJ) according to standard procedures.
[0297]
One band with the expected Mr was identified in conditioned medium from 293 cells transfected with pFGF-CX (FIG. 11A, lane 1). Conditioned media from cells transfected with the control vector did not react with the antibody (FIG. 11A, lane 5). After suramin treatment, it was found that significant amounts of FGF-CX could indeed be released from the cell surface / ECM, indicating that HSPG probably plays a role in the separation of this protein (FIG. 11A). , Lane 2). These results indicate that FGF-CX can be secreted without the classical signal peptide.
[0298]
Recombinant FGF-CX protein has effects mediated by DNA synthesis and cell proliferation (possibly via high affinity binding of FGF-CX to cell surface receptors and regulated via low affinity interactions with HSPGs). Stimulate). Suramin extraction data suggests that FGF-CX binds to HSPGs present on the cell surface and / or in the ECM.
[0299]
b) Expression with signal peptide
For the purpose of enhancing protein secretion, a construct (pCEP4 / Sec-FGF-CX) in which the FGF-CX cDNA was fused in frame with a cleavable amino-terminal secretory signal sequence derived from the Igκ gene was made. The resulting protein also contained a carboxy-terminal V5 and a polyhistidine tag as described above for pFGF-CX. After transfection into 293 cells, a protein product with an expected Mr of about 31 kDa was obtained, and it was also found that suramin releases significant amounts of isolated FGF-CX protein (FIG. 11A; lanes 3 and 4). . As expected, pCEP4 / Sec-FGF-CX produced more soluble FGF-CX protein than pFGF-CX. [0300]
Results similar to those described above for 293 cells were obtained with NIH 3T3 cells (FIG. 11B).
[0301]
(Example 8. Real-time quantitative expression analysis of FGF-CX nucleic acid by PCR) Quantitative expression of various clones was performed by real-time quantitative PCR (TAQMAN (TAQMAN) registered in Perkin-Elmer Biosystems ABI PRISM (registered trademark) 7700 Sequence Detection System). (Trademark) analysis) in 41 normal and 55 tumor samples (in most cases, the samples shown in panels A and B of FIG. 15 are the samples identified in Table 3). In Table 3, the following abbreviations are used:
ca. = Carcinoma,
^* = Established from metastasis,
met = metastasis,
s cell var = small cell mutant,
non-s = non-sm = non-small,
squam = squamous epithelium,
pl. eff = pl effusion = pleural effusion,
glio = glioma,
astro = astrocytoma, and
neuro = neuroblastoma.
[0302]
First, 96 RNA samples were normalized to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RNA (total of about 50 ng or about 1 ng poly A +) was converted to a random hexamer according to the TAQMAN® reverse transcriptase kit (PE Biosystems, Foster City, CA; Catalog No. N808-0234) and according to the manufacturer's protocol. Was used to convert to cDNA. Reactions were performed in 20 μl and incubated at 48 ° C. for 30 minutes. The cDNA (5 μl) was then combined with β-actin and GAPDH TAQMAN® assay reagents (PE Biosystems; catalog numbers 4310881E and 4310884E, respectively) and TAQMAN® universal PCR master mix (PE Biosystems) according to the manufacturer's protocol. Transferred to a separate plate for the TAQMAN® reaction using catalog number 430447). Reactions were performed in 25 μl using the following parameters: 50 ° C. for 2 minutes; 95 ° C. for 10 minutes; 95 ° C. for 15 seconds / 60 ° C. for 1 minute (40 cycles). The results are recorded as CT values using the log scale (cycles where a given sample exceeds a threshold level of fluorescence) and the difference in RNA concentration between a given sample and the sample with the lowest CT value is It is shown as 2 to the power of ΔCT. The relative percent expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100. Mean CT values obtained for β-actin and GAPDH were used to normalize RNA samples. The RNA sample that produced the highest CT value did not require further dilution, while all other samples were diluted relative to this sample according to the average CT value of β-actin / GAPDH.
[0303]
Normalized RNA (5 μl) was converted to cDNA and analyzed by One Step RT-PCR Master Mix Reagents (PE Biosystems; Cat. No. 4309169) and TAQMAN® using gene specific primers according to the manufacturer's instructions. did. Probes and primers were set up for each assay according to the Perkin Elmer Biosystem's Primer Express Software package (Version I for Macintosh Power PC from Apple Computer) using the sequence of clone 103266230.0.38 as input. The default settings were used for the reaction conditions and the following parameters were designed before selecting the primers: primer concentration = 250 nM, primer melting temperature (T _m ) Range = 58 ° C to 60 ° C, optimal T of primer _m = 59 ° C, maximum difference between primers = 2 ° C, probe has no 5'G, probe T _m Is the primer T _m Amplicon size 75 bp to 100 bp, which must be 10 ° C. higher. Selected probes and primers (see below) were synthesized by Synthegen (Houston, TX, USA). The probe was purified twice by HPLC to remove unreacted dye and evaluated by mass spectrometry to demonstrate the coupling of reporter and quencher dyes to the 5 'and 3' ends of the probe, respectively. did. The final concentration of the forward and reverse primers was 900 nM each, and the concentration of the probe was 200 nM.
[0304]
For PCR, normalized RNA from each tissue and each cell line was spotted into each well of a 96-well PCR plate (Perkin Elmer Biosystems). A PCR cocktail containing two probes (a probe specific for FGF-CX and a second gene specific probe to serve as an internal standard) was prepared using 5 mM MgCl 2 ₂ , DNTPs (dA, dG, dC, dU at a ratio of 1: 1: 1: 2), 0.25 U / ml AmpliTaq Gold ^TM 1 × TaqMan with (PE Biosystems), and 0.4 U / μl RNase inhibitor, and 0.25 U / μl reverse transcriptase. ^TM PCR Master Mix was used to set up for PE Biosystems 7700. Reverse transcription was performed at 48 ° C. for 30 minutes, followed by PCR amplification as follows: 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute.
[0305]
(Table 3. Tissue samples used in TaqMan expression analysis)
Number Tissue sample
1 endothelial cells
2 endothelial cells (treatment)
3 pancreas
4 Pancreas ca. CAPAN 2
5 fat
6 adrenal glands
7 Thyroid
8 Salivary glands
9 Pituitary
10 brain (fetus)
11 Brain (whole)
12 brain (cerebellar tonsils)
13 Brain (cerebellum)
14 Brain (hippocampus)
15 brain (hypothalamus)
16 Brain (black matter)
17 Brain (thalamus)
18 spinal cord
19 CNS ca. (Glio / astro) U87-MG
CNS ca. (Glio / astro) U-118-
20 MG
21 CNS ca. (Astro) SW1783
CNS ca. ^* (Neuro; met) SK-N
22 AS
23 CNS ca. (Astro) SF-539
24 CNS ca. (Astro) SNB-75
25 CNS ca. (Glio) SNB-19
26 CNS ca. (Glio) U251
27 CNS ca. (Glio) SF-295
28 Heart
29 Skeletal muscle
30 bone marrow
31 Thymus
32 spleen
33 lymph nodes
34 colon (ascending)
35 stomach
36 Small intestine
37 colon ca. SW480
Colon ca. ^* (SW480
38 met) SW620
39 colon ca. HT29
40 colon ca. HCT-116
41 colon ca. CaCo-2
42 colon ca. HCT-15
43 colon ca. HCC-2998
Stomach ca. ^* (Liver met) NCI-
44 N87
45 bladder
46 trachea
47 kidney
48 kidney (fetus)
49 kidney ca. 786-0
50 kidney ca. A498
51 kidney ca. RXF 393
52 kidney ca. ACHN
53 kidney ca. UO-31
54 kidney ca. TK-10
55 liver
56 Liver (fetus)
57 liver ca. (Hepatoblastoma) HepG2
58 lungs
59 lungs (fetus)
60 lungs ca. (Small cell) LX-1
61 lung ca. (Small cell) NCI-H69
62 lungs ca. (S. Cell var.) SHP-77
63 lung ca. (Large cell) NCI-H460
64 lungs ca. (Non-sm.cell) A549
65 lung ca. (Non-s. Cell) NCI-H23
66 lung ca. (Non-s. Cell) HOP-62
67 lung ca. (Non-s.cl) NCI-H522
68 lungs ca. (Squam.) SW 900
69 lung ca. (Squam.) NCI-H596
70 Mammary gland
71 chest ca. ^* (Pl. Effusion) MCF-7
72 chest ca. ^* (Pl.ef) MDA-MB-231
73 chest ca. ^* (Pl. Effusion) T47D
74 chest ca. BT-549
75 chest ca. MDA-N
76 Ovary
77 ovary ca. OVCAR-3
78 ovary ca. OVCAR-4
79 ovary ca. OVCAR-5
80 ovary ca. OVCAR-8
81 ovary ca. IGROV-1
82 ovary ca. ^* (Ascites) SK-OV-3
83 Myometrium
84 Uterus
85 Placenta
86 prostate
87 prostate ca. ^* (Bone met) PC-3
88 Testicle
89 Melanoma Hs688 (A). T
90 Melanoma ^* (Met) Hs688 (B). T
91 Melanoma UACC-62
92 Melanoma M14
93 Melanoma LOX IMVI
94 Melanoma ^* (Met) SK-MEL-5
95 Melanoma SK-MEL-28
96 Melanoma UACC-257
The following primers and probes were designed. Each carries a minimum of three mismatches in the corresponding regions of the highly homologous human FGF-9 and human FGF-16 genes, as is specific for FGF-CX. Set Ag81b covers the region from base 270 to base 343 in FIG. 1 (SEQ ID NO: 1). They should not detect other known FGF family members. The primers and probes used were as follows.
Ag81b (F): 5′-GGACCACAGCCTCTCTCGGTA-3 ′ (SEQ ID NO: 18);
Ag81b (R): 5'-TGTCCACACCTCTAATACTGACCAG-3 '(SEQ ID NO: 19);
Ag81b (P): 5'-FAM-CCCACTGCCCACTGATGAATTCCAA-TAMRA-3 '(SEQ ID NO: 20).
[0306]
The results from a representative experiment are shown in FIG. 15 Panel A and FIG. 15 Panel B. Expression is plotted as a percentage of the sample showing the highest level of expression. Quadruple runs were performed and are represented by variously shaded bars. In 39 normal human tissues tested, FGF-CX was found to be most highly expressed in the brain, especially the cerebellum (FIG. 15 panel A and FIG. 15 panel B). Other tissues in the central nervous system expressed much weaker levels of FGF-CX. Of the 54 human tumor cell lines tested, FGF-CX was a lung carcinoma cell line (LX-1), a colon carcinoma cell line (SW-480), a colon carcinoma cell line and metastasis (SW480), and a gastric cancer It was found to be most highly expressed in tumor cell lines (NCI-N87; see FIG. 15 panel A and FIG. 15 panel B).
[0307]
Further real-time (real-time) expression analysis was performed on an extensive panel of tumor tissues obtained during surgery. These tissues contain portions from the actual tumor itself, as well as a portion called "normal adjacent tissue (NAT)", which is typically already inflamed and shows histological evidence of dysplasia. . A primer-probe set (Ag81) selected to be specific for FGF-CX was used in such TaqMan experiments with such surgical tissue samples. Here, duplicate runs were performed:
Ag81 (F): 5'-AGGCAGAAGCGGGAGATAGAT-3 '(SEQ ID NO: 21);
Ag81 (R): 5'-AGCAGCTTTTACCTCATTCCAAATG-3 '(SEQ ID NO: 22); and
Ag81 (P): TET-5'-CCATCTACCATCCACCACCAGTTGCAGAA-3'-TAMRA (SEQ ID NO: 23).
Set Ag81 covers the region from base 477 to base 554 in FIG. 1 (SEQ ID NO: 1). This copy is shown as gray and black shaded bars in FIG. 15 Panel C and FIG. 15 Panel D. This result indicates that for many matched pairs of tumors and their dysplastic NAT samples, FGF-CX is highly expressed in NAT rather than the tumor itself; more specifically, in parenchymal cells adjacent to the tumor. It shows dramatically. Examples that produce this consistent pattern include ovarian, bladder, uterine, lung, prostate, and liver cancers.
[0308]
Without being limited by theory, from the results of FIG. 15 panel C and FIG. 15 panel D, FGF-CX was found to inhibit tumor epithelium and / or Alternatively, it is considered that paracrine stimulation of other components may contribute to tumor progression. Similarly, FGF-CX can function to stimulate components in host tissues that synthesize or secrete FGF-CX in an autocrine fashion. These host component cells can subsequently act on the tumor fraction.
[0309]
An increase in the expression profile of FGF-CX compared to non-matched normal tissues suggests that FGF-CX plays a prospective or promoting role in tumor progression. Thus, a number of targeting approaches (as non-limiting examples, monoclonal antibodies, ribozymes, antisense oligonucleotides, peptides that neutralize the interaction of FGF-CX with cognate receptors, and FGF-CX) Therapeutic targeting of FGF-CX using any of these (including small molecule drugs that modulate the unidentified receptor of) is expected to have a positive therapeutic impact on disease progression. Similarly, the use of such agents to modulate the biological activity of FGF-CX in tumor progression is expected to synergize or enhance conventional chemotherapy and radiation therapy. Particular disease applications to which therapeutic targeting of FGF-CX may be applied include adenocarcinoma of the colon, prostate, lung, kidney, uterus, breast, bladder, ovary.
[0310]
(Example 9. Stimulation of bromodeoxyuridine taken up by recombinant FGF-CX)
293-EBNA cells (Invitrogen) were transfected using Lipofectamine 2000 according to the manufacturer's protocol (Life Technologies, Gaithersburg, MD). Cells were supplemented with 10% fetal calf serum (FBS; Life Technologies) 5 hours after transfection. Cells were washed and fed with Dulbecco's modified Eagle's medium (DMEM; Life Technologies) 18 hours after transfection to produce BrdU and protein for the proliferation assay (Example 10). After 48 hours, the medium was discarded and the cell monolayer was incubated with 100 μM suramin (Sigma, St. Louis, MO) in 0.5 ml of DMED for 30 minutes at 4 ° C. The suramin extraction conditioned medium is then removed, clarified by centrifugation (5 min; 2000 × g), and subjected to TALON metal affinity chromatography utilizing a carboxy-terminated polyhistidine tag, according to the manufacturer's instructions (Clontech, Palo Alto). Alto, CA). The retained fusion protein was released by washing the column with imidazole.
[0311]
FGF-CX protein concentration was estimated by Western analysis using a calibration curve generated with known concentrations of V5-tagged protein. For Western analysis, conditioned media was collected 48 hours after transfection, and cell monolayers were then incubated with 0.5 ml DMEM containing 100 μM suramin at 4 ° C. for 30 minutes. The suramin-containing conditioned medium was then collected.
[0312]
To generate control proteins, 293-EBNA cells were transfected with the pCEP4 plasmid (Invitrogen) and subjected to the purification procedures outlined above.
[0313]
Recombinant FGF-CX was tested for its ability to induce DNA synthesis in a bromodeoxyuridine (BrdU) uptake assay. NIH 3T3 cells (ATCC No. CRL-1658, American Type Culture Collection, Manassas, Va.), CCD-1070Sk cells (ATCC No. CRL-2091), or MG-63 cells (ATCC No. CRL-1427) in a 96-well plate. , Cultured to approximately 100% confluence, washed with DMEM, and serum-starved in DMEM for 24 hours (NIH 3T3) or 48 hours (CCD-1070Sk and MG-63). Then, recombinant FGF-CX or control protein was added to the cells for 18 hours. The BrdU assay was performed according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN) using a 5 hour BrdU incorporation time.
[0314]
FGF-CX was found to induce DNA synthesis in NIH 3T3 mouse fibroblasts at a half-maximal concentration of about 5 ng / ml (FIG. 16A). In contrast, proteins purified from cells transfected with the control vector did not induce DNA synthesis. When measured by BrdU incorporation, FGF-CX was expressed in various human cell lines (CCD-1070Sk normal human dermal fibroblasts (FIG. 16B), CCD-1106 keratinocytes (FIG. 16C), MG-63 osteosarcoma cells (data (Not shown) and including breast epithelial cells) were also found to induce DNA synthesis at comparable dose levels.
[0315]
(Example 10. Induction of cell proliferation by recombinant FGF-CX)
To determine whether recombinant FGF-CX induces cell growth, NIH3T3 cells were cultured in 6-well plates to approximately 50% confluence, washed with DMEM, and treated with recombinant FGF-CX or control protein. Were fed for 48 hours in DMEM containing, and then counted. Cell numbers were determined by trypsinizing cells and counting them on a Beckman Coulter Z1 series counter (Beckman Coulter, Fullerton, Calif.). FGF-CX was found to induce an approximately 3-fold increase in cell number in this assay, relative to the control protein (FIG. 17).
[0316]
To detail the morphological change events in growth, NIH 3T3 cells were treated with recombinant FGF-CX or control protein in DMEM / 2% calf serum for 48 hours and Zeiss Axiovert 100 microscope (Carl Zeiss). , Inc., Thornwood, NY).
[0317]
In addition to achieving higher cell densities (FIG. 17), NIH 3T3 cells cultured in the presence of FGF-CX prepared as described in Example 9 showed a disordered growth pattern. This indicates a lack of contact inhibition (FIG. 18). In addition, individual cells were found to be elongated and flexible. These results indicate that FGF-CX acts as a growth factor and suggest that recombinant FGF-CX mediates the morphological transformation of NIH 3T3 cells.
[0318]
(Example 11. Tumor formation by ectopic FGF-CX transfected NIH 3T3 cells in nude mice)
NIH 3T3 cells were transfected with pCEP4 / Sec-FGF-CX or a control vector using Lipofectamine Plus according to the manufacturer's protocol (Life Technologies). Cells were supplemented with 10% calf serum (CS; Life Technologies) 5 hours after transfection. pCEP4 / Sec-FGF-CX transfected cells have been morphologically transformed by 48 hours post-transfection and the morphological transformation has been selected after 2 weeks of selection in growth medium containing hygromycin. Maintained. In contrast, cells transfected with the control vector maintained their normal morphology (data not shown). Thus, transfected cells behave as expected, for example, based on the experiments reported in Example 10.
[0319]
To study the induction of ectopic tumors, NIH 3T3 cells were transfected with various experimental and control vectors. Two days after transfection, cells were transfected with DMEM / 5% CS (for pFGF-CX transfected cells) or DMEM / 10% CS supplemented with 500 μg / ml hygromycin B (pCEP4 / Sec-FGF-CX transfected). (For cells). After two weeks of culture, subconfluent cells were trypsinized, neutralized with DMEM / 10% CS, washed with PBS, and counted. One million cells in PBS were injected into the lateral subcutaneous tissue of female athymic nude mice (Jackson Laboratories, Bar Harbor, ME).
[0320]
NIH 3T3 cells were transfected with FGF-CX expression plasmids (pFGF-CX and pIgκ-FGF-CX) or their appropriate control vectors. We found that cells transfected with any of the FGF-CX expression vectors were morphologically transformed by 48 hours post-transfection (data not shown) and recombinant FGF-CX Was found to have a phenotype similar to the phenotype that occurs after exposure of NIH 3T3 cells to E. coli (Figure 17). In contrast, cells transfected with the control vector maintained their normal morphology (data not shown).
[0321]
To determine whether ectopic expression of FGF-CX in vivo induces tumorigenicity of NIH 3T3 cells, stable transfectants were generated and injected subcutaneously into nude mice. By day 11, all animals injected with either pFGF-CX or pIgκ-FGF-CX transfected cells had a rapidly growing tumor that increased in size by day 14. However, none of the animals injected with control cells developed tumors by week 2 (FIG. 19). These results indicate that cells transformed by transfection with a vector carrying the FGF-CX gene promote tumor development and growth in vivo.
[0322]
(Example 12. Method for identifying nucleic acid encoding fibroblast growth factor-20-like protein)
A further fibroblast growth factor-20-like protein sequence (FGF20X) of the present invention (also referred to herein as clone CG53135-02) was derived by in silico prediction of this sequence by experimental cloning of a cDNA fragment. A cDNA fragment covering either the full length of the DNA sequence or a portion of the sequence, or both, was cloned. In silico predictions were based on sequences available in CuraGen's own sequence database, or in the public human sequence database, and were provided with either the full-length DNA sequence or some portion thereof.
[0323]
Experimental cloning was performed using one or more of the methods summarized below:
SeqCalling ^TM Technology: cDNA is derived from a variety of human samples that exhibit multiple tissue types, normal and disease states, pathological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissues, cultured primary cells or cell lines. Cells and cell lines may be treated with biochemical or chemical factors (compounds) that regulate gene expression (eg, growth factors, chemokines or steroids). The derived cDNA was then sequenced using CraGen's in-house SeqCalling technology. Sequence traces were evaluated manually and edited for correction as needed. Using bioinformatics programs to generate consensus sequences for each assembly, cDNA sequences from all samples, sometimes including public human sequences, were assembled together. Each assembly is included in the CuraGen Corporation database. If the degree of identity with another component was at least 95% over 50 bp, the sequence was included as a component for assembly. Each assembly represents a gene or portion thereof and contains information about the variant, such as splice forms, single nucleotide polymorphisms (SNPs), insertions, deletions, and other sequence variations.
[0324]
Exon Linking: The cDNA code for the CG53135-02 sequence was cloned by the polymerase chain reaction (PCR) using the following primers: 5'-AGGTCACATCATGCTGTTATTGGC-3 '(SEQ ID NO: 24), and 5'-CTGTCTGTTCCTCAGAAGAAGTTCTTGATCC -3 '(SEQ ID NO: 25). Primers were designed based on in silico predictions of the full length portion or several portions (one or more exons) of the cDNA / protein sequence of the invention. Using these primers, cDNA was amplified from pools containing expressed human sequences from the following tissues: adrenal gland, bone marrow, brain-tonsil, brain-cerebellum, brain-hippocampus, brain-nigral, brain- Thalamus, brain-whole brain, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma-large, mammary gland, pancreas, pituitary, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach , Testis, thyroid, trachea, and uterus.
[0325]
Multiple clones, sometimes containing public human sequences, were sequenced using a bioinformatics program to generate consensus sequences for each assembly, and these fragments were assembled together. Each assembly is included in the CuraGen Corporation database. If the degree of identity with another component was at least 95% over 50 bp, the sequence was included as a component for assembly. Each assembly represents a gene or portion thereof and contains information about the variant, such as splice forms, single nucleotide polymorphisms (SNPs), insertions, deletions, and other sequence variations.
[0326]
Physical clone: The PCR product (covering the entire open reading frame) induced by exon ligation was cloned into the pCR2.1 vector from Invitrogen, and 137627 :: 160883874.1403010. A9 was obtained.
[0327]
Variant sequences are also included in the present application. Variant sequences may include single nucleotide polymorphisms (SNPs). A SNP may be referred to in some cases as a "cSNP" to indicate that the nucleotide sequence containing the SNP is derived from cDNA. SNPs can occur in several ways. For example, SNPs can result from the substitution of one nucleotide for another at various sites. Such substitutions can be either transitions or transversions. SNPs can also result from nucleotide deletions or insertions relative to a reference allele. In this case, the sites at which one allele carries a gap for a particular nucleotide in another allele are diverse sites. A SNP that occurs within a gene can result in a change in the amino acid encoded by the gene at the location of the SNP. A SNP within a gene can also be silent if the codon containing the SNP encodes the same amino acid as a result of the redundancy of the genetic code. SNPs that occur outside of the region of the gene, or in introns within the gene, do not result in a change in any amino acid sequence of the protein, but may result in altered regulation of the expression pattern. Examples include changes in transient expression, regulation of physiological responses, regulation of cell type expression, intensity of expression, and stability of transcribed messages.
[0328]
The DNA and protein sequences of the novel fibroblast growth factor-20-like gene were obtained by exon ligation and are reported herein as clone CG53135-02 and are shown in Tables 4 and 5 below.
[0329]
(Table 4. Nucleotide sequence encoding the fibroblast growth factor-20-like protein of the present invention (SEQ ID NO: 26))
[0330]
Embedded image

(Table 5. Protein sequence encoded by SEQ ID NO: 26 (SEQ ID NO: 27))
[0331]
Embedded image

A novel 540 nucleotide nucleic acid (clone CG53135-02) encoding a novel fibroblast growth factor-20-like protein is shown in Table 4. An open reading frame beginning at nucleotides 1-3 and ending at nucleotides 538-540 was identified. This polypeptide represents a novel functional fibroblast growth factor-20-like protein. The start and stop codons of the open reading frame are highlighted in bold. Putative untranslated regions (underlined) (if present) are found upstream of the start codon and downstream of the stop codon. The encoded protein having 179 amino acid residues is shown in Table 5 using the one-letter code.
[0332]
(Similarity)
In the search of the sequence database, for example, if the nucleic acid of the present invention is 506 bases derived from Homo sapiens, gb: GENBANK-ID: AB044277 | acc: AB044277.1 mRNA (Homo sapiens mRNA for FGF-20: complete cds) Was found to have 495 bases (97%) identical to The full-length amino acid sequence of the protein of the present invention comprises ptnr of 211 amino acid residues derived from Homo sapiens (human) (fibroblast growth factor-20 (FGF-20)) out of 162 amino acid residues: SWISSSNEW-ACC: Q9NP95 protein It was found to have 160 residues (98%) identical to and 160 residues (98%) similar to this among 162 amino acid residues. Further homology is provided in Table 6 below.
[0333]
[Table 1]

A multiple sequence alignment is given in Table 7 and the protein of the invention is shown in the first column in a ClustalW analysis comparing the protein of the invention with the relevant protein sequence. Note that this sequence represents a splice form of fibroblast growth factor-20, as shown at positions 20-51. In the alignments shown above, the amino acid residues in black outline indicate residues that are also conserved between sequences (ie, residues that may need to preserve structural or functional properties); Amino acid residues having a gray background, which retain equivalent physical and / or chemical properties without altering the structure or function of the protein (eg, groups L, V, I, and M are similar Are similar to each other between the sequences; and amino acid residues having a white background are not conserved or similar between the sequences.
[0334]
(Table 7. ClustalW alignment of CG53135-02 protein with related proteins)
[0335]
[Table 2]

The presence of an identifiable domain in the proteins disclosed herein is determined by a search against a domain database such as Pfam, PROSITE, ProDom, Block or Print, and then identified by the Interpro domain accession number. The important domains are summarized in Table 8.
[0336]
[Table 3]

Peparin-binding growth factors I and II (HBGF) [1,2] (also known as acidic and basic fibroblast growth factor (FGF)) have a wide variety of mesoderm or neuroectoderm origins Are structurally related mitogens that stimulate the proliferation or differentiation of cells. These two proteins belong to the family of growth factors and oncogenes, which are also members of the superfamily that also include the interleukin-1 protein, the Kunitz-type soybean trypsin inhibitor (STI) (see) and histactophilin. All have very similar structures and the HBGF and interleukin-1 families share some sequence similarity (about 25% [1]), but they show quite similarity to STI Absent.
[0337]
HBGF is involved in a number of different processes involved in controlling cell differentiation and proliferation [1]. HBGF1 and HBFG2 have similar effects: they induce mesoderm formation in embryonic development and mediate wound repair, angiogenesis and nerve growth; they also induce fibroblasts, endothelial cells and large cells. Induces glial cell proliferation and migration. HBGF3 (int-2) and HBGF4 (hst / ks) are known oncogenes from gastric tumors and Kaposi's sarcoma, respectively. HBGF5 and HBGF6 are also oncogene products. HBGF7 (keratinocyte growth factor) is probably the major paracrine effector of normal epithelial cell proliferation.
[0338]
These growth factors cause dimerization of these tyrosine kinase receptors that result in intracellular signaling. Currently, there are four known tyrosine kinase receptors for fibroblast growth factor. Each of these receptors can bind to several different members of this family [3].
[0339]
The crystal structures of HBGF1 and HBGF2 were analyzed [4] and shown to have the same 12-chain β-sheet structure as both interleukin-1 and Kunitz-type soybean trypsin inhibitor [5]; Leukin-1 was found to be similar, and they were presumed to have similar structures [6]. The β-sheet is arranged in three similar leaves around a central axis, with six chains forming anti-parallel β-barrels. Several regions of HBGF1 (primarily the beta chains 1-3 and the loop between chains 8 and 9) are involved in receptor binding. The loop between chains 10 and 11 is thought to be involved in binding pepperin.
[0340]
(Table 9: PSORT, SignalP and hydropathy results for CuraGen accession number CG53135-02)
[0341]
[Table 4]

The novel FGF20X nucleic acids or fragments thereof encoding the FGF20X proteins of the present invention may be further useful in diagnostic applications where the presence or amount of the nucleic acid or protein is evaluated. These substances are further useful in the production of antibodies that immunospecifically bind to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies can be produced according to methods known in the art using inferences from the hydrophobicity chart, as described in the section “Anti-FGF20X Antibodies” below. The disclosed FGF20X protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated FGF20X epitopes are from about amino acids 20-65. In another specific embodiment, the FGF20X epitope is from about amino acids 80-175.
[0342]
This indicates that the FGF20X sequences of the present invention have properties similar to those of other proteins known to contain this / these domains and properties similar to those of these domains.
[0343]
(Chromosome information)
The FGF20X gene disclosed in the present invention maps to chromosome 8p21.3-p22. This assignment was made using genomic clones, public genes and information associated with ESTs, and the CuraGen Corporation's Electronic Northern Bioinformatics tool, which share sequence identity with the disclosed sequences.
[0344]
(Tissue expression)
The fibroblast growth factor-20-like gene disclosed in the present invention is expressed in at least the following tissues: mammalian tissues, colon, lung, brain, liver, kidney, and stomach. The expression information is derived from the tissue source of the sequence contained in the derivative of the sequence of clone CG53135-02.
[0345]
(Equation 1)

(Equivalent)
From the foregoing detailed description of certain embodiments of the invention, it is apparent that certain novel compositions and methods, detections and treatments, including nucleic acids, polypeptides, and antibodies, have been described. Although these specific embodiments have been disclosed in detail herein, this is done by way of example only, and is not intended to be limiting with respect to the appended claims above. In particular, various substitutions, alterations, and modifications may be made to the invention, as are conventional events for one skilled in the art, without departing from the spirit and scope of the invention as defined by the appended claims. Is intended by the present inventors. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
[Brief description of the drawings]
FIG.
FIG. 1 is a description of the nucleotide sequence of the novel FGF-CX polynucleotide of the present invention (SEQ ID NO: 1) and the translated amino acid sequence of the novel FGF-CX protein of the present invention (SEQ ID NO: 2).
FIG. 2
FIG. 2 is a BLASTN alignment between the nucleic acid sequence of SEQ ID NO: 1 and an FGF-9-like glial activator (GAF) sequence (SEQ ID NO: 5).
FIG. 3
FIG. 3 shows the complementary strand of the nucleic acid sequence of SEQ ID NO: 1 and three discontinuous segments of human chromosome 8 long genomic fragment (GenBank accession number AB020858) (SEQ ID NOS: 6 to 8 in AC, respectively). BLASTN alignment.
FIG. 4
FIG. 4 is a ClustalW alignment of four vertebrate FGF-like proteins (SEQ ID NOs: 9-12) with the FGF-CX protein of the invention (SEQ ID NO: 2). Black, gray, and white representations are identical, conserved, and non-conserved residues, respectively, in the alignment.
FIG. 5
FIG. 5 is ClustalW of FGF-CX and three other FGF family members. FGF-CX was aligned with human FGF-9, human FGF-16, and Xenopus FGF-CX (accession numbers D14838, AB009391 and AB012615, respectively).
FIG. 6
FIG. 6 is a BLASTP alignment of the FGF-CX polypeptide sequence (SEQ ID NO: 2) with human FGF-9 (SEQ ID NO: 9), showing residues that are identical (“|”) and positive (“+”). Show.
FIG. 7
FIG. 7 is a BLASTX alignment between the FGF-CX polypeptide sequence (SEQ ID NO: 2) and mouse FGF-9 (SEQ ID NO: 10), with identical (“|”) and positive (“+”) residues Is shown.
FIG. 8
FIG. 8 is a BLASTX alignment of the FGF-CX polypeptide sequence (SEQ ID NO: 2) with rat FGF-9 (SEQ ID NO: 11), showing residues that are identical (“|”) and positive (“+”) Is shown.
FIG. 9
FIG. 9 is a BLASTX alignment between the FGF-CX polypeptide sequence (SEQ ID NO: 2) and Xenopus XFGF-CX (SEQ ID NO: 12), identical (“|”) and positive (“+”) residues Is shown.
FIG. 10
FIG. 10 is a representation of a hydropathy of the FGF-CX polypeptide of SEQ ID NO: 2 generated in a 19 residue window.
FIG. 11
FIG. 11 shows a Western analysis of FGF-CX. Samples from 293 cells (panel A) or NIH 3T3 cells (panel B) transiently transfected with the indicated constructs were examined by Western analysis using an anti-V5 antibody. CM = training medium, SE = suramin extraction training medium. Molecular weight markers are shown on the left.
FIG.
FIG. 12 shows a Western analysis of FGF-CX protein secreted by 293 cells.
FIG. 13
FIG. 13 depicts an analysis of the FGF-CX gene, which contains the nucleotide and deduced amino acid sequence of FGF-CX (SEQ ID NO: 2). The start and stop codons are in bold and the in-frame stop codon present in the 5'UTR is underlined.
FIG. 14
FIG. 5 shows a Western analysis of the FGF-CX (SEQ ID NO: 2) protein expressed in E. coli.
FIG.
FIG. 15 depicts an analysis of FGF-CX expression obtained by real-time quantitative PCR using FGF-CX-specific TaqMan reagents. Results for normalized RNA from normal human tissue samples are shown in Panel A and results for normalized RNA from tumor cell lines are shown in Panel B. The results obtained using the tumor tissue obtained directly during the surgery are shown in Panels C and D.
FIG.
FIG. 16 presents the biological activity of recombinant FGF-CX, as represented by the effect of recombinant FGF-CX on DNA synthesis. Cells were serum starved, incubated with the indicated factors for 18 hours, and analyzed by BruU incorporation assay. Samples were performed in triplicate. Panel A is NIH 3T3 mouse fibroblasts. Panel B is a CCD-1070 human fibroblast. Panel C is CCD-1106 human keratinocytes.
FIG.
FIG. 17 shows the biological activity of recombinant FGF-CX, as indicated by the effect of recombinant FGF-CX on cell growth. NIH 3T3 cells were incubated with serum-free medium supplemented with the indicated factors and counted after 48 hours. The samples were performed in duplicate.
FIG.
FIG. 18 depicts the biological activity of recombinant FGF-CX as demonstrated by the effect of recombinant FGF-CX on cell morphology. NIH 3T3 cells were incubated with FGF-CX protein or control protein for 48 hours and photographed at a magnification of × 25.
FIG.
FIG. 19 shows a graph showing the tumorigenic activity of FGF-CX. NIH 313 cells stably transfected with the indicated constructs were injected subcutaneously into athymic nude mice and examined for tumor formation over a two week period. A minimum of 4 animals was used as each data point.

Claims (62)

An isolated polypeptide, comprising:
(A) an amino acid sequence defined by SEQ ID NO: 2 or 27;
(B) a variant of the amino acid sequence defined by SEQ ID NO: 2 or 27, wherein any amino acid specified in the selected sequence has been changed to a different amino acid, provided that A variant in which no more than 15% of the amino acid residues are altered in this way;
(C) the mature form of the amino acid sequence defined by SEQ ID NO: 2 or 27;
(D) a variant of the mature form of the amino acid sequence defined by SEQ ID NO: 2 or 27, wherein any amino acid in the mature form of the selected sequence has been changed to a different amino acid sequence, A variant wherein no more than 15% of the amino acid residues of the mature form of the sequence are so altered; and (e) fragments of the amino acid sequences described in paragraphs (a)-(d). ,
A polypeptide comprising an amino acid sequence selected from the group consisting of: A fragment of the polypeptide of claim 1. 2. The polypeptide of claim 1, wherein said polypeptide is a naturally occurring allelic variant of SEQ ID NO: 2 or 27. 4. The polypeptide of claim 3, wherein the variant is a translation product of a single nucleotide polymorphism. 2. The polypeptide of claim 1, wherein the polypeptide is a variant polypeptide, wherein one or more of the optional amino acids identified in SEQ ID NOs: 2 or 27 has conservative substitutions. A polypeptide that is altered to provide. An isolated nucleic acid molecule, wherein the nucleic acid molecule comprises:
(A) a polypeptide contained in SEQ ID NO: 2 or 27;
(B) a variant of SEQ ID NO: 2 or 27, wherein any amino acid specified in the selected sequence has been changed to a different amino acid, provided that no more than 15% of the amino acids in the sequence A variant in which the residues are thus altered;
(C) the mature form of the amino acid sequence defined by SEQ ID NO: 2 or 27;
(D) a variant of the mature form of the amino acid sequence defined by SEQ ID NO: 2 or 27, wherein any amino acid in the mature form of the selected sequence has been changed to a different amino acid, A variant wherein no more than 15% of the amino acid residues in the sequence of the form are so altered.
(E) fragments of the amino acid sequences described in (a) to (d); and (f) the complement of any of the nucleic acid molecules described in the paragraphs (a) to (e). A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising a more selected amino acid sequence. 7. The nucleic acid molecule of claim 6, wherein said nucleic acid molecule comprises a nucleotide sequence of a naturally occurring variant of an allelic nucleic acid. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule encodes a variant polypeptide comprising the polypeptide sequence of a naturally occurring polypeptide variant. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule comprises a single nucleotide polymorphism that encodes the variant polypeptide. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule is:
(A) a nucleotide sequence defined by SEQ ID NO: 1 or 26;
(B) a nucleotide sequence, wherein one or more nucleotides in the nucleotide sequence defined by SEQ ID NO: 1 has changed from a nucleotide provided by the selected sequence to a different nucleotide, provided that 20% A nucleotide sequence in which the following nucleotides are thus altered;
(C) a nucleic acid fragment of the sequence according to (a);
(D) a nucleic acid fragment of the sequence of (b); and (e) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of any complement of the nucleic acid molecule. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule comprises a nucleotide sequence, wherein any nucleotide identified in the coding sequence of the selected nucleotide sequence is A nucleic acid molecule that varies from a nucleotide defined by a selected sequence to a different nucleotide, provided that no more than 20% of the nucleotides in the selected coding sequence are so varied. An isolated nucleic acid molecule encoding a fragment of an FGF-CX polypeptide. 13. The nucleic acid molecule according to claim 12, wherein said FGF-CX polypeptide is a variant of SEQ ID NO: 2 or 27. 13. The nucleic acid molecule of claim 12, wherein said FGF-CX polypeptide is a mature FGF-CX polypeptide. 13. The nucleic acid molecule of claim 12, wherein the FGF-CX polypeptide is a variant of the mature form of SEQ ID NO: 2 or 27. A vector comprising the nucleic acid molecule according to claim 6. A cell comprising the vector of claim 16. An antibody that immunospecifically binds to the polypeptide of claim 1. 19. The antibody according to claim 18, wherein said antibody is a monoclonal antibody. 19. The antibody according to claim 18, wherein said antibody is a humanized antibody or an antibody. A method for determining the presence or amount of a polypeptide according to claim 1 in a sample, the method comprising:
(A) providing the sample;
(B) contacting the sample with an antibody that immunospecifically binds to the polypeptide; and (c) determining the presence or amount of the antibody bound to the polypeptide, thereby determining the amount of poly in the sample. Determining the presence or amount of the peptide,
A method comprising: 7. A method for determining the presence or amount of a nucleic acid molecule according to claim 6 in a sample, said method comprising:
(A) providing the sample;
(B) contacting the sample with a probe that binds to the nucleic acid molecule; and (c) determining the presence or amount of the probe bound to the nucleic acid molecule, thereby determining the presence of the nucleic acid molecule in the sample. Determining the presence or amount, thereby determining the presence or amount of the polypeptide in the sample;
A method comprising: 2. A method for identifying an agent that binds to a polypeptide according to claim 1, comprising:
(A) contacting the polypeptide with a candidate substance; and (b) determining whether the candidate substance binds to the polypeptide, wherein the binding candidate substance is the factor;
A method comprising: 24. The method of claim 23, wherein the candidate substance has a molecular weight of about 1500 Da or less. 2. A method for modulating the activity of a polypeptide according to claim 1, wherein the method binds the polypeptide and the polypeptide in an amount sufficient to modulate the activity of the polypeptide. A method comprising the step of contacting with a compound. A method for identifying a potential therapeutic agent for use in treating a pathology, wherein said pathology comprises aberrant expression, aberrant processing or aberrant expression of a polypeptide according to claim 1. Associated with various physiological interactions, the method comprising:
(A) providing a cell that expresses the polypeptide and has a property or function attributed to the polypeptide;
(B) contacting the cells provided in step (a) with a test agent; and (c) determining whether the test agent alters a property or function attributable to the polypeptide, Altering the property or function of the polypeptide as observed in the presence of the test agent, thereby indicating that the test agent is a potential therapeutic agent. 27. The method of claim 26, further comprising providing the potential therapeutic agent to a further test to identify the therapeutic agent. 27. The method of claim 26, wherein the candidate substance is an antibody or has a molecular weight of about 1500 Da or less. 27. The method of claim 26, wherein said property or function comprises cell growth or proliferation. 30. The method of claim 29, wherein said test agent binds to said polypeptide. A therapeutic agent identified according to the method of claim 26. A therapeutic agent identified using the method of claim 27. 32. The therapeutic agent of claim 31, wherein said agent is an antibody or has a molecular weight of about 1500 Da or less. 2. A method for treating or preventing a polypeptide-related disorder according to claim 1, wherein the disorder is characterized by insufficient or inefficient proliferation of cells or tissues, the method comprising: Administering the polypeptide of claim 1 to the subject in an amount and for a time sufficient to treat or prevent the polypeptide-related disorder in the subject, wherein the subject is treated with the disorder. The method is considered to be affected or prone to the disorder. 35. The method of claim 34, wherein said subject is a human. A method of treating or preventing a disorder associated with abnormal expression, abnormal processing or abnormal physiological interaction of a protein according to claim 1, wherein said disorder comprises insufficient cells or tissues. Alternatively, the method comprises administering to the subject the nucleic acid of claim 6 in an amount and for a period of time sufficient to treat or prevent the disorder in the subject. A method wherein the subject is considered to be suffering from or susceptible to the disorder. 37. The method of claim 36, wherein said subject is a human. A method of treating or preventing a disorder associated with abnormal expression, abnormal processing or abnormal physiological interaction of a protein according to claim 1, wherein the disorder comprises hyperplasia of a cell or tissue. Alternatively, the method comprises administering to the subject a therapeutic agent in an amount sufficient to treat or prevent the disorder in the subject, wherein the subject comprises: A method wherein the subject is susceptible to or is susceptible to the disorder. 39. The method of claim 38, wherein said therapeutic agent is the antibody of claim 18. 39. The method of claim 38, wherein said subject is a human. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the nucleic acid molecule of claim 6 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the antibody of claim 18 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the therapeutic agent of claim 31 and a pharmaceutically acceptable carrier. 45. The pharmaceutical composition of claim 44, wherein said therapeutic agent has a molecular weight of about 1500 Da or less. 42. A kit comprising the pharmaceutical composition of claim 41 in one or more containers. 43. A kit comprising the pharmaceutical composition of claim 42 in one or more containers. A kit comprising the pharmaceutical composition of claim 43 in one or more containers. A method for screening a modulator or potential predisposition to a disorder associated with aberrant expression, aberrant processing or aberrant physiological interactions of a polypeptide according to claim 1, wherein the method comprises: , The following steps:
a) providing a test animal having an increased risk for said disorder, wherein said test animal recombinantly expresses the polypeptide of claim 1;
b) administering a test compound to the test animal;
c) measuring the activity of the polypeptide in the test animal after administering the compound of step (a); and d) measuring the activity of the protein in the test animal in a control animal not receiving the compound. A change in the activity of the polypeptide in the test animal, relative to the control animal, as compared to the activity of the polypeptide, wherein the change in the test compound is a modulator of potential or predisposition to the disorder. Showing process,
A method comprising: 50. The method of claim 49, wherein the test animal expresses a test protein transgene or expresses the transgene under the control of a promoter at increased levels relative to a wild-type test animal. A recombinant test animal, wherein the promoter is not the native gene promoter of the transgene. A method for determining the presence or predisposition of a disease associated with an altered level of a polypeptide of claim 1 in a first mammalian subject, comprising:
a) measuring the expression level of the polypeptide in a sample from the first mammalian subject; and b) determining the amount of the polypeptide in the sample of step (a), Comparing the amount of the polypeptide present in a control sample from a second mammalian subject, which is known not to be present or known to be less susceptible to the disease,
Wherein a change in expression level of said polypeptide in said first subject as compared to said control sample is indicative of the presence or predisposition of said disease. 7. A method for determining the presence or predisposition of a disease associated with an altered level of a nucleic acid molecule of claim 6 in a first mammalian subject, comprising:
a) measuring the amount of the nucleic acid in a sample from the first mammalian subject; and b) determining the amount of the nucleic acid in the sample of step a) without the disease. Or comparing to the amount of said nucleic acid present in a control sample from a second mammalian subject, which is known to be less susceptible to said disease,
Wherein the change in the level of the nucleic acid in the first subject as compared to the control sample is indicative of the presence or predisposition of the disease. A method of treating a pathological condition in a mammal, wherein said pathology is associated with aberrant expression, aberrant processing or aberrant physiological interaction of the polypeptide of claim 1, The method comprises administering a polypeptide to the mammal in an amount sufficient to alleviate the pathological condition, wherein the polypeptide comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 27, or A method which is a polypeptide having an amino acid sequence at least 95% identical to a biologically active fragment thereof. A method of treating a pathological condition in a mammal, wherein said pathology is associated with aberrant expression, aberrant processing, or aberrant physiological interaction of an FGF-CX polypeptide, wherein said method comprises: 20. A method comprising administering the antibody of claim 18 to the mammal in an amount and for a time sufficient to alleviate the pathological condition. A method of promoting cell proliferation in a subject, comprising administering to a subject in need of administration of the polypeptide of claim 1 an amount of the polypeptide effective to promote cell proliferation. Administering for a period of time. 56. The method of claim 55, wherein said subject is a human. 56. The method of claim 55, wherein the cells whose growth is promoted include: cells in the vicinity of a wound, cells of the vasculature, cells of hematopoiesis, cells of erythropoiesis, the inside of the gastrointestinal tract. A method selected from the group consisting of a cell and a cell in a hair follicle. A method of inhibiting cell growth in a subject, wherein said growth is associated with expression of a polypeptide according to claim 1, wherein said composition is administered to said subject, and cell growth in said subject is reduced. Administering a sufficient amount to inhibit. 59. The method of claim 58, wherein said composition inhibits cleavage of FGF-CX polypeptide. 59. The method of claim 58, wherein said composition comprises an antFGF-CX antibody or an FGF-CX therapeutic agent. 59. The method of claim 58, wherein said subject is a human. 59. The method of claim 58, wherein the cells whose growth is inhibited are selected from the group consisting of: transformed cells, hyperplastic cells, tumor cells, and neoplastic cells.

Images