JP3621883B2 - Semaphorin protein receptor DNA and polypeptide encoded by virus - Google Patents

Description

下流Ｎｏｔ１プライマー：
【００８９】
【化２】

Ｂａｕｍら， Ｃｉｒ． Ｓｈ． ４４：３０（１９９４）に記載されるような、免疫グロブリンの突然変異タンパク質（ｍｕｔｅｉｎ）ヒトＦｃ領域を含むＢｇｌ ＩＩからＮｓｌ Ｉの制限断片を、ネズミＩＬ−７シグナルペプチドおよび米国特許第５，０１１，９１２号に記載されるようなＦＬＡＧ^ＴＭオクタペプチドを含む発現ベクター（ｐＤＣ３０４）内に連結した。その後、エクトロメリアＡ３９Ｒのアミノ酸１５−３９９をコードするＰＣＲ増幅ＤＮＡを、突然変異タンパク質ヒトＦｃ領域、ネズミＩＬ−７シグナルペプチドおよびＦＬＡＧ^ＴＭペプチドを含む発現ベクター内に二方向連結で連結した。生じたＤＮＡ構築物をサル腎臓細胞株ＣＶ−１／ＥＢＮＡに（ｐｓｖ３ｎｅｏの共トランスフェクションと共に）トランスフェクションした。０．５％低免疫グロブリンウシ血清を含む培地で７日間培養した後、０．２％アジ化物溶液を上清に添加し、そして０．２２μｍフィルターを通し上清を濾過した。その後、およそ１ｌの培養上清を、１０ ｍｌ／分で、４．６ ｘ １００ ｍｍプロテインＡカラム（ＰｅｒＳｅｐｔｉｖｅ ＢｉｏｓｙｓｔｅｍｓのＰＯＲＯＳ ２０Ａ）を用い、ＢｉｏＣａｄ プロテインＡ ＨＰＬＣタンパク質精製系を通過させた。プロテインＡカラムは、上清中の融合タンパク質のＦｃ部分に結合し、融合タンパク質を固定し、そして上清中の他の構成要素がカラムを通過するのを可能にする。カラムを３０ ｍｌのＰＢＳ溶液で洗浄し、そして結合している融合タンパク質をｐＨ ３．０に調整したクエン酸でＨＰＬＣカラムから溶出させた。溶出した精製融合タンパク質は、溶出の際、ｐＨ ７．４の１Ｍ ＨＥＰＥＳ溶液を用い、中和した。
実施例２
エクトロメリアセマフォリン／ポリＨｉｓ融合タンパク質の調製
以下は、エクトロメリアＡ３９Ｒ／ポリＨｉｓ融合タンパク質（Ａ３９Ｒ／ポリＨｉｓ）の調製を記載する。該方法は、融合タンパク質をコードするＤＮＡ構築物を調製し、該ＤＮＡ構築物で細胞株をトランスフェクションし、そしてトランスフェクションされた細胞から上清を採取することを含んだ。
【００９０】
エクトロメリアＡ３９Ｒ（Ａ３９Ｒ ＯＲＦ、配列番号８のアミノ酸１−３９９）をコードするＤＮＡを、ＰＣＲ技術および合成オリゴヌクレオチドプライマーを用い、エクトロメリアウイルスゲノムＤＮＡから単離し、そして増幅した。該プライマーは、５’端にＮｏｔ １部位および３’端に精製過程に用いるモチーフＧｌｙ−Ｓｅｒ−６ｘＨｉｓを添加した。該プライマーは、Ｇｌｙ−Ｓｅｒ−６ｘＨｉｓモチーフの後に、インフレーム終止コドンおよびＢｇｌ ２部位を添加した。ＰＣＲ産物を切断し、ｐＤＣ４０９発現ベクター（ＭｃＭａｈｏｎら， ＥＭＢＯ Ｊ． １０：２８２１， １９９１）にクローンした。
【００９１】
生じたＤＮＡ構築物を、サル細胞株ＣＯＳ−１（ＡＴＣＣ ＣＲＬ−１６５０）に一過性にトランスフェクションした。０．５％低免疫グロブリンウシ血清を含む培地で７日間培養した後、細胞上清を採取し、そして０．２％アジ化ナトリウム溶液を上清に添加した。０．２２μｍフィルターを通し上清を濾過し、調製スケール濃縮装置（Ｍｉｌｌｉｐｏｒｅ；マサチューセッツ州ベッドフォード）で１０倍に濃縮し、そして、ニッケルＮＴＡスーパーフローセルフパック樹脂カラム（Ｑｉａｇｅｎ、カリフォルニア州サンタクラリタ）を備えたＢｉｏＣａｄ ＨＰＬＣタンパク質精製上で精製した。上清がカラムを通過した後、カラムを緩衝液Ａ（２０ ｍＭ ＮａＰＯ_４、ｐＨ ７．４；３００ ｍＭ ＮａＣｌ；５０ ｍＭ イミダゾール）で洗浄した。その後、勾配溶出技術を用い、結合しているタンパク質をカラムから溶出させた。タンパク質を含む分画を集め、４−２０％ ＳＤＳ−ＰＡＧＥ還元ゲル上で解析した。Ａ３９Ｒ／ポリＨｉｓ融合タンパク質を含むピークをプールし、２倍に濃縮し、そしてその後ＰＢＳ中で透析した。生じたＡ３９Ｒ／ポリＨｉｓ融合タンパク質をその後、０．２２μｍ無菌フィルターを通し濾過した。
実施例３
Ａ３９Ｒに対する結合に関する細胞株のスクリーニング
実施例１に記載されるように調製されたＡ３９Ｒ／Ｆｃ融合タンパク質を用い、標準的フローサイトメトリー方法論にしたがい、定量的結合研究を用い、結合に関し細胞株をスクリーニングした。スクリーニングされる各細胞株に関し、該方法は、およそ１００，０００の細胞をインキュベーションし、ＰＢＳ中の２％ ＦＣＳ（ウシ胎児血清）、５％正常ヤギ血清および５％ウサギ血清で１時間ブロッキングすることを含んだ。その後、ブロッキングされた細胞をＡ３９Ｒ／Ｆｃ融合タンパク質５μｇ／ｍｌと、ＰＢＳ中の２％ ＦＣＳ、５％ヤギ血清および５％ウサギ血清中でインキュベーションした。インキュベーション後、試料をＦＡＣＳ緩衝液（ＰＢＳ中の２％ ＦＣＳ）で２回洗浄し、そしてその後、マウス抗ヒトＦｃ／ビオチン（Ｊａｃｋｓｏｎ Ｒｅｓｅａｒｃｈより購入）およびＳＡＰＥ（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓから購入したストレプトアビジン−フィコエリトリン）で処理した。この処理は、抗ヒトＦｃ／ビオチンをすべての結合しているＡ３９Ｒ／Ｆｃに結合させ、そしてＳＡＰＥを抗ヒトＦｃ／ビオチンに結合させ、細胞に結合しているＡ３９Ｒ／Ｆｃ上に蛍光同定標識を生じさせる。蛍光検出フローサイトメトリーを用い、すべての結合しているタンパク質に関し細胞を解析した。結果により、Ａ３９Ｒセマフォリンは、ヒトＮＫ細胞、ネズミ脾臓Ｂ細胞、ヒトＰＢ Ｔ細胞、ヒトＴ、Ｂ、赤芽球、リンパ芽球および骨髄前駆細胞、繊維芽細胞および上皮細胞系譜によく結合することが示された。表１は、フローサイトメトリー研究の結果を列挙する。「＋」は細胞表面およびＡ３９Ｒの間に結合が検出されたことを示す。「−」は細胞表面およびＡ３９Ｒの間に結合が検出されなかったことを示す。
【００９２】
【表１】

【００９３】
実施例４
推定上のセマフォリン受容体の同定
Ａ３９Ｒに対する結合に関し陽性に試験されたＣＢ２３細胞（ヒト臍帯血Ｂ細胞株）およびヒトＰＢ Ｔ細胞を、推定上の受容体の発現に関し、そしていくらかの受容体が膜結合分子、可溶性分子または両方として発現しているか決定するため、試験した。概して、該解析は、ＣＢ２３およびヒトＰＢ Ｔ細胞表面を放射標識し、採取し、そして細胞上清および溶解物をＡ３９Ｒ／Ｆｃ融合タンパク質で処理し、すべての推定上の受容体を沈澱させ、そしてその後、電気泳動ゲル上で免疫沈降物を視覚化することを含んだ。
【００９４】
特に、該方法は、Ｂｅｎｊａｍｉｎら， Ｂｌｏｏｄ ７５：２０１７−２０２３（１９９０）に記載されるように、まず、［^１２５Ｉ］で、およそ１ ｘ １０^７のＣＢ２３またはＰＢ Ｔ細胞を放射標識することを含んだ。培養細胞上清を採取し、そして１４，０００ ｒｐｍ、３０分間の遠心分離により、清澄にした。細胞溶解物は、プロテアーゼ阻害剤、フェニルメチルスルホニルフロリド、ペプスタチン−Ａ、およびロイペプチンを含む１％ Ｔｒｉｔｏｎ−Ｘ １００を含む１ ｍｌのリン酸緩衝生理食塩水中で、細胞を氷上で３０分間インキュベーションすることにより生成した。溶解物を１４，０００ ｒｐｍ、３０分間の遠心分離により清澄にした。溶解物および／または上清に存在するすべての受容体を沈澱させるため，細胞上清または溶解物２００μｌを実施例１に記載されるように調製したＡ３９Ｒ／Ｆｃ融合タンパク質２μｇとインキュベーションした。インキュベーションは４℃で穏やかに揺らしながら１時間行った。同様にＦｃタンパク質コントロール試料を調製し、そしてインキュベーションした。インキュベーション後、プロテインＡセファロースビーズ（＃１７−０７８０−０１、Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ Ｉｎｃ．、ニュージャージー州ピスカタウェイ）を溶解物および上清に添加し、そして混合物を４℃で穏やかに揺らしながら１時間インキュベーションした。ＰＢＳ １％ Ｔｒｉｔｏｎ−Ｘ １００溶液で、ビーズをよく洗浄した。結合しているタンパク質を溶出させ、そしてＳＤＳ ＰＡＧＥにより解析した。タンパク質バンドをオートラジオグラフィーで視覚化し、そして単一のおよそ２００ ｋＤａのバンドが、Ａ３９Ｒ／Ｆｃに結合するが、コントロールＦｃタンパク質には結合しないことを見出した。セマフォリン受容体は細胞溶解物および細胞上清に存在し、膜結合タンパク質としての、そして分泌される可溶性タンパク質としてのその発現が確認された。
実施例５
セマフォリン受容体の単離および配列決定
実施例１に記載されるように調製されたＡ３９Ｒ／Ｆｃ融合タンパク質を用い、ヒトセマフォリン受容体ポリペプチドを単離し、そして単離ポリペプチド精製のための方法を確認した。ＣＢ２３細胞ペレットを、ホモジェナイズ緩衝液（１０ ｍＭリン酸、３０ ｍＭ ＮａＣｌ、ｐＨ ７．４）中にＰＭＳＦ、ロイペプチン、アプロチニン、ペプスタチンＡを各々１ ｍＭ、１０μｇ／ｍｌ ＡＰＭＳＦ、および１ ｍＭ ＥＤＴＡを含むプロテアーゼ阻害剤溶液に懸濁することにより、セマフォリン受容体を単離した。細胞をダウンス（ｄｏｕｎｃｅ）ホモジェナイズし、そしてホモジェナイズ緩衝液中の４１％ショ糖溶液に上層し、そして２５，０００ ｒｐｍ、４℃で４５分間、Ｂｅｃｋｍａｎ ＳＷ−２８ローターで回転させ落とした。中間相を集め、そして冷たいホモジェナイズ緩衝液で希釈し、ダウンスし、そして回転させた。生じた透明な膜ペレットを−８０℃で貯蔵した。
【００９５】
充填細胞２４０ ｍｌから調製した膜ペレットを、２０ ｍＭ Ｔｒｉｓ、１５０ ｍＭ ＮａＣｌ、上記に同定されるプロテアーゼ阻害剤、１％ Ｔｒｉｔｏｎ Ｘ−１００およびＣａＣｌ_２、ＭｇＣｌ_２、およびＭｃＣｌ_２塩０．１ ｍＭの水性溶液（緩衝液Ａ）１００ ｍｌと合わせた。懸濁ペレットをダウンスし、そしてＳＷ−２８ローターで、２５，０００ ｒｐｍ、４℃で３０分間回転させた。上清を１００ ｍｌコムギ胚（ｗｈｅａｔ ｇｅｒｍ）凝集素カラムに入れ、そして１０カラム体積の緩衝液Ａを用い１ ｍｌ／分の速度で溶出させた。カラムに特異的に結合しているタンパク質をその後、０．２Ｍ Ｎ−アセチルグルコサミンを含む緩衝液Ａで溶出させた。
【００９６】
タンパク質に関し陽性に試験された分画をプールし、そしてＡ３９Ｒ／Ｆｃ融合タンパク質１００μｇと４℃で１時間インキュベーションした。インキュベーションした混合物をセファロースカラムに通し、特異的に結合しない成分を除去し、そしてその後、プロテインＡ／セファロース固体支持体の０．５ ｍｌカラムに通過させた。プロテインＡ／セファロース固体支持体を、１％ Ｔｒｉｔｏｎ Ｘ−１００を含むＰＢＳ ２０カラム体積で洗浄し、その後、ＰＢＳで洗浄し、いかなる非結合成分も洗い落とした。その後、ｐＨ ３．０の５０ ｍＭクエン酸の０．３５ ｍｌ分画で、段階的方式で、プロテインＡ／セファロースカラム上に保持されているタンパク質を溶出した。タンパク質に関し陽性に試験された分画を合わせ、そして１０ ｋＤ ＭＷＣＯ Ｃｅｎｔｒｉｃｏｎ濃縮装置を用い、５０μｌに濃縮した。生じた濃縮試料中のタンパク質を還元し、そしてその後、標準的ＤＴＴおよびヨード酢酸法を用い、アルキル化した。アルキル化タンパク質をその後、８％ゲル上で電気泳動した。ゲル上のタンパク質を、５％酢酸を含む５０％ ＭｅＯＨ中のクーマシーＧで視覚化し、そしてその後、５０％ ＭｅＯＨ中で脱染した。
【００９７】
タンパク質標準に比較することにより位置決定された、およそ２００ ｋＤバンドを、かみそりで切り出し、そして１００ ｍＭ炭酸アンモニウム中で一晩洗浄した。ゲル切片を乾燥するまで高速蒸発させ、そして１００ ｍＭ炭酸アンモニウム中のトリプシンの１：１０溶液を乾燥スライドに添加した。スライドを３７℃で１６時間インキュベーションし、そしてその後切片中のタンパク質を、各抽出と共に３０分間インキュベーションしながら、５％ギ酸を含む５０％アセトニトリルで３回抽出した。
【００９８】
トリプシン消化ペプチド断片を凍結乾燥し、０．１％トリフルオロ酢酸５０μｌに溶き、そしてＣ−１８逆相パッキングを充填した５００μ ｉｄ（内径） ｘ ２５ ｃｍキャピラリーカラム上でＲＰ−ＨＰＬＣにより分離した。ＨＰＬＣ液相は、５分後１０％、１０５分後８５％のアセトニトリル／水勾配であった。溶出タンパク質は２１５ ｎｍで検出した。各タンパク質が溶出すると、別個の分画に集め、そして分画中のペプチドのＮ末端配列解析を、製造者の指示にしたがい、４９４ Ｐｒｏｃｉｓｅ配列決定装置上で行った。
【００９９】
上述のように得られたＲＰ−ＨＰＬＣ分画を、真空遠心機で乾燥し、０．５％酢酸を含む５０％メタノール６μｌに分画中のペプチドを溶解した。各ペプチド溶液２μｌをナノスプレーチップ（Ｐｒｏｔｅｉｎ Ａｎａｌｙｓｉｓ Ｃｏｍｐａｎｙ、デンマーク、オーゼンセ）に装填した。データは、ナノスプレー供給源を備えたＦｉｎｎｉｇａｎ ＴＳＱ７００ 三重四重極質量分析計（カリフォルニア州サンホセ）で得た。質量スペクトルは、単位分解能で得た。直列質量分析には、第一の四重極を３−４Ｄａの幅を通過するのに十分な分解能で作動させ、そして第三の四重極を単位分解能（ｕｎｉｔ ｒｅｓｏｌｕｔｉｏｎ）で作動させた。衝突ガスは、４ ｍトールの圧で供給した。標準的エステル化法を用い、メチルエステル化を行った。
【０１００】
トリプシン生成ペプチドの直列質量分析解析により、精製タンパク質の単離部分に関するアミノ酸配列情報が提供された。直列質量スペクトルデータを、部分的ＳＥＱＵＥＳＴアルゴリズム検索ツール（Ｅｎｇ， Ｊ．Ｋ．ら， Ｊ Ａｍ Ｓｏｃ． Ｍａｓｓ １９９４）を用い、コンピューターが補助する非重複性タンパク質データベースおよびＥＳＴデータベースのスクリーニングで用いた。配列番号２のアミノ酸４２１−４２８に対応するペプチド照会（ｑｕｅｒｙ）配列ＧｌｕＧｌｕＴｈｒＰｒｏＶａｌＰｈｅＴｙｒＬｙｓ、およびアミノ酸４３６−４４５に対応するＡｓｎＩｌｅＴｙｒＩｌｅＴｙｒＬｅｕＴｈｒＡｌａＧｌｙＬｙｓは、ＥＳＴ第２４８５３４号（寄託番号Ｎ７８２２０号）が、照会ペプチド配列に対し１００％同一性を有する含有ペプチド配列を含むと同定した。アミノ酸３８８−４０１に対応するＴｈｒＶａｌＬｅｕＰｈｅＬｅｕＧｌｙＴｈｒＧｌｙＡｓｐＧｌｙＧｌｎＬｅｕＬｅｕＬｙｓは、ＥＳＴ第Ｒ０８９４６号が、照会に対し１００％同一性を含むと同定した。
【０１０１】
ＥＳＴ ２４８５３４の部分および精製タンパク質の３つのペプチド断片の間の１００％同一性は、ＥＳＴ ２４８５３４内に含まれるｃＤＮＡは、精製タンパク質のコード領域に対するヌクレオチド配列の部分を代表することを、強く示唆した。セマフォリン受容体ｃＤＮＡの供給源は、ファージライブラリースクリーニング法およびＥＳＴ ２４８５３４に基づくＰＣＲプライマーを用い、同定した。
【０１０２】
オリゴヌクレオチドプライマーは、以下のヌクレオチド配列を有した：

ヒト組織ｃＤＮＡファージライブラリーの一団をＰＣＲ反応のテンプレートとして用い、ＰＣＲ単離および増幅方法論を実行した。ＰＣＲ反応混合物は、最終反応体積３０μｌ中に、ファージライブラリーストック１μｌ、０．３μＭの最終濃度のＰＣＲオリゴヌクレオチドプライマー、１ ｘ ＰＣ２緩衝液（Ａｂ Ｐｅｐｔｉｄｅｓ， Ｉｎｃ．、ミズーリ州セントルイス）、ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）各０．２ ｍＭ、１６：１混合Ｋｌｅｎ−Ｔａｑ／Ｖｅｎｔポリメラーゼ（Ｋｌｅｎ−Ｔａｑポリメラーゼ、Ａｂ Ｐｅｐｔｉｄｅｓ， Ｉｎｃ．およびＶｅｎｔポリメラーゼ、Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ、マサチューセッツ州ビバリー）０．２μｌを含んだ。ＰＣＲ反応サイクルは、Ｓｔｒａｔａｇｅｎｅ、カリフォルニア州ラホヤのＲｏｂｏｃｙｃｌｅｒ ９６を用い、９８℃５分間での１サイクル；９８℃４５秒間での３０サイクル；６８℃４５秒間での３０サイクル；７２℃４５秒間での３０サイクル；および７２℃５分間での１サイクルを含んだ。いくつかのライブラリー中のｃＤＮＡが、電気泳動ＰＣＲ産物中の適切なサイズのＤＮＡバンドの出現に基づき、精製ＶＥＳＰＲタンパク質をコードするＤＮＡを含むとして、陽性に同定された。
【０１０３】
ファージライブラリーの２つ、ヒト包皮繊維芽細胞およびヒト真皮繊維芽細胞を、さらなる解析のため選択した。ライブラリーを確立された方法にしたがって蒔き、そしてＥＳＴ ２４８５３４をテンプレートとして用いたＰＣＲ増幅産物由来の放射標識ランダムプライマープローブで探査した（ｐｒｏｂｅｄ）。増幅産物を得るために用いたＰＣＲ条件は、上述の通りであり、そしてプローブはＳｔｒａｔａｇｅｎｅ、カリフォルニア州ラホヤのＰｒｉｍｅ−ＩＴ ＩＩランダムプライマー標識キットを用い、生成した。およそ１ ｘ １０^６ ｃｐｍ／ｍｌの精製プローブを用い、ナイロン膜フィルター上のヒト包皮ファージライブラリーを、１０ ｘ デンハルト（Ｄｅｎｈａｒｄｔｓ）溶液、ｐＨ ７．５の５０ ｍＭ Ｔｒｉｓ、０．９Ｍ ＮａＣｌ、０．０１％ピロリン酸ナトリウム、１％ドデシル硫酸ナトリウム、および２００μｇ／ｍｌの変性断片化サケ精子ＤＮＡのハイブリダイゼーション緩衝液中で、６３℃で一晩探査した。探査後、探査した膜を、６３℃で、一度、６ ｘ ＳＳＣ、０．１％ ＳＤＳで２０分間、一度、２ ｘ ＳＳＣ、０．１％ ＳＤＳで２０分間、一度、１ ｘ ＳＳＣ、０．１％ ＳＤＳで２０分間、そして一度、０．１ ｘ ＳＳＣ、０．１％ ＳＤＳで２０分間、洗浄した。探査しそして洗浄したフィルターをＸ−ｏｍａｔ ＡＲ Ｘ線フィルム（Ｅａｓｔｍａｎ−Ｋｏｄａｋ Ｃｏｒｐ．）に一晩曝露した。４つのオーバーラップしているｃＤＮＡを同定した。オーバーラップしているＤＮＡを、配列決定されたトリプシン消化生成タンパク質断片と共に用い、配列番号１に示されるようなＶＥＳＰＲのコード配列および配列番号２に提示されるアミノ酸配列を完了し、そして確認した。
実施例６
Ａ３９Ｒセマフォリンに対するモノクローナル抗体
本実施例は、Ａ３９Ｒセマフォリンに対する抗体を調製するための方法を例示する。精製Ａ３９Ｒ／Ｆｃを上の実施例１に記載されるように調製した。精製タンパク質を用い、米国特許第４，４１１，９９３号に記載されるようにＡ３９Ｒセマフォリンに対する抗体を生成した。簡潔には、マウスをＡ３９Ｒ／Ｆｃで１０μｇで、０、２および６週に免疫した。初回免疫は、Ｖａｘｃｅｌｌ， Ｉｎｃ．のＴＩＴＥＲＭＡＸアジュバントと共に調製し、そして続く免疫は、不完全フロイントアジュバント（ＩＦＡ）と共に調製した。１１週目に、ＰＢＳ中のＡ３９Ｒ／Ｆｃ ３−４μｇでマウスをＩＶ追加免疫した。ＩＶ追加免疫の３日後、脾臓細胞を採取し、そして５０％水性ＰＥＧ １５００溶液を用い、Ａｇ８．６５３骨髄腫融合パートナーと融合させた。ハイブリドーマ上清を、Ａ３９Ｒ／Ｆｃおよび不適切なＦｃタンパク質に対するドットブロットアッセイにより、Ａ３９Ｒ抗体に関しスクリーニングした。
実施例７
セマフォリン受容体を発現している組織に関するノーザンブロット解析
以下は、本発明のＶＥＳＰＲポリペプチドを発現する組織および細胞種を同定するため行われたノーザンブロット実験を記載する。該結果は、フローサイトメトリー解析およびＡ３９Ｒ／Ｆｃ融合タンパク質を用いて得られた細胞結合結果を確認する。
【０１０４】
実施例５に記載されるように、ＥＳＴデータベース検索の結果、配列番号２のＶＥＳＰＲの部分的クローンと考えられるＥＳＴ（ＥＳＴ ２４８５３４）が発見された。ＰＣＲ技術およびＥＳＴ ２４８５３４のヌクレオチド１−３７２に基づくオリゴヌクレオチドプライマーを用い、リボプローブテンプレートを生成した。ＥＳＴ ２４８５３４のヌクレオチド１−３７２を含む上流および下流プライマーは以下の配列を有した：

下線部分は、Ｔ７部位である。
【０１０５】
２つのプライマーを用い、リボプローブの生成に用いるため、ＥＳＴ２４８５３４からＰＣＲ産物を単離し、そして増幅した。ＡｍｂｉｏｎのＭＡＸＩｓｃｒｉｐｔ ＳＰ６／Ｔ７キットを用い、３μｌのＲＮＡｓｅ不含水、２μｌの１０ ｘ転写緩衝液、１０ ｍＭ ｄＡＴＰ／ｄＣＴＰ／ｄＧＴＰ各１μｌ、５μｌの５’／３’ ＥＳＴ ２４８５３４ ＰＣＲ産物、５μｌのＡｍｅｒｓｈａｍ［α^３２Ｐ］ＵＴＰ １０ ｍＣｉ／ｍｌ、２μｌのＴ７ ＲＮＡポリメラーゼを室温で合わせることにより、リボプローブを生成した。組み合わせたものを微量遠心分離し簡潔に回転させ、そして３７℃で３０分間インキュベーションした。その後、１μｌのＤＮＡｓｅを混合物に添加し、そして３７℃で１５分間反応させた。反応産物をＧ−２５パッキング（Ｂｏｅｈｒｉｎｇｅｒ）の２カラム体積に通過させた。リボプローブ１μｌをシンチレーションカウンターで１分間カウントし、ｃｐｍ／ｍｌを測定した。
【０１０６】
多様な細胞株由来のポリアデニル化ＲＮＡを、１．２％アガロースホルムアルデヒドゲル上に分画し、そして該ＲＮＡをＨｙｂｏｎｄナイロン膜（Ａｍｅｒｓｈａｍ、イリノイ州アーリントンハイツ）上にブロットすることにより、ノーザンブロットを生成した。Ｍａｎｉａｔｉｓ（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂ． Ｐｒｅｓｓ， １９８９）に記載されるような標準的ノーザンブロット生成法を用いた。総ＲＮＡ多数組織ノーザンブロットは、商業的に購入した（ＢｉｏＣｈａｉｎ Ｉｎｓｔｉｔｕｔｅ， Ｉｎｃ．、カリフォルニア州サンレアンドロ、カタログ番号０２１００１、０２１００２、０２１００３）。
【０１０７】
ノーザンブロットを、５０％ホルムアミドハイブリダイゼーション溶液（３０ ｍｌの２０ ｘ ＳＳＣ、２ ｍｌの１００ ｘ デンハルト試薬、１ ｍｌの１０ ｍｇ／ｍｌ変性断片化サケ精子ＤＮＡ、５０ ｍｌの１００％ホルムアミドおよび２０ ｍｌの１０％ ＳＤＳ）中でプレハイブリダイゼーションした。総ＲＮＡブロットは４２℃で４時間プレハイブリダイゼーションし、そしてポリＡ＋ ＲＮＡブロットは６３℃で４時間プレハイブリダイゼーションした。リボプローブを、１０^６ ｃｐｍ／ｍｌのカウントで、清浄なハイブリダイゼーション溶液（プレハイブリダイゼーション溶液と同じ）に添加した。プレハイブリダイゼーション溶液をブロットから除去し、そしてハイブリダイゼーション溶液およびリボプローブをブロットに添加した。ハイブリダイゼーションを一晩、穏やかに震蕩しながら進めた。総ＲＮＡブロットは６３℃でハイブリダイズさせ、そしてポリＡ＋ ＲＮＡブロットは６３℃でハイブリダイズさせた。
【０１０８】
探査した総ＲＮＡブロットを、４２℃で、一度、０．０５％ ＳＤＳを含む２ ｘ ＳＳＣで３０分間、そして５５℃で、一度、０．０５％ ＳＤＳを含む２ ｘ ＳＳＣで３０分間；６３℃で、二度、０．１％ ＳＤＳを含む０．１ ｘ ＳＳＣで３０分間；三度、０．１％ ＳＤＳを含む０．１ ｘ ＳＳＣで３０分間、洗浄し、そしてその後Ｘ線フィルムに曝露した。ポリＡ＋ブロットを、６３℃で、一度、０．０５％ ＳＤＳを含む２ ｘ ＳＳＣ溶液で３０分間、そして一度、０．１％ ＳＤＳを含む１ ｘ ＳＳＣで３０分間、洗浄し、そしてその後Ｘ線フィルムに曝露した。
【０１０９】
ノーザンブロットを探査した結果および陽性結合プローブに関する生じたＸ線フィルムの視覚化により、ＶＥＳＰＲがフローサイトメトリー実験において陽性結合が示されたものと同一の細胞に発現されていることが確認される。ハイブリダイズしているＲＮＡはＭＰ−１、ＨＦＦおよびＣＢ２３細胞で検出された。陽性ＲＮＡを示した初代組織には、心臓、脳、肺、脾臓および胎盤が含まれた。ＲＡＪＩ細胞にはＲＮＡは検出されなかった。
実施例８
ＡＨＶセマフォリンＦｃ融合タンパク質の生成
以下は、ＡＨＶセマフォリン／免疫グロブリン融合タンパク質（ＡＨＶセマ／Ｆｃ）を調製することを記載する。該方法は、融合タンパク質をコードするＤＮＡ構築物を調製し、該ＤＮＡ構築物で細胞株をトランスフェクションし、そしてトランスフェクションされた細胞から上清を採取することを含んだ。
【０１１０】
ＡＨＶ−セマをコードするＤＮＡは、Ｅｎｓｓｅｒら， Ｊ． Ｇｅｎ． Ｖｉｒ． ７６：１０６３−１０６７， １９９５に記載される。アルセラフィン・ヘルペスウイルスＤＮＡ株ＷＣ１１（Ｐｌｏｗｒｉｇｈｔ， Ｗ．ら， Ｎａｔｕｒｅ １８８：１１６７−１１６９， １９６０）から、ＰＣＲ技術、および配列が公表されているＡＨＶ−セマ配列に基づく合成オリゴヌクレオチドプライマーを用い、ＡＨＶ−セマアミノ酸７０−６５３をコードするＤＮＡを単離し、そして増幅した。上流オリゴヌクレオチドプライマーにより、Ｓｐｅ １部位を導入した。下流オリゴヌクレオチドプライマーにより、終止コドンの下流にＮｏｔ １部位を導入した。可溶性ＡＨＶセマを単離するのに用いた一般的な方法は、Ｓｐｒｉｇｇｓら， Ｊ． Ｖｉｒｏｌｏｇｙ， ７０：５５５７（１９９６）に記載されている。
【０１１１】
Ｇｏｏｄｗｉｎら， Ｃｅｌｌ ７３：４４７−４５６， １９９３に記載されるような免疫グロブリンの突然変異タンパク質Ｆｃ領域を含む制限断片を、ネズミＩＬ−７シグナルペプチドおよび米国特許第５，０１１，９１２号に記載されるようなＦＬＡＧ^ＴＭオクタペプチドを含む発現ベクター（ｐＤＣ４０９）内に連結した。その後、コードする、ＰＣＲ増幅ＡＨＶセマＤＮＡを、突然変異タンパク質ヒトＦｃ領域、ネズミＩＬ−７シグナルペプチドおよびＦＬＡＧ^ＴＭペプチドを含む発現ベクター内に二方向連結で連結した。生じたＤＮＡ構築物をサル腎臓細胞株ＣＶ−１／ＥＢＮＡに（ｐＳＶ３ｎｅｏの共トランスフェクションと共に）トランスフェクションした。０．５％低免疫グロブリンウシ血清を含む培地で７日間培養した後、０．２％アジ化物溶液を上清に添加し、そして０．２２μｍフィルターを通し上清を濾過した。その後、およそ１ｌの培養上清を、１０ ｍｌ／分で、４．６ ｘ １００ ｍｍプロテインＡカラム（ＰｅｒＳｅｐｔｉｖｅ ＢｉｏｓｙｓｔｅｍｓのＰＯＲＯＳ ２０Ａ）を用い、ＢｉｏＣａｄ プロテインＡ ＨＰＬＣタンパク質精製系を通過させた。プロテインＡカラムは、上清中の融合タンパク質のＦｃ部分に結合し、融合タンパク質を固定し、そして上清中の他の構成要素がカラムを通過するのを可能にする。カラムを３０ ｍｌのＰＢＳ溶液で洗浄し、そして結合している融合タンパク質をｐＨ ３．０に調整したクエン酸でＨＰＬＣカラムから溶出させた。溶出した精製融合タンパク質は、溶出の際、ｐＨ ７．４の１Ｍ ＨＥＰＥＳ溶液を用い、中和した。
実施例９
組換えセマフォリン受容体の発現
実施例５に記載されるように精製されたタンパク質のセマフォリン受容体（ＶＥＳＰＲ）アミノ酸配列、並びに、やはり実施例５に記載されるような、ＥＳＴデータベース検索から得られた情報および放射標識プローブでのハイブリダイゼーション方法論を用いて得られたｃＤＮＡを用い、ｃＤＮＡを生成し、そして該ｃＤＮＡで細胞をトランスフェクションし、組換えＶＥＳＰＲポリペプチドの発現を可能にする。
【０１１２】
ｐＤＣ４０６由来のＤＣ４０９発現ベクター中のｃＤＮＡを、標準的技術（ＭｃＭａｈｏｎら， ＥＭＢＯ Ｊ． １０：２８２１， １９９１）を用いＣＶ１／ＥＢＮＡ細胞にトランスフェクションする。より詳細には、ＣＶ１ ＥＢＮＡ細胞を、１０％ウシ胎児血清を補ったダルベッコの最小必須培地（培地）１０ ｍｌ中に、１０ ｃｍプレート当たり２ ｘ １０^６細胞の密度で蒔く。細胞を３７℃で一晩付着させる。６６．７μＭクロロキンおよびＶＥＳＰＲをコードするｃＤＮＡ ５μｇを含むＤＮＡ混合物を含む培地１．５ ｍｌで、培地を置換する。１７５μｌおよび２５μｌのＤＥＡＥデキストランを含む培地を細胞に添加する。細胞およびｃＤＮＡを３７℃で５時間インキュベーションする。ｃＤＮＡ混合物を除去し、そして１０％ ＤＭＳＯを含む新鮮な培地１ ｍｌで、細胞に２．５分間ショックを与える。新鮮な培地で培地を置換し、そして細胞を少なくとも３日間増殖させる。
【０１１３】
ＶＥＳＰＲの可溶性型を回収するため、可溶性型を含む上清を集め、そしてＨＰＬＣ技術またはアフィニティークロマトグラフィー技術を用い、ＶＥＳＰＲタンパク質を回収する。ＶＥＳＰＲの膜結合している型を回収するため、トランスフェクション細胞を採取し、１％ パラホルムアルデヒドで固定し、洗浄し、そしてその損なわれていない型で用いる。
実施例１０
ＶＥＳＰＲ結合研究
本発明の受容体ポリペプチドの結合特性を調べるため、膜結合ＶＥＳＰＲ細胞外ドメインを発現している細胞を、Ｇｏｏｄｗｉｎら， Ｃｅｌｌ ７３：４４７−４５６（１９９３）およびＳｐｒｉｇｇｓら， Ｊ Ｖｉｒｏｌ ７０：５５５７（１９９６）に記載されるスライド結合アッセイに供することにより、結合研究を行った。
【０１１４】
ｐＤＣ４０６（ＭｃＭａｈｏｎら， ＥＭＢＯ Ｊ． １０：２８２１， １９９１）由来であるが、単一のＢｇｌ ２を有するｐＤＣ４０９発現ベクターを、クローニング法のため、選択した。アミノ酸１９−１１００をコードするＶＥＳＰＲ ｃＤＮＡを、Ｓａｌ １（５’）およびＮｏｔ １（３’）部位を通じ、ｐＤＣ４０９発現ベクターにサブクローンし、ＤＮＡ構築物を形成した。
【０１１５】
ｐＤＣ４０９中の２μｇのＶＥＳＰＲ ｃＤＮＡ（アミノ酸１９−１１００をコードする）と共にＤＥＡＥ／デキストランを介し、ＣＶ−１／ＥＢＮＡ細胞をトランスフェクションした（Ｇｉｒｉら， ＥＭＢＯ Ｊ． １３：２８２２， １９９４）。トランスフェクション細胞を３日間培養し、そしてＣＶ−１／ＥＢＮＡ細胞単層を、Ａ３９Ｒ／Ｆｃ、ＡＨＶセマ／Ｆｃ、またはコントロールＦｃタンパク質１μｇ／ｍｌとインキュベーションした。その後、インキュベーション細胞を洗浄し、そして^１２５Ｉ標識マウス抗ヒトＩｇＧ（Ｊａｃｋｓｏｎ Ｉｍｍｕｎｏｒｅｓｅａｒｃｈ、ペンシルバニア州ウェストグローブ）とインキュベーションした。よく洗浄した後、細胞を固定し、Ｇｅａｒｉｎｇら， ＥＭＢＯ Ｊ ８：３６６７−３６７６（１９８９）に記載されるように写真感光乳剤に浸し、そして現像した。陽性結合は、Ｆｃタンパク質に結合しているＶＥＳＰＲを発現している細胞を覆う、露出されたまたは黒ずんだ銀粒の存在により、決定した。
実施例１１
フローサイトメトリーおよび阻害結合研究
以下は、Ａ３９Ｒ／Ｆｃ融合タンパク質（実施例１）およびＡＨＶセマ／Ｆｃ融合タンパク質（実施例９）に対する結合に関するＣＢ２３細胞のフローサイトメトリー解析を記載する。やはり以下に記載されるのは、実施例２に記載されるように調製されたＡ３９Ｒ／ポリＨｉｓ融合タンパク質の過剰量でのＡＨＶセマおよびＡ３９Ｒ結合の阻害を測定することに向けられた研究である。
【０１１６】
フローサイトメトリー解析は、まず約１ ｘ １０^６のＣＢ２３細胞を、３％正常ヤギ血清および３％正常ウサギ血清を含むＦＡＣＳ緩衝液中で、氷上で３０分間インキュベーションし、非特異的結合をブロッキングすることにより、行った。Ａ３９Ｒ／Ｆｃ、ＡＨＶセマ／ＦｃおよびコントロールＦｃタンパク質の一部を多様な濃度で添加し、そしてインキュベーションを３０分間続けた。細胞を洗浄し、そしてその後、ＦＡＣＳ緩衝液中のフィコエリトリン結合Ｆｃ特異的抗ヒトＩｇＧとインキュベーションした。細胞を洗浄し、そしてＢｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ、マサチューセッツ州ベッドフォードのＦＡＣＳｃａｎ上で解析した。結果は、ＡＨＶセマフォリンおよびＡ３９Ｒセマフォリンの陽性結合を示した。
【０１１７】
結合阻害研究は、約１ ｘ １０^６のＣＢ２３細胞を、ＦＡＣＳ緩衝液中で、氷上で３０分間インキュベーションすることにより、行った。Ａ３９Ｒ／ポリＨｉｓおよびコントロールＨｉｓタンパク質を多様な濃度で異なる試料に添加し、そしてインキュベーションをもう３０分間続けた。その後、Ａ３９Ｒ／ＦｃまたはＡＨＶセマ／Ｆｃを多様な濃度でインキュベーション細胞に添加し、そしてインキュベーションをもう３０分間続けた。細胞を洗浄し、そしてその後、ＦＡＣＳ緩衝液中のフィコエリトリン結合Ｆｃ特異的抗ヒトＩｇＧとインキュベーションした。細胞を再び洗浄し、そしてＦＡＣＳｃａｎ上で解析した。結果は、Ａ３９Ｒ／ポリＨｉｓを用い、Ａ３９ＲおよびＡＨＶセマが完全に阻害されるが、異種性Ｈｉｓ含有タンパク質では阻害されないことを立証した。
実施例１２
Ａ３９ＲセマフォリンでのヒトＢ細胞凝集
Ａ３９Ｒセマフォリンに対するヒトＢ細胞反応を調べるため、Ｓｐｒｉｇｇｓら， Ｊ Ｅｘｐ Ｍｅｄ １７６：１５４３（１９９２）に記載されるように、ヒト扁桃腺Ｂ細胞を精製した。Ａ３９Ｒ／ポリＨｉｓ融合タンパク質を実施例２に記載されるように調製した。Ａ３９Ｒ／ポリＨｉｓ融合タンパク質の溶液は、最終Ａ３９Ｒ濃度が１μｇ／ｍｌになるよう調製し、そしてＡ３９Ｒ／ポリＨｉｓ融合タンパク質溶液を、約１０^５の精製Ｂ細胞のｉｎ ｖｉｔｒｏ培養中でインキュベーションした。インキュベーションを約２４時間続けると、細胞凝集が生じた。融合タンパク質を培養に添加する前に、実施例６に記載されるように調製された、Ａ３９Ｒに対するモノクローナル抗体の１０倍モル過剰量を融合タンパク質調製に添加すると、細胞凝集は遮断された。さらに、Ａ３９Ｒセマフォリンを、培養に添加する前に、熱不活化すると、凝集は遮断された。
【０１１８】
本研究は、ＶＥＳＰＲがＢ細胞上に発現され、そしてＡ３９ＲおよびＶＥＳＰＲの間の相互作用がＢ細胞凝集を生じることを確認する。Ｂ細胞凝集は、Ｂ細胞の活性化を示す。活性化Ｂ細胞は、サイトカインを分泌し、抗体を産生し、または抗原提示細胞になることが知られる。
実施例１３
Ａ３９Ｒセマフォリンでのマウス樹状細胞およびマクロファージ凝集
Ａ３９Ｒに対する樹状細胞およびマクロファージ反応を調べるため、マウス細胞培養をＡ３９Ｒセマフォリンと接触させ、そして該組み合わせの影響を観察した。マクロファージを含むマウス樹状細胞培養は、Ｍａｒａｓｋｏｖｓｋｙら， Ｊ Ｅｘｐ Ｍｅｄ １８４：１９５３（１９９６）に記載されるように、マウスをＦｌｔ−３で免疫し、そして細胞を単離しそして精製することにより、得た。
【０１１９】
簡潔には、メスＣ５７Ｂｌ／６マウスに、連続９−１０日間、ＰＢＳ １００μｌ中のＦｌｔ３Ｌ １０μｇおよびマウス血清アルブミン１μｇの溶液を、毎日一度注射した。免疫後、ＮＨ_２Ｃｌの存在下で、すりガラスのスライドの間で脾臓組織を破壊し、赤血球を枯渇させることにより、脾臓の単一細胞懸濁物を調製した。残存細胞を、Ｔｈｙ−１、Ｂ２２０、ＮＫ１．１、およびＴＥＲ１１９に対するｍＡｂとインキュベーションし、そしてその後、１０％ウサギ補体とインキュベーションした。その後、インキュベーション細胞を洗浄し、そして抗免疫グロブリン（Ｉｇ）被覆磁気ビーズを用い、残ったｍＡｂ被覆細胞を除去した。残存濃縮（ｅｎｒｉｃｈｅｄ）細胞を培養し、または多様な細胞集団に関し分類した。
【０１２０】
分類のため選択した細胞を、Ｍａｒａｓｋｏｖｓｋｙら， Ｊ Ｅｘｐ Ｍｅｄ １８４：１９５３−１９６２， １９９６に記載されるように、抗ＣＤ１１ｃおよび抗ＣＤ１１ｂで染色し、そしてＣおよびＤ／Ｅ集団に関し分類した。
【０１２１】
Ａ３９Ｒ／ポリＨｉｓ融合タンパク質を実施例２に記載されるように調製した。Ａ３９Ｒ／ポリＨｉｓ融合タンパク質溶液は、最終Ａ３９Ｒ濃度１μｇ／ｍｌで、約１０^５の分類または枯渇マウス細胞と、ｉｎ ｖｉｔｒｏ培養中でインキュベーションした。４−６時間以内に、細胞は凝集し始めた。融合タンパク質をマウス細胞培養に添加する前に、実施例６に記載されるように調製された、Ａ３９Ｒに対するモノクローナル抗体の１０倍モル過剰量をＡ３９Ｒ／ポリＨｉｓ融合タンパク質調製に添加すると、凝集は遮断された。
【０１２２】
本研究は、ＶＥＳＰＲが樹状細胞およびマクロファージ上に発現され、そしてＡ３９ＲおよびＶＥＳＰＲの間の相互作用が樹状細胞およびマクロファージ凝集を生じることを確認する。
実施例１４
Ａ３９ＲセマフォリンはＣＤ６９活性化抗原を上方制御する
培養樹状細胞に対するＡ３９Ｒセマフォリンの影響を調べるため、マウスに９日間、毎日Ｆｌｔ３−Ｌ調製を注射した。マウス樹状細胞を採取し、そしてその後、１０％ ＦＢＳおよび２０ ｎｇ／ｍｌのＧＭ−ＣＳＦを含む培地中で５日間培養した。
【０１２３】
５日目、Ａ３９Ｒ／ポリＨｉｓ融合タンパク質１μｇ／ｍｌを培養に添加した。６日目、細胞を診断抗体で染色した。診断抗体染色実験の結果により、ＣＤ１１ｃ^＋、ＣＤ１１ｂ^＋細胞（樹状細胞）が発現するＣＤ６９活性化抗原量が増加していることが示され、したがって、Ａ３９Ｒセマフォリンおよびその受容体の相互作用がＣＤ６９発現を上方制御することが立証された。
【０１２４】
融合タンパク質が熱で不活性化されると、融合タンパク質はＣＤ６９抗原に対し、影響を持たなかった。非染色および染色細胞の間の平均蛍光強度の典型的な変化は、およそ５００チャンネルないし２５００チャンネルの間だった。再び、これらの結果は、細胞活性化の一過性でそして初期の発現マーカーである、ＣＤ６９活性化抗原の制御に対する、Ａ３９Ｒセマフォリンおよびその膜結合受容体の間の相互作用の有意な影響を立証する。
実施例１５
ＩＬ−１２産生におけるＡ３９Ｒの影響の評価
マウス脾臓細胞からのＩＬ−１２の産生におけるＡ３９Ｒの役割を研究するため、実施例１３に記載されるように、マウスをｆｌｔ３−Ｌで免疫し、そして樹状細胞を生成し、採取しそして精製した。
【０１２５】
およそ５ ｘ １０^５細胞／０．５ ｍｌの精製された未分類樹状細胞を、以下のもう１つの存在下で修飾ＤＭＥＭ培地中でインキュベーションした（１ ｘ １０^６／ｍｌで５００μｌ）：２０ ｎｇ／ｍｌ ｍｕＧＭ−ＣＳＦ（Ｉｍｍｕｎｅｘ、ワシントン州シアトル）、２０ ｎｇ／ｍｌ γ−ＩＦＮ（Ｇｅｎｚｙｍｅ、マサチューセッツ州ボストン）、１０μｇ／ｍｌ ＳＡＣ（ＣａｌＢｉｏｃｈｅｍ、カリフォルニア州ラホヤ）。各細胞調製をさらに、１μｇ／ｍｌのＡ３９Ｒ／ポリＨｉｓ融合タンパク質単独で、あるいは１μｇ／ｍｌまたは０．１μｇ／ｍｌのｍｕＣＤ４０Ｌ三量体（Ｉｍｍｕｎｅｘ、ワシントン州シアトル）と組み合わせて処理した。培養は、加湿３７℃、空気中の１０％ ＣＯ_２中で、１６−１８時間インキュベーションした。インキュベーション後、培養細胞の各群の生存率を測定し、そして上清を集め、そしてＥＬＩＳＡアッセイキット（Ｇｅｎｚｙｍｅ、マサチューセッツ州ボストン）を用い、ｍｕＩＬ１２（Ｐ７０）に関しアッセイした。組換えサイトカインで構築した標準曲線を参考にし、ｍｕＩＬ１２レベルを計算した。
【０１２６】
ＥＬＩＳＡ試験により、特に、Ａ３９Ｒはその受容体と相互作用し、未分類マウス樹状細胞からのＩＬ−１２の産生においてインターフェロンおよびＳＡＣと相乗作用することが立証された。このｉｎ ｖｉｖｏ ＩＬ−１２誘導はナチュラルキラー細胞活性化およびガンマインターフェロン産生を促進し、そしてガンマインターフェロン感受性サイトカインを上方制御するのに寄与する。
実施例１６
単球上のＭＨＣクラスＩＩおよびＣＤ８６の制御に対するＡ３９Ｒの影響の試験
以下の実験は、Ａ３９Ｒとその膜結合受容体との相互作用による、ＭＨＣクラスＩＩおよびＣＤ８６の上方制御を記載する。健常ドナーからの末梢血を、低内毒素ＰＢＳで、ｐＨ ７．４および室温で１：１に希釈した。その後、希釈血３５ ｍｌをＩｓｏｌｙｍｐｈ（Ｇａｌｌａｒｄ ａｎｄ Ｓｃｈｌｅｓｉｎｇｅｒ Ｉｎｄｕｓｔｒｉｅｓ， Ｉｎｃ．、ニューヨーク州カールプレース）１５ ｍｌに上層し、そして室温で、２２００ ｒｐｍで２５分間遠心分離した。血漿層を保存した。ＰＢＭＣ層を採取し、そして３回洗浄し、Ｉｓｏｌｙｍｐｈを除去した。洗浄したＰＢＭＣをＸ−Ｖｉｖｏ １５血清不含培地（ＢｉｏＷｈｉｔｔａｋｅｒ、メリーランド州ウォーカーズビル）に再懸濁し、そしてＴ１７５フラスコに加えた。フラスコは、先に２％ゼラチン（Ｓｉｇｍａ、ミズーリ州セントルイス）で被覆し、そして保存した血漿層で３０分間前処理してあった。ＰＢＭＣを３７℃、５％ ＣＯ_２で９０分間付着させ、そしてその後、低内毒素ＰＢＳの１０ ｍｌ洗浄で、穏やかに３回リンスした。酵素不含解離緩衝液（Ｇｉｂｃｏ， ＢＲＬ）中で細胞をインキュベーションし、そして細胞を何度もＰＢＳ中で洗浄することにより、付着単球細胞を採取した。単球を２５００ ｒｐｍで５分間遠心分離し、計数し、そして１ ｍｌ中に５ ｘ １０^５細胞／ウェルで２４ウェルプレートに並べた。培養は９５％純粋だった。
【０１２７】
細胞をより樹状細胞様表現型に分化させるため，精製単球細胞を、２０ ｎｇ／ｍｌ ＧＭ−ＣＳＦおよび１００ ｎｇ／ｍｌ ＩＬ−４の存在下で、７−９日間培養した。７−９日目に、１μｇ／ｍｌのＡ３９Ｒ／ポリＨｉｓまたはコントロールポリＨｉｓ含有タンパク質で培養を処理し、そして翌日、解析のため、細胞および上清を採取した。
【０１２８】
単球由来樹状細胞表面マーカーを調べるためのフローサイトメトリー実験において、特定のタンパク質に対して向けられる結合体化ｍＡｂで細胞を染色した。染色により、試験した末梢血ドナーのほとんどで、Ａ３９Ｒ処理はこれらの細胞上のＣＤ８６およびＭＨＣクラスＩＩ発現を下方制御することが示された。ＣＤ８６およびＭＨＣクラスＩＩ分子は、樹状細胞による亢進した抗原提示のマーカーであるため、これらの下方制御により、Ａ３９Ｒとこの細胞集団上のその受容体との相互作用の免疫抑制性の影響が示唆される。
実施例１７
ＣＤ５４の上方制御
以下は、Ａ３９Ｒセマフォリンおよび精製単球上のその受容体の間の相互作用の影響、およびより詳細には、セマフォリンとのインキュベーション後の単球上のＣＤ５４発現の影響を記載する。Ａ３９Ｒ／ポリＨｉｓまたはコントロールタンパク質の存在下で、一晩培養に置いた以外は、実施例１６に記載されるように、末梢血ドナーから新たに単離された単球を精製した。
【０１２９】
一晩培養の後、培養細胞および単球特異的細胞表面マーカーに対して向けられるｍＡｂを用い、フローサイトメトリーを行った。試験されたドナーすべてで、ＣＤ５４表面発現のレベルは、Ａ３９Ｒの存在下で亢進されたが、熱不活化Ａ３９Ｒの存在化では亢進されなかった。同様に、コントロールタンパク質を含む培養では、ＣＤ５４表面発現は亢進されなかった。
【０１３０】
ＣＤ５４はＩＣＡＭ−１としても知られるが，付着分子であり、その増加した発現は、細胞活性化を示すとみなされている。これらのデータにより、Ａ３９Ｒのその受容体との相互作用を促進することにより、新たに単離されたヒト単球を活性化することが可能であることが示される。
実施例１８
新たに単離されたヒト単球からのサイトカイン誘導
実施例１６に記載されるように、新たに単離されたヒト単球を精製し、そして実施例１７に記載されるように培養した。一晩Ａ３９Ｒ／ポリＨｉｓとインキュベーションした後、炎症誘発性サイトカインの存在に関し、単球上清を調べた。試験したドナーすべてで、ＩＬ−６およびＩＬ−８がＡ３９Ｒタンパク質により誘導された。熱不活化Ａ３９Ｒおよびコントロールタンパク質は、ＩＬ−６またはＩＬ−８を誘導しなかった。さらに、サイトカイン産生は、Ａ３９Ｒに対して向けられるｍＡｂを含むことにより遮断された。
【０１３１】
本実験の結果は、Ａ３９Ｒ、または本タンパク質の相同体がその受容体との相互作用により、新たに単離された単球によるサイトカイン産生を誘導する可能性があることを立証する。都合のよいことに、ＶＥＳＰＲの可溶性型は、Ａ３９Ｒまたはその相同体に反応し、単球の炎症誘発性活性を阻害するのに用いることが可能である。
実施例１９
単球凝集研究
セマフォリンのその単球上の受容体に対する相互作用に対する、ヒト単球の反応を調べるため、実施例１７に記載されるように単球を精製し、そして実施例２に記載されるようにＡ３９Ｒ／ポリＨｉｓ融合タンパク質を調製した。該融合タンパク質および精製された培養単球をインキュベーションした。インキュベーションを２０時間続けると、単球凝集を生じた。実施例１７に立証される結果により、観察された単球凝集はＣＤ５４上方制御の結果として起こると示唆された。しかし、他の要因もまた、凝集に寄与している可能性がある。
【０１３２】
本研究は、本発明のセマフォリン受容体が単球上に発現し、そしてＡ３９ＲおよびＶＥＳＰＲの間の相互作用は単球凝集を生じることを確認する。Ｂ細胞同様、単球凝集は、単球の活性化を示す。
実施例２０
ＶＥＳＰＲに対するモノクローナル抗体
本実施例は、ＶＥＳＰＲポリペプチドに対する抗体を調製するための方法を例示する。精製ＶＥＳＰＲポリペプチドを実施例１０に記載されるように調製する。精製タンパク質を用い、米国特許第４，４１１，９９３号に記載されるように、ＶＥＳＰＲに対する抗体を生成する。簡潔には、マウスを０、２および６週間目にＶＥＳＰＲで１０μｇで免疫する。初回免疫は、Ｖａｘｃｅｌｌ， Ｉｎｃ．のＴＩＴＥＲＭＡＸアジュバントと共に調製し、そして続く免疫は、不完全フロイントアジュバント（ＩＦＡ）と共に調製する。１１週目に、ＰＢＳ中のＶＥＳＰＲ ３−４μｇでマウスをＩＶ追加免疫する。ＩＶ追加免疫の３日後、脾臓細胞を採取し、そして５０％水性ＰＥＧ １５００溶液を用い、Ａｇ８．６５３骨髄腫融合パートナーと融合させる。ハイブリドーマ上清を、ＶＥＳＰＲおよび不適切なＦｃタンパク質に対するドットブロットアッセイにより、ＶＥＳＰＲ抗体に関しスクリーニングする。
【配列表】

[0001]
Field of Invention
The present invention relates to semaphorin receptor polypeptides, nucleic acids encoding such semaphorin receptor polypeptides, methods for producing recombinant semaphorin receptor polypeptides, and pharmaceutical compositions comprising such polypeptides About.
[0002]
Background of the Invention
The semaphorin gene family includes a number of molecules that encode related transmembrane and secreted glycoproteins known to be neurological regulators. Semaphorins are generally well conserved in their extracellular domain, which is typically about 500 amino acids in length. Semaphorin family proteins have been observed in neuronal and non-neuronal tissues and have been extensively studied for their role in nerve growth cone guidance. For example, secreted semaphorins, known as collapsin-1 and Drosophila semaphorin II, are selectively involved in repulsive growth cone guidance during development. Flies with semaphorin II that are mutated to decrease function exhibit unusual behavioral characteristics.
[0003]
Alternative semaphorin genes have been identified in several strains of poxvirus. This semaphorin is found in vaccinia virus (Copenhagen strain) and is encoded in an open reading frame (ORF) known as A39R. The A39R-encoded protein has no transmembrane domain and potential membrane binding and is known as a secreted protein. The variola virus ORF also contains sequences that share homology with the vaccinia virus ORF A39R at the nucleotide and amino acid level. Another virus semaphorin, AHV-sema, has been found in Alceraphine Herpesvirus (AHV).
[0004]
Based on similarities to insect semaphorins, genes encoding mammalian (human, rat, and mouse) semaphorins have been identified. Functional studies of these semaphorins suggest that embryonic and adult neurons require semaphorins to establish operable connections. Importantly, the fast reaction time of growth cone cultures against the appropriate semaphorin suggests that semaphorin signaling involves a receptor-mediated signaling mechanism. To date, a single semaphorin receptor, called neuropilin, has been isolated using rat spinal cord-derived mRNA. Another receptor has been suggested, termed neuropilin-2 (Kolodkin et al., Cell 90: 753-762, 1997).
[0005]
Semaphorin ligands secreted into the extracellular environment transmit information through receptor-producing cells in a local and systemic manner. In order to further study the nature of cellular processes controlled by such local and systemic signaling, it would be beneficial to identify additional semaphorin receptors and ligands. In addition, semaphorins encoded by viruses are produced by infected cells and are present in viruses that are lytic (poxviruses) and viruses that are not known to be neurotropic (AHV), and thus are major It is unlikely that the primary function is to modify the neurological response. Virus-encoded semaphorins are more likely to function to modify the immunological response of the infected host, and mammalian homologues to the virus-encoded semaphorin have an immunological response. It is likely to function to be modified. Given the suggestion that viral semaphorins may function in the immune system as natural immune regulators, it is beneficial to identify semaphorin receptors as therapeutic agents to enhance or downregulate immune responses. I will.
[0006]
Summary of the Invention
The present invention relates to semaphorin receptors as isolated or homogeneous proteins. In particular, the present invention includes, but is not limited to, a semaphorin receptor termed VESPR (virus encoded semaphorin protein receptor) that binds to semaphorins including A39R vaccinia semaphorins and AHV semaphorins. A polypeptide is provided. Also within the scope of the present invention is an expression vector comprising DNA encoding a VESPR polypeptide and DNA encoding a VESPR polypeptide. The present invention also produces host cells that have been transfected or transformed with an expression vector comprising DNA encoding a VESPR polypeptide, and culturing such host cells under conditions that direct expression to produce a VESPR polypeptide. A method for doing so is also included. The invention further includes antibodies directed against the VESPR polypeptide.
[0007]
Further within the scope of the present invention are methods for purifying or isolating semaphorins or cells expressing semaphorins to which the VESPR polypeptides of the invention bind. In such methods, at least one VESPR polypeptide is bound to a solid phase matrix and a mixture comprising a semaphorin polypeptide to which the VESPR polypeptide binds, or a mixture of semaphorin-expressing cells, is bound to a VEPSR polypeptide. And then separating the contact surface and solution.
[0008]
Furthermore, the present invention provides methods for treating inflammation and inflammatory diseases. Such methods include administering a therapeutically effective amount of a soluble VESPR polypeptide to a human or other mammal suffering from a disease associated with the proinflammatory activity of a semaphorin ligand.
[0009]
Detailed Description of the Invention
The present invention relates to a novel semaphorin receptor polypeptide called semaphorin protein receptor (VESPR) encoded by a virus, a DNA encoding a VESPR polypeptide, and a recombinant expression vector comprising DNA encoding a VESPR polypeptide. provide. The invention further provides a method for isolating VESPR polypeptides, as well as culturing and expressing host cells transfected with a recombinant expression vector under conditions suitable for expressing a semaphorin receptor. A method is provided for producing a recombinant VESPR polypeptide by recovering the processed receptor polypeptide.
[0010]
In particular, the present invention provides VESPR polypeptides that bind to semaphorins, including but not limited to vaccinia virus A39R semaphorins and AHV semaphorins. The native VESPR polypeptide described herein uses an Ectromeleria virus A39R semaphorin / Fc fusion protein (A39R / Fc) to recover VESPR from the membrane of human cells that express the receptor, Isolated. As described in the following examples, the VESPR polypeptide of the present invention was found to be B cell line, monocytic cell line, T cell line, dendritic cell, NK cell, lung epithelial cell, stroma by flow cytometry experiment. Proven to be expressed in intestinal epithelial cells and lymphoma cells.
[0011]
Furthermore, as demonstrated in the Examples below, the VESPR polypeptides of the present invention bind to their ligands and are involved in the upregulation of CD69 activating antigen on dendritic cells. The properties of the semaphorin receptor also described herein are that it interacts with its ligand, synergizes with interferon and SAC, up-regulates IL-12 production on mouse dendritic cells, and the MHC class The ability to down regulate II and CD86 expression. The VESPR polypeptides of the invention are also involved in increased expression of CD54 on monocytes, suggesting cellular activation as a result of interactions between semaphorins and their receptors. Among the uses of VESPR polypeptides that arise from the aforementioned biological properties of receptor-ligand interactions may induce IL-12 production and subsequent natural killer cell activation. VESPR polypeptides find additional use to treat diseases and adverse conditions associated with inflammation. In particular, soluble VESPR polypeptides may be used to antagonize the proinflammatory activity associated with the interaction of semaphorin ligands and their receptors. Rheumatoid arthritis, a disease associated with chronic inflammation of the synovial tissue, has been linked to the upregulation of the human semaphorin E gene (Mangasser-Stephan et al., Biochem and Biophys Res Com, 234: 153-156, 1997). ). Thus, the soluble VESPR polypeptides of the present invention may be useful for downregulating semaphorin activity that mediates this inflammatory disease.
[0012]
VESPR, the natural semaphorin receptor of the present invention, was isolated using a viral semaphorin ligand known as ectromelia A39R. Example 1 below was used to isolate an A39R semaphorin ligand and to identify cell lines that bind to the ligand and to determine the impact of the interaction between A39R and its cell-bound receptor. The preparation of an A39R / Fc fusion protein is described.
[0013]
Examples 4 and 5 describe identifying a native VESPR polypeptide of the invention and isolating and purifying human VESPR polypeptide. The amino acid sequence of a human VESPR polypeptide isolated as described in Example 5 is disclosed in SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 is an isolated and purified receptor using tandem mass spectrometry of peptides produced by trypsin digestion in combination with adjacent EST sequences and identified cDNAs. Obtained by sequencing the body. The amino acid sequence shown in SEQ ID NO: 2 has a predicted extracellular domain of amino acids 1-944, including a signal peptide with a predicted cleavage site at amino acid 34. The predicted transmembrane domain of SEQ ID NO: 2 contains amino acids 945-965, and the cytoplasmic domain of SEQ ID NO: 2 spans amino acids 966-1568.
[0014]
The DNA encoding amino acids 19-1100 of human VESPR in E. coli DH10B was deposited at the American Type Culture Collection, Rockville, Maryland, USA, and deposited The number ____ was obtained. The deposit was made under the conditions of the Budapest Treaty. The DNA construct of the deposit differs from that of SEQ ID NO: 1 in that nucleotide 172 is C. The resulting encoded amino acid 58 is leu.
[0015]
Amino acid sequence searches were performed on proteins and polypeptides that share homology with full-length VESPR or its domains in available databases. Searches for polypeptides that share homology with VESPR were performed using the BLAST algorithm described in Altschul et al., J Mol Bio 215: 403-410 (1990). Using this program, VESPR amino acid sequences were compared to protein and DNA sequences found in databases obtained from the National Center for Biotechnology Information. The similarity score obtained as a result of these searches identified a group of polypeptides having various degrees of homology with VESPR. The highest degree of similarity was found between the VESPR and a group of proteins known as the “plexin gene family” (Maestrini et al., 1996 and Kameyama et al., 1996). Using the Smith-Waterman algorithm, as performed in the Genetics Computer Group (GCG) programs “BESTFIT” and “PILEUP” (Wisconsin Package, 9.0), VESPR and members of the human and murine plexin family Inter-pair and multiple sequence alignments were made. The average versus percent identity of the aligned protein sequences was calculated using the GCG program “DISTANCES”.
[0016]
Due to the pairwise sequence alignment between each of the VESPR polypeptide and several members of the plexin gene family, an average identity of 39% to 40% of its cytoplasmic domain (amino acids 966-1568) and 24%-for each of the total proteins An average identity of 25% was revealed. The higher degree of homology of the cytoplasmic domain suggests that the cytoplasmic domain has a similar signaling mechanism.
[0017]
To identify regions of similarity between protein sequences that were found to have some overall homology, the BLI XEM and MSPCRUNCH programs (Sonhammer and Durbin (1944a, b)) were used to Homology analysis was performed. Homology analysis revealed novel subdomains with similarity to regions of the semaphorin domains of several members of the semaphorin gene family described in Korodkin et al. (1993). The novel subdomain includes amino acids 380-482 of the VESPR sequence of the present invention. This subdomain can be further divided into two separate, smaller regions, containing residues 388-402 and 454-482, respectively. The C proximal half subdomain contains several highly conserved cysteine and tryptophan residues, and the consensus sequence Cx (5) -Cx (2) -Cx (7) -C- xW-C-x (5) -C, where x can be any amino acid. This total subdomain is different from the standard semaphorin domain described for the semaphorin gene family: (a) the subdomain is smaller (the subdomain is 100 amino acid residues out of 500 total semaphorin domains) Group), (b) the subdomain is also present in the plexin gene family and the MET-hepatocyte growth factor receptor family, both of which are not members of the standard semaphorin gene family, and (c) the subdomain is They are not themselves members of the semaphorin gene family, but differ in that they are present in genes that interact with a member of the semaphorin family (A39R). These subdomain sequences thus represent peptides that are potentially capable of further identifying other receptors that interact with semaphorins.
[0018]
The cDNA sequence encoding the VESPR polypeptide of SEQ ID NO: 2 is assembled as a composite of flanking ESTs and cloned cDNA nucleotide sequences and is disclosed in SEQ ID NO: 1. As described in Example 5, by identifying the cDNA encoding the amino acid sequence of SEQ ID NO: 2, it becomes possible to construct an expression vector containing the encoded cDNA. Host cells that are then transfected with a recombinant expression vector containing a cDNA encoding a VESPR polypeptide are cultured under conditions suitable for expressing the VESPR polypeptide, and the expressed VESPR polypeptide is recovered. Provides a method for producing a VESPR polypeptide of the invention.
[0019]
Because VESPR polypeptides are found in B cell lines, T cell lines and dendritic cells, it is possible to treat a variety of conditions associated with hypersensitivity or hypoactive immune regulation. Furthermore, the ligand and receptor complex may be involved in nerve growth, development, and / or maintenance. Although not limited to these, specific uses of VESPR are described below.
[0020]
The terms “VESPR” and “VESPR polypeptide” of the present invention include a polypeptide having the amino acid sequence of SEQ ID NO: 2 and a protein encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1. Further, the term is a polypeptide having a high degree of similarity or high degree of identity to the amino acid sequence of SEQ ID NO: 2, a biologically active molecule that is a member of the semaphorin family or The polypeptide comprises a polypeptide that binds to at least one of the fragments of the molecule. Furthermore, the term VESPR refers to the biologically active gene product of the DNA of SEQ ID NO: 1. Also included in the term VESPR are soluble or truncated proteins that primarily contain the binding portion of the protein, retain biological activity, and can be secreted. Particular examples of such soluble proteins are those comprising the sequence of amino acids 1-944 of SEQ ID NO: 2.
[0021]
When referring to a VESPR or semaphorin receptor polypeptide, the term “biologically active” means that the VESPR or semaphorin receptor polypeptide is capable of binding to at least one semaphorin. To do. Suitable assays for measuring VESPR binding are described herein and may include standard flow cytometry tests and slide binding tests.
[0022]
“Isolated” means that the VESPR is free of other proteins or polypeptides that are substantially a residue of the expression process, eg, as a purified product of recombinant host cell culture, or as a purified extract. means.
[0023]
The VESPR variants discussed herein are substantially homologous to native VESPR, but differ in amino acid sequence from that of native VESPR due to one or more deletions, insertions or substitutions Means a polypeptide having The variant amino acid sequence is preferably at least 80% identical to the native VESPR amino acid sequence, and most preferably at least 90% identical. Percent identity is described, for example, using the GAP computer program, version 8.1, described in Devereux et al. (Nucl. Acids Res. 12: 387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). It may be determined by comparing the information. Preferred default parameters for the GAP program include: (1) a single comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), and supervised by Schwartz and Dayhoff, Atlas of Protein Sequence and Structure , National Biomedical Research Foundation, pp. 353-358, 1979, Gribskov and Burgess, Nucl. Acids Res. 14: 6745, 1986 weighted comparison matrix; (2) 3.0 penalty for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) No penalty for end gaps . Variants may contain conservative substitution sequences, meaning that a given amino acid residue is replaced by a residue having similar physicochemical properties. Examples of conservative substitutions are those that replace one aliphatic residue with each other, eg, Ile, Val, Leu, or Ala with each other, or one between Lys and Arg; Glu and Asp; or between Gln and Asn. Substitution from one polar residue to another is included. Other such conservative substitutions are well known, for example, substitution of entire regions having similar hydrophobic properties. Naturally occurring VESPR variants or alleles are also included in the present invention. Examples of such variants are proteins that retain binding properties resulting from selective mRNA events or from proteolytic cleavage of the VESPR protein. Alternative splicing of mRNA can result in a partially excised but biologically active VESPR polypeptide, such as a naturally occurring soluble form of the protein. Variants believed to result from proteolysis include, for example, proteolysis of one or more terminal amino acids (generally 1-5 terminal amino acids) from a VESPR polypeptide upon expression in different types of host cells. N-terminal differences due to selective removal are included.
[0024]
As described above, Example 1 describes the construction of a novel viral A39R / Fc fusion protein useful for studying VESPR binding. Other antibody Fc regions may be substituted for the human IgG1 Fc region described in this example. Suitable Fc regions are those capable of binding with high affinity to protein A or protein G, and fragments of human IgG1 or fragments of human or murine IgG1 Fc regions, such as interchain disulfide bonds are formed. As will be understood, the fragment comprises at least the hinge region. The viral A39R: Fc fusion protein offers the advantage of being easily purified. In addition, a disulfide bond is formed between the Fc regions of two separate fusion protein chains, producing a dimer.
[0025]
As mentioned above, in one aspect, the invention includes a soluble VESPR polypeptide. Soluble VESPR polypeptides contain all or part of the extracellular domain of native VESPR but lack the transmembrane region that would result in retention of the polypeptide on the cell membrane. A soluble VESPR polypeptide conveniently includes a natural (or heterologous) signal peptide to facilitate secretion when initially synthesized, but the signal is secreted when the VESPR polypeptide is secreted from the cell. The peptide is cleaved. Soluble VESPR polypeptides included in the present invention retain the ability to bind to semaphorin ligands. Indeed, a soluble VESPR polypeptide may also include part of the signal or part of the cytoplasmic domain or other sequences, provided that the soluble VESPR protein can be secreted.
[0026]
Soluble VESPR is identified by separating intact cells expressing the desired protein from the medium, for example by centrifugation, and assaying the medium (supernatant) for the presence of the desired protein (and It may be distinguished from its insoluble membrane-bound counterpart). The presence of VESPR in the medium indicates that the protein is secreted from the cells and is therefore a soluble form of the desired protein.
[0027]
Soluble forms of VESPR polypeptides have many advantages over native membrane-bound VESPR proteins. Since soluble proteins are secreted from cells, they are suitable for the purification of proteins from recombinant host cells. In addition, soluble proteins are generally more suitable for intravenous administration.
[0028]
Examples of soluble VESPR polypeptides include those that include a substantial portion of the extracellular domain of a native VESPR polypeptide. An example of a soluble VESPR polypeptide is amino acids 1-944 of SEQ ID NO: 2. In addition, partially excised soluble VESPR containing less than the entire extracellular domain is included in the present invention, for example amino acids 35-944. When first expressed in a host cell, the soluble VESPR polypeptide may further comprise one of the heterologous signal peptides described below that function in the host cell used. Alternatively, the protein may comprise a natural signal peptide. In one embodiment of the invention, the soluble VESPR (from N-terminus to C-terminus) is a yeast α-factor signal peptide, the FLAG® peptide described below and in US Pat. No. 5,011,912, And a fusion protein comprising a soluble VESPR polypeptide consisting of amino acids 1-944 or 35-944 of SEQ ID NO: 2. The recombinant fusion protein is expressed in and secreted from yeast cells. The FLAG® peptide facilitates protein purification and can subsequently be cleaved from soluble VESPR using bovine mucosal enterokinase. Isolated DNA sequences encoding soluble VESPR proteins are included in the present invention.
[0029]
Truncated VESPR polypeptides, including soluble polypeptides, may be prepared by any of a number of conventional techniques. Desired DNA sequences may be chemically synthesized using techniques known per se. DNA fragments may also be produced by restriction endonuclease digestion of full length clonal DNA sequences and isolated by electrophoresis on an agarose gel. A linker containing a restriction endonuclease cleavage site may be used to insert the desired DNA fragment into the expression vector, or the fragment may be digested with a naturally occurring cleavage site therein. Well known polymerase chain reaction methods may also be used to amplify the DNA sequence encoding the desired protein fragment. As a further alternative, known mutagenesis techniques may be used to insert a stop codon at a desired point, eg, immediately downstream of the codon for the last amino acid of the binding domain.
[0030]
As mentioned above, the present invention provides isolated or homogeneous VESPR polypeptides, both recombinant and non-recombinant. Variants and derivatives of the native VESPR protein that retain the desired biological activity (eg, ability to bind semaphorin) may be obtained by mutation of the nucleotide sequence encoding the native VESPR polypeptide. Modification of the natural amino acid sequence may be accomplished by any of several conventional methods. Mutations may be introduced at a particular locus by synthesizing an oligonucleotide containing the mutated sequence that is flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion.
[0031]
Alternatively, oligonucleotide-designated site-directed mutagenesis may be used to provide a modified gene in which a predetermined codon may have been modified by substitution, deletion or insertion. Typical examples of making the above modifications are all incorporated herein by reference: Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985). 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82: 488, 1985); 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462.
[0032]
A native VESPR polypeptide may be modified to produce a VESPR derivative by forming a covalent or aggregated conjugate with other chemical moieties such as glycosyl groups, lipids, phosphates, acetyl groups and the like. . Covalent derivatives of VESPR polypeptides can also be prepared by linking a chemical moiety to a functional group on the VESPR amino acid side chain, or on the N-terminus or C-terminus of the VESPR polypeptide, or on its extracellular domain. Good. Other VESPR derivatives within the scope of the present invention include covalent or aggregated binding of VESPR polypeptides or fragments thereof to other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Contains the body. For example, the conjugate may comprise a signal or leader polypeptide sequence (eg, an α-factor leader of Saccharomyces) at the N-terminus of the VESPR polypeptide. The signal or leader peptide directs delivery of the conjugate from the synthesis site to a site inside or outside the cell membrane or cell wall, either simultaneously with or after translation.
[0033]
VESPR polypeptide fusions may include peptides added to facilitate purification and identification of VESPR. Such peptides include, for example, the antigenic identification peptides described in PolyHis or US Pat. No. 5,011,912 and Hopp et al., Bio / Technology 6: 1204, 1988.
[0034]
The invention further includes VESPRs with or without linking natural pattern glycosylation. A VESPR polypeptide expressed in a yeast or mammalian expression system (eg, COS-7 cells) may be similar to the native VESPR polypeptide in molecular weight and glycosylation pattern based on the choice of expression system, or It can be significantly different. Expression of the VESPR polypeptide in a bacterial expression system such as E. coli provides a non-glycosylated molecule.
[0035]
Equivalent DNA constructs encoding various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences not required for biological activity or binding are included in the present invention. For example, the N-glycosylation site of the VESPR extracellular domain may be modified to prevent glycosylation, which allows expression of reduced carbohydrate analogs in mammalian and yeast expression systems. The N-glycosylation site of a eukaryotic polypeptide is characterized by the amino acid triplet Asn-XY, where X can be any amino acid other than Pro, and Y is Ser or Thr. Natural human VESPR proteins are amino acids 86-88, 141-143, 149-151, 241-243, 252-254, 386-388, 407-409, 548-550, 553-555, 582-584 of SEQ ID NO: 2. 588-590, 591-593, 653-655, 686-688, 692-694, 715-717, 741-743, 771-773, 796-798, 821-823, 871-873, 890-892, 895 -2497 and 920-922 contain 24 such triplets. Appropriate substitutions, additions or deletions to the nucleotide sequences encoding these triplets will result in prevention of carbohydrate residue attachment at the Asn side chain. For example, a single nucleotide modification selected such that Asn is replaced by a different amino acid is sufficient to inactivate the N-glycosylation site. Known methods for inactivating the N-glycosylation site of proteins include those described in US Pat. No. 5,071,972 and EP 276,846, incorporated herein by reference. .
[0036]
In another example, a sequence encoding a Cys residue that is not essential for biological activity is modified so that the Cys residue is deleted or replaced with another amino acid, and upon regeneration, an incorrect intramolecular It may prevent the formation of disulfide bridges. Other equivalents are prepared by modifying adjacent dibasic amino acid residues to enhance expression in yeast systems where KEX2 protease activity is present. EP 212,914 discloses the use of site-directed mutagenesis to inactivate the KEX2 protease processing site of a protein. The KEX2 protease processing site modifies Arg-Arg, Arg-Lys, and Lys-Arg pairs and deletes, adds, or replaces residues to eliminate the occurrence of these adjacent basic residues Is inactivated. The Lys-Lys pair is much less sensitive to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivate the KEX2 site. Human VESPR contains 11 KEX2 protease processing sites.
[0037]
Nucleic acid sequences that are within the scope of the present invention include those that hybridize and are biologically active under moderate or highly stringent conditions to the VESPR nucleotide sequences disclosed herein. Isolated DNA and RNA sequences encoding certain VESPRs are included. Moderately stringent conditions include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Vol. 1, pp 101-104, Cold Spring Harbor Laboratory Press, (1989) as used in pre-wash solution of 5 × SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) And about 55 ° C., 5 × SSC, overnight hybridization conditions. Very stringent conditions include higher temperature hybridizations and washes. One skilled in the art will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as the length of the nucleic acid molecule and the relative amounts of A, T / U, C and G nucleotides. Let's go.
[0038]
Because of the known degeneracy of the genetic code, where one or more codons may encode the same amino acid, the DNA sequence differs from that shown in SEQ ID NO: 1 and still has the amino acid sequence of SEQ ID NO: 2 May encode a VESPR polypeptide. Such variant DNA sequences may result from silent mutations (eg, occurring during PCR amplification) or may be the product of intentional mutagenesis of native sequences.
[0039]
The present invention provides an equivalent isolated DNA sequence encoding a biologically active VESPR: (a) a cDNA comprising the nucleotide sequence shown in SEQ ID NO: 1; (b) moderately stringent conditions DNA encoding a VESPR polypeptide that is capable of hybridizing with (a) below and biologically active; (c) as defined by (a) or (b) as a result of the genetic code A DNA that is degenerate and that encodes a biologically active VESPR polypeptide; and (d) a DNA complementary to the DNA of (a), (b) or (c), The DNA sequence is selected from the above. VESPR polypeptides encoded by such DNA equivalent sequences are included in the present invention.
[0040]
A DNA equivalent to the DNA sequence of SEQ ID NO: 1 will hybridize under moderate and very stringent conditions to a DNA sequence encoding a polypeptide comprising the sequence of SEQ ID NO: 2. Examples of VESPR proteins encoded by such DNA include, but are not limited to, VESPR fragments and inactivated N glycosylation sites, inactivated KEX2 protease processing sites, or conservative amino acid substitutions as described above. VESPR proteins containing are included. Also included are VESPR polypeptides encoded by DNA from other species and said protein that will hybridize to the cDNA of SEQ ID NO: 1.
[0041]
Variants with the essential ability to bind semaphorins may be identified by any suitable assay. The biological activity of a VESPR polypeptide may be measured, for example, by competition for binding of semaphorins to receptor binding domains (ie, competitive binding assays).
[0042]
One type of competitive binding assay for VESPR polypeptides uses radiolabeled soluble VESPR and intact semaphorin expressing cells. Instead of intact cells, soluble semaphorin: Fc fusion protein, through interaction of protein A, protein G or antibodies against the semaphorin or Fc part of the molecule and the Fc part of the fusion protein, The fusion protein bound to the solid phase may be substituted. Another type of competitive binding assay utilizes radiolabeled soluble semaphorins, such as fusion proteins, and intact cells expressing VESPR.
[0043]
Competitive binding assays can also be performed according to conventional methodologies. In one embodiment, a soluble VESPR polypeptide may be generated and competed with a fixed receptor. For example, radiolabeled soluble semaphorin ligands may be antagonized by soluble VESPR in assays for binding activity to surface-bound semaphorin receptors. Qualitative results may be obtained by a competitive autoradiographic plate binding assay, or a Scatchard plot may be utilized to generate quantitative results.
[0044]
Alternatively, semaphorin binding proteins such as VESPR or anti-semaphorin antibodies, column chromatography matrices or similar supports suitable for identifying, isolating or purifying cells expressing semaphorin on the surface, etc. It may be bound to a solid phase. The binding of the semaphorin binding protein to the solid phase contact surface may be accomplished by any means, for example, constructing a VESPR: Fc fusion protein and using such a solid phase through protein A or protein G interaction. It may be combined with A variety of other means for immobilizing proteins on a solid phase are well known in the art and are suitable for use in the present invention. For example, magnetic microspheres may be coated with VESPR and held in an incubation container through a magnetic field. A suspension of the cell mixture containing semaphorin expressing cells is contacted with a solid phase having a VESPR polypeptide thereon. Cells with semaphorin on their surface bind to the immobilized VESPR and then wash away unbound cells. This affinity binding method is useful for purifying, screening, or separating such semaphorin expressing cells from solution. Methods for releasing positively selected cells from the solid phase are known in the art and include, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-toxic to cells and are preferably directed to cleave cell surface binding partners. In the case of a semaphorin-VESPR interaction, the enzyme preferably cleaves the semaphorin, thereby releasing the resulting cell suspension from the “foreign” semaphorin receptor component. The purified cell population may then be used to repopulate mature (adult) tissue.
[0045]
Alternatively, a cell mixture suspected of containing semaphorin positive cells may be first incubated with biotinylated VESPR. The incubation period is typically at least 1 hour continuous to ensure binding to semaphorin. The resulting mixture is then passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides cell binding to the beads. The use of avidin-coated beads is known in the art. Berenson et al., J. MoI. Cell. Biochem. , 10D: 239 (1986). Washing of unbound components and release of bound cells is performed using conventional methods.
[0046]
As noted above, VESPR may be used to isolate cells that express semaphorin. In an alternative method, VESPR or its extracellular domain or fragment is used to detect semaphorin expressing cells,¹²⁵It may be combined with a detectable moiety such as I.¹²⁵Radiolabeled with I function labeled with high specific activity¹²⁵This may be done by any of several standard methodologies that result in I-VESPR molecules. Alternatively, iodinated or biotinylated antibodies against semaphorin receptors may be used. Another detectable moiety such as an enzyme capable of catalyzing a colorimetric or fluorescence spectroscopic reaction, biotin, or avidin may be used. Cells to be tested for semaphorin expression may be contacted with a labeled VESPR polypeptide. Following incubation, unbound labeled VESPR is removed and binding is measured using a detectable moiety.
[0047]
The binding properties of VESPR (including mutants) are also shown in conjugated semaphorins (eg,¹²⁵I-semaphorin: Fc) may be used for determination. However, in this case, a sample containing a putative VESPR variant competes for binding to the conjugated semaphorin using intact cells expressing semaphorin bound to a solid support. Measure the degree.
[0048]
Other means of assaying for VESPR include the use of anti-VESPR antibodies, cell lines that grow in response to VESPR, or recombinant cell lines that express semaphorin and grow in the presence of VESPR.
[0049]
The VESPR proteins disclosed herein may also be used to measure the biological activity of a semaphorin protein in terms of binding affinity for VESPR. As one example, the VESPR polypeptide of the present invention may be used to determine whether biological activity is retained after modification (eg, chemical modification, partial excision, mutation, etc.) of a semaphorin protein. Good. In this way, the biological activity of a semaphorin protein can be determined prior to use, for example, in research studies or clinically.
[0050]
The VESPR polypeptides of the present invention find use as reagents that may be used, for example, by performing “quality assurance” studies to monitor the shelf life and stability of semaphorin proteins under different conditions . For example, VESPR polypeptides may be used for binding affinity studies to measure the biological activity of semaphorin proteins stored at different temperatures or produced in different cell types. The binding affinity of the modified semaphorin protein to VESPR is compared to that of the unmodified semaphorin protein to detect any adverse effect of the modification on the biological activity of the semaphorin.
[0051]
VESPR polypeptides also find use as carriers for delivering binding agents to cells expressing semaphorins. As described in Example 7 below, putative human semaphorins are expressed in cells found in the placenta, testis, ovary and spleen. Thus, VESPR polypeptides are used to deliver diagnostic or therapeutic agents to these cells (or other cell types that are found to express semaphorin on the cell surface) in vitro or in vivo. May be.
[0052]
Diagnostic and therapeutic agents that may be conjugated to a VESPR polypeptide include, but are not limited to drugs, toxins, radionuclides, chromophores, enzymes that catalyze colorimetric or fluorescent spectroscopy, and the like Comparable with the specific agent selected according to the intended application. Examples of agents include those used to treat various types of cancer, such as nitrogen mustards such as L-phenylalanine nitrogen mustard or intercalating agents such as cyclophosphamide, cis-diaminodichloroplatinum. ), Antimetabolites such as 5-fluorouracil, vinca alkaloids such as vincristine, and antibiotics such as bleomycin, doxorubicin, daunorubicin, and derivatives thereof. Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosome inactivating protein, mycotoxins such as trichothecene, and derivatives and fragments thereof (eg single chain). Radionuclides suitable for diagnostic use are not limited to:¹²³I,¹³¹I,^99mTc,¹¹¹In, and⁷⁶There is Br. Radionuclides suitable for therapeutic use are not limited to:¹³¹I,²¹¹At,⁷⁷Br,¹⁸⁶Re,¹⁸⁸Re,²¹²Pb,²¹²Bi,¹⁰⁹Pd,⁶⁴Cu, and⁶⁷There is Cu.
[0053]
Such agents may be bound to the semaphorin receptor by any suitable conventional method. A VESPR is a protein, including, for example, a functional group on an amino acid side chain that can react with a functional group of a desired agent to form a covalent bond. Alternatively, the protein or agent may be derivatized to produce or attach the desired reactive functional group. Derivatization may involve one linkage of a bifunctional coupling reagent (Pierce Chemical Company, Rockford, Ill.), Available to attach a variety of molecules to proteins. Several techniques are known for radiolabeling proteins. The radionuclide metal may be bound to the receptor, for example by using a suitable bifunctional chelator.
[0054]
Conjugates (preferably covalently linked) comprising VESPR and an appropriate diagnostic or therapeutic agent are thus prepared. The conjugate may be administered or otherwise used in an amount suitable for the particular application.
[0055]
Another use of the VESPR of the present invention is as a research tool to study the role that receptors may play in immune regulation and viral infections in conjunction with semaphorins. The VESPR polypeptides of the present invention may also be used in in vitro assays for the detection of semaphorins or VESPR bound by the polypeptides, or their interactions.
[0056]
As described in Example 16, semaphorin interacts with the membrane-bound receptor of the present invention, interferon and Staphylococcus aureus (type C) (SAC) in IL-12 production from dendritic cells. ) And synergistic action. The use of VESPR and its semaphorin ligand to induce IL-12 production promotes natural killer cell and T cell production and induces cytokine production (mainly γ-interferon). IL-12 and IL-12 induced gamma interferon production supports Th1 cell differentiation and down-regulates the production of cytokines associated with Th2 cell differentiation. IL-12 is known to act as both a pro-inflammatory cytokine and an immune regulator. Thus, soluble VEPSR may be used to antagonize IL-12 production and down regulate the Th1 cell differentiation of the organism. Similarly, soluble VESPR may be used to promote the production of cytokines associated with Th2 cell differentiation and thus inhibit pro-inflammatory activity. Alternatively, VESPR may be used in combination with its semaphorin ligand to increase IL-12 production in combination with vaccine administration against pathogens for which cellular immunity is effective. An enhanced amount of IL-12 in this manner acts as an adjuvant in vaccine administration and induces a more sustained Th1-type immunological memory.
[0057]
Furthermore, administration of IL-12 to animals with tumors is known to result in tumor regression and establishment of tumor-specific immune responses. Thus, using a semaphorin ligand that binds to VESPR to enhance or promote IL-12 can induce a therapeutic immune response against aggressive micrometastatic tumors.
[0058]
Furthermore, as described in Example 18, the receptor of the present invention binds to its semaphorin ligand and increases CD54 expression on monocytes. This observation suggests that semaphorin / semaphorin receptor interactions mediate cellular activation that contributes to pro-inflammatory activities typically involved in monocyte activation. Such activities include phagocytosis, phagocytosis, nitric oxide production and increased cytokine production. In order to antagonize or reverse the pro-inflammatory activity resulting from the interaction between the semaphorin ligand and its membrane-bound receptor, a pharmaceutical composition comprising a therapeutically effective amount of the soluble VESPR of the present invention, The organism may be administered parenterally. Soluble VESPR binds to the semaphorin ligand, thus preventing the ligand from binding to the membrane-bound receptor and contributing to pro-inflammatory activity. A therapeutically effective amount of VESPR is an amount sufficient to antagonize pro-inflammatory activity.
[0059]
Despite increased expression of CD54 on monocytes, microphysiometer data suggests that cell signaling through VESPR does not activate cells in the classical immunological sense. Indicated. More specifically, microphysiology instrument data suggests that when VESPR binds to its ligand, a decrease in the rate of media pH change occurs. This is the opposite of what happens during cell activation. Such a decrease is experienced with drugs that inhibit protein kinases in cells or viral infections that can metabolically numb the cells. Inhibitory signals delivered through VESPR can be antagonized by administration of a soluble form of VESPR. Such soluble forms bind effectively to VESPR binding partners and antagonize inhibitory signaling, thus preventing the inactivated state of the cell. Alternatively, and in accordance with the present invention, an anti-VESPR antibody that communicates information may be used as a therapeutic agent to treat autoimmune diseases where inflammation is the result of presentation of self antigens to T cells. More specifically, such anti-VESPR antibodies may be targeted to be taken up by antigen presenting cells and inactivate antigen presenting cells as well as protein kinase inhibitors. Since the antigen presentation of the antigen-presenting cells responsible for inflammation is inferior, autoimmunity is cured.
[0060]
Semaphorin ligand binds to VESPR and down-regulates the expression of CD86, a MHC class II molecule and a costimulatory molecule, on dendritic cells cultured with GM-CSF and IL-4 (Example 17 ) Suggests that the interaction between the semaphorin ligand and the receptor of the present invention is involved in immunosuppression of mature dendritic cells. In order to antagonize or reverse the immunosuppressive activity resulting from the interaction between the semaphorin ligand and its membrane-bound receptor, a pharmaceutical composition comprising a therapeutically effective amount of the soluble VESPR of the present invention is It may be administered parenterally. Soluble VESPR binds to the semaphorin ligand, thus preventing the ligand from binding to membrane-bound receptors and contributing to immunosuppressive activity. Alternatively, administering an appropriate semaphorin ligand to a patient or organism damaged by the effects of chronic inflammation will contribute to suppressing the pro-inflammatory activity of differentiated macrophages.
[0061]
The data show that VESPR ligand is found on T cells and that VESPR is involved in the migration of dendritic cells from the T cell zone of lymph nodes. Furthermore, the data suggest that VESPR is involved in blocking the T cell immune response as soon as the pathogen is removed.
[0062]
The VESPR polypeptides of the present invention may be formulated according to known methods used to prepare pharmaceutically useful compositions. VESPR is a pharmaceutically suitable diluent (eg, Tris-HCl, acetic acid, phosphate), preservative (eg, thimerosal, benzyl alcohol, parabens) as a single active ingredient or in combination with other known active ingredients. ), Emulsifiers, solubilizers, adjuvants and / or carriers may be mixed and combined. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th edition, 1980, Mack Publishing Co. It is described in. In addition, such compositions may be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymer compounds such as polyacetic acid, polyglycolic acid, hydrogels, or liposomes, microemulsions, micelles, single cells. VESPR polypeptides incorporated into lamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts may be included. Such compositions will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate of VESPR. The VESPR polypeptide may also be bound to an antibody directed against a tissue-specific receptor, ligand or antigen, or may be coupled to a ligand for a tissue-specific receptor.
[0063]
The VESPR polypeptide may be administered topically, parenterally, or by inhalation. The term “parenteral” includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. These compositions will typically include an effective amount of VESPR, alone or in combination with an effective amount of any other active ingredient. The dosage and desired drug concentration contained in the composition can vary depending on many factors, including the intended use, the patient's weight and age, and the route of administration. Preliminary doses may be determined according to animal studies and dosage estimates for human administration may be performed according to practices recognized in the art.
[0064]
VESPR polypeptides may exist as oligomers, such as dimers or trimers that are covalently or non-covalently linked. The oligomers may be linked by disulfide bonds formed between cysteine residues on different VESPR molecules. In one embodiment of the present invention, a VESPR dimer is generated by fusing VESPR to the Fc region of an antibody (eg, IgG1) in a manner that does not interfere with binding of VESPR to the semaphorin ligand binding domain. . The Fc polypeptide is preferably fused to the C-terminus of soluble VESPR (including only the ligand binding moiety). The general preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is described, for example, by Ashkenazi et al. (PNAS USA 88), incorporated herein by reference. : 10535, 1991) and Byrn et al. (Nature 344: 677, 1990). A gene fusion encoding a VESPR: Fc fusion protein is inserted into an appropriate expression vector. Allows VESPR: Fc fusion proteins to assemble in a manner that closely resembles antibody molecules so that interchain disulfide bonds are formed between Fc polypeptides, resulting in divalent products. If the fusion protein is made with both heavy and light chains of an antibody, it is possible to form a VESPR oligomer containing as many as four VESPR extracellular regions. Alternatively, two soluble VESPR domains may be connected by a peptide linker.
[0065]
Suitable host cells for expression of the VESPR polypeptide include prokaryotic, yeast or higher order eukaryotic cells. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY (1985). Cell-free translation systems may also be used to produce VESPR polypeptides using RNA from the DNA constructs disclosed herein.
[0066]
Prokaryotes include gram negative or gram positive organisms such as E. coli or Bacillus. Prokaryotic host cells suitable for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas, Streptomyces, and S. A variety of other species within are included. In prokaryotic host cells such as E. coli, the VESPR polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal methionine may be cleaved from the expressed recombinant VESPR polypeptide.
[0067]
VESPR polypeptides may be expressed in yeast host cells, preferably from the genus Saccharomyces (eg, S. cerevisiae). Other genera of yeast, such as Pichia, K. Lactis (K. lactis) or Kluyveromyces may also be used. Yeast vectors will often contain an origin of replication sequence derived from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a sequence for polyadenylation, a sequence for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073, 1980), or enolase, glyceraldehyde-3-phosphate dehydrogenase, Other glycolytic enzymes such as hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase ( Hess et al., J. Adv. Enzyme Reg. 7: 149, 1968; and Holland et al., Biochem. 17: 4900, 1978). Is included. Other vectors and promoters suitable for use in yeast expression are Hitzeman, EPA-73,657 or Freeer et al., Gene, 107: 285-195 (1991); and van den Berg et al., Bio / Technology, 8: 135- 139 (1990). Another alternative is the glucose repressible ADH2 promoter described in Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). A shuttle vector capable of replicating in both yeast and E. coli is a DNA sequence derived from pBR322 (Amp) for selection and replication in E. coli.^rThe gene and origin of replication may be constructed by inserting into the yeast vector described above.
[0068]
A yeast α-factor leader sequence may be used to secrete the VESPR polypeptide directly. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, for example, Kurjan et al., Cell 30: 933, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those skilled in the art. The leader sequence may be modified to include one or more restriction sites near its 3 'end. This will facilitate the fusion of the leader sequence to the structural gene.
[0069]
Yeast transformation protocols are known to those skilled in the art. One such protocol is Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929, 1978. The Hinnen et al. Protocol is Trp.⁺Transformants are selected in selective medium, where the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acid, 2% glucose, 10 μg / ml adenine and 20 μg / ml uracil.
[0070]
Yeast host cells that have been transformed with a vector containing an ADH2 promoter sequence may be grown in “rich” media to induce expression. An example of a rich medium consists of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg / ml adenine and 80 μg / ml uracil. Derepression of the ADH2 promoter occurs when glucose is depleted from the medium.
[0071]
Mammalian or insect host cell culture systems may also be used to express recombinant VESPR polypeptides. A baculovirus system for producing heterologous proteins in insect cells is reviewed in Luckow and Summers, Bio / Technology 6:47 (1988). Established cell lines of mammalian origin may also be used. Examples of suitable mammalian host cell lines include monkey kidney cell COS-7 strain (ATCC CRL 1651) (Gluzman et al., Cell 23: 175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163). , Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and African green monkey kidney cells as described in McMahan et al. (EMBO J. 10: 2821, 1991). A CV-1 / EBNA-1 cell line derived from strain CVI (ATCC CCL 70) is included.
[0072]
Transcriptional and translational regulatory sequences for mammalian host cell expression vectors may be excised from the viral genome. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early and late promoters, enhancers, splicing, and polyadenylation sites may also be used to provide other genetic elements for expression of structural gene sequences in mammalian host cells. Good. Viral early and late promoters are particularly useful because both are easily obtained from the viral genome as fragments that may also contain viral origins of replication (Fiers et al., Nature 273: 113, 1978). Smaller or larger SV40 fragments may also be used provided that they contain approximately 250 bp of the sequence from the Hind III site to the Bgl I site located at the SV40 viral origin of replication site.
[0073]
Exemplary expression vectors for use in mammalian host cells may be constructed as disclosed in Okayama and Berg (Mol. Cell. Biol. 3: 280, 1983). A system useful for stable high level expression of mammalian cDNA in C127 murine mammary epithelial cells may be constructed substantially as described in Cosman et al. (Mol. Immunol. 23: 935, 1986). A useful high expression vector, PMLSV N1 / N4, described in Cosman et al., Nature 312: 768, 1984, has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and US patent application Ser. No. 07 / 701,415, filed May 16, 1991, incorporated herein by reference. The vector may be derived from a retrovirus. Instead of the natural signal sequence and in addition to the initiator methionine, a heterologous signal sequence such as the signal sequence of IL-7 described in US Pat. No. 4,965,195; Cosman et al., Nature 312: 768 (1984) IL-2 receptor signal sequence as described in); IL-4 signal peptide as described in EP 367,566; type I IL-1 receptor signal peptide as described in US Pat. No. 4,968,607 And a type II IL-1 receptor signal peptide as described in EP 460,846 may be added.
[0074]
A VESPR polypeptide as an isolated, purified or homogeneous protein according to the present invention may be produced by a recombinant expression system as described above or purified from a naturally occurring cell. Good. VESPR can be purified substantially homogeneously as indicated by a single protein band upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
[0075]
One method for producing VESPR involves culturing host cells transformed with an expression vector comprising a DNA sequence encoding a VESPR polypeptide under conditions sufficient to promote expression of the VESPR polypeptide. Including that. The receptor is then recovered from the medium or cell extract depending on the expression system used. As known to those skilled in the art, the method for purifying the recombinant protein will vary depending on factors such as the host cell used and whether the recombinant protein is secreted into the medium.
[0076]
For example, when using an expression system that secretes recombinant protein, the medium may be first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. Following the concentration step, the concentrate may be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin such as a matrix or support having diethylaminoethyl (DEAE) side chains may be used. The matrix may be acrylamide, agarose, dextran, cellulose or other types commonly used for protein purification. Alternatively, a cation exchange step may be used. Suitable cation exchangers include a variety of insoluble matrices containing sulfopropyl or carboxymethyl groups. A sulfopropyl group is preferred. Finally, one or more reverse phase high performance liquid chromatography using a hydrophobic reverse phase high performance liquid chromatography (RP-HPLC) medium (eg, silica gel with side chain methyl or other aliphatic groups). The (RP-HPLC) step may be used to further purify the VESPR polypeptide. Methods using some or all of the above purification steps in various combinations are well known and can be used to provide substantially homogeneous recombinant proteins.
[0077]
An affinity column containing the receptor binding domain of semaphorin that binds to VESPR can be utilized to affinity purify the expressed VESPR polypeptide. VESPR polypeptides can be prepared using conventional techniques, for example, by dialysis in a high salt elution buffer and then in a lower salt buffer for use, or depending on the affinity matrix utilized. Or it may be removed from the affinity column by changing other components. Alternatively, the affinity column may contain an antibody that binds to VESPR. Example 20 describes a method for using the VESPR of the present invention to generate monoclonal antibodies directed against VESPR.
[0078]
Recombinant protein produced in bacterial culture is first disrupted by host cells, centrifuged, extracted from cell precipitates for insoluble polypeptides or from supernatant fluid for soluble polypeptides, then one Alternatively, it may be isolated by performing further concentration, salting out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC may be used for the final purification step. Microbial cells may be destroyed by any convenient method, including freeze-thaw cycles, ultrasound, mechanical disruption, or the use of cytolytic agents.
[0079]
Transformed yeast host cells are preferably used to express VESPR as a secreted polypeptide to simplify purification. Recombinant polypeptides secreted from yeast host cell fermentation may be purified by methods similar to those disclosed in Urdal et al. (J. Chromatog. 296: 171, 1984). Urdal et al. Describe two consecutive reverse phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.
[0080]
Useful fragments of VESPR nucleic acids include antisense or sense oligonucleotides, including single stranded nucleic acid sequences (RNA or DNA) capable of binding to target VESPR mRNA (sense) or VESPR DNA (antisense) sequences. included. The antisense or sense oligonucleotide comprises a fragment of the coding region of the VESPR cDNA according to the present invention. Such fragments generally comprise at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. Based on cDNA sequences encoding a given protein, the ability to obtain antisense or sense oligonucleotides is described, for example, by Stein and Cohen (Cancer Res. 48: 2659, 1988) and van der Krol et al. (BioTechniques 6: 958, 1988). It is described in.
[0081]
Binding of the antisense or sense oligonucleotide to the target nucleic acid sequence is by one of several means, including by enhanced degradation of duplexes, immature termination of transcription or translation, or by other means, This results in the formation of a duplex that blocks transcription or translation of the target sequence. Thus, antisense oligonucleotides may be used to block VESPR protein expression. Antisense or sense oligonucleotides further include oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages such as those described in WO 91/06629), and such sugar linkages are endogenous Resistant to nucleases. Oligonucleotides with such resistant sugar linkages are stable in vivo (ie capable of resisting enzymatic degradation), but retain sequence specificity capable of binding to the target nucleotide sequence. Other examples of sense or antisense oligonucleotides include organic moieties such as those described in WO 90/10448, and other that increase the affinity of the oligonucleotide for target nucleic acid sequences such as poly (L-lysine). Oligonucleotides that are covalently attached to the moiety are included. In addition, intercalating agents such as ellipticine, and alkylating agents or metal complexes may bind to the sense or antisense oligonucleotide to modify the binding specificity of the antisense or sense oligonucleotide to the target nucleotide sequence.
[0082]
Antisense or sense oligonucleotides are, for example, CaPO₄It may be introduced into cells containing the target nucleic acid sequence by any gene transfer method, including mediated DNA transfection, electroporation, or by using a gene transfer vector such as Epstein-Barr virus. Preferably, the antisense or sense oligonucleotide inserts the antisense or sense oligonucleotide into a suitable retroviral vector, and then contacts the cell with the retroviral vector comprising the insert sequence in vivo or ex vivo. Thus, it is introduced into a cell containing the target nucleic acid sequence. Suitable retroviral vectors include, but are not limited to, murine retroviruses M-MuLV, N2 (M-MuLV derived retrovirus) or double copies designated DCT5A, DCT5B and DCT5C Vectors (see PCT application US 90/02656) are included.
[0083]
Sense or antisense oligonucleotides may also be introduced into cells containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or the sense or antisense oligonucleotide or its conjugate form is intracellularly present. Do not block entering.
[0084]
Alternatively, sense or antisense oligonucleotides may be introduced into cells containing the target nucleic acid sequence by formation of oligonucleotide-lipid complexes, as described in WO 90/10448. Sense or antisense oligonucleotide-lipid complexes are preferably separated intracellularly by endogenous lipases.
[0085]
In addition to the above, the following examples are provided to illustrate certain embodiments and are not intended to limit the scope of the invention.
[0086]
【Example】
Example 1
Preparation of ectromelia semaphorin / Fc fusion protein
The following describes the preparation of ectromelia semaphorin A39R / immunoglobulin fusion protein (A39R / Fc). The method involved preparing a DNA construct encoding the fusion protein, transfecting a cell line with the DNA construct, and collecting the supernatant from the transfected cells. The A39R / Fc fusion protein was used as described in Examples 3, 4, 5 and 6 to study VESPR binding properties and to isolate VESPR.
[0087]
DNA encoding A39R semaphorin was isolated and amplified from ectromelia virus genomic DNA using PCR techniques and synthetic oligonucleotide primers whose sequences were based on the published A39R sequence in the Copenhagen strain of vaccinia virus. Copenhagen strain A39R DNA sequence is described by Goebel, S .; J. et al. Et al., Virology 179: 247, 1990. Isolated ectromelia A39R DNA is presented in SEQ ID NO: 7, and the protein encoded by the DNA is presented in SEQ ID NO: 8. A Spe1 site was introduced upstream of amino acid 15 of the A39R polypeptide by an upstream oligonucleotide primer. A Notl site was introduced downstream of the stop codon of Ectromelia A39R, after amino acid 399, with a downstream oligonucleotide primer. The primer sequences were as follows:
Upstream Spe1 primer:
[0088]
[Chemical 1]

Downstream Not1 primer:
[0089]
[Chemical 2]

Baum et al., Cir. Sh. 44:30 (1994), a Bgl II to Nsl I restriction fragment comprising an immunoglobulin mutein human Fc region, the murine IL-7 signal peptide and US Pat. No. 5,011. FLAG as described in No. 912^TMLigated into an expression vector (pDC304) containing the octapeptide. A PCR amplified DNA encoding amino acids 15-399 of ectromelia A39R was then ligated to the mutant protein human Fc region, murine IL-7 signal peptide and FLAG.^TMTwo-way ligation was performed in an expression vector containing the peptide. The resulting DNA construct was transfected into the monkey kidney cell line CV-1 / EBNA (with co-transfection of psv3neo). After 7 days in culture with medium containing 0.5% low immunoglobulin bovine serum, 0.2% azide solution was added to the supernatant and the supernatant was filtered through a 0.22 μm filter. Subsequently, approximately 1 liter of culture supernatant was passed through a BioCad Protein A HPLC protein purification system using a 4.6 x 100 mm Protein A column (POROS 20A from PerSeptive Biosystems) at 10 ml / min. The Protein A column binds to the Fc portion of the fusion protein in the supernatant, immobilizes the fusion protein, and allows other components in the supernatant to pass through the column. The column was washed with 30 ml PBS solution and the bound fusion protein was eluted from the HPLC column with citric acid adjusted to pH 3.0. The eluted purified fusion protein was neutralized with 1M HEPES solution at pH 7.4 at the time of elution.
Example 2
Preparation of ectromelia semaphorin / polyHis fusion protein
The following describes the preparation of an ectromelia A39R / polyHis fusion protein (A39R / polyHis). The method involved preparing a DNA construct encoding the fusion protein, transfecting a cell line with the DNA construct, and collecting the supernatant from the transfected cells.
[0090]
DNA encoding ectromelia A39R (A39R ORF, amino acids 1-399 of SEQ ID NO: 8) was isolated and amplified from ectromelia virus genomic DNA using PCR techniques and synthetic oligonucleotide primers. The primer was added with Not 1 site at the 5 'end and the motif Gly-Ser-6xHis used for the purification process at the 3' end. The primer added an in-frame stop codon and a Bgl 2 site after the Gly-Ser-6xHis motif. The PCR product was cleaved and cloned into the pDC409 expression vector (McMahon et al., EMBO J. 10: 2821, 1991).
[0091]
The resulting DNA construct was transiently transfected into the monkey cell line COS-1 (ATCC CRL-1650). After culturing in a medium containing 0.5% low immunoglobulin bovine serum for 7 days, the cell supernatant was collected and 0.2% sodium azide solution was added to the supernatant. The supernatant is filtered through a 0.22 μm filter, concentrated 10-fold with a preparative scale concentrator (Millipore; Bedford, Mass.), And equipped with a nickel NTA superflow self-packed resin column (Qiagen, Santa Clarita, CA). Purified on BioCad HPLC protein purification. After the supernatant passes through the column, the column is washed with buffer A (20 mM NaPO₄, PH 7.4; 300 mM NaCl; 50 mM imidazole). The bound protein was then eluted from the column using a gradient elution technique. Fractions containing protein were collected and analyzed on a 4-20% SDS-PAGE reducing gel. The peak containing the A39R / polyHis fusion protein was pooled, concentrated 2 fold and then dialyzed in PBS. The resulting A39R / polyHis fusion protein was then filtered through a 0.22 μm sterile filter.
Example 3
Screening of cell lines for binding to A39R
A39R / Fc fusion proteins prepared as described in Example 1 were used to screen cell lines for binding using quantitative binding studies according to standard flow cytometry methodology. For each cell line to be screened, the method involves incubating approximately 100,000 cells and blocking with 2% FCS (fetal calf serum), 5% normal goat serum and 5% rabbit serum in PBS for 1 hour. Included. The blocked cells were then incubated with 5 μg / ml of A39R / Fc fusion protein in 2% FCS, 5% goat serum and 5% rabbit serum in PBS. Following incubation, samples were washed twice with FACS buffer (2% FCS in PBS) and then mouse anti-human Fc / biotin (purchased from Jackson Research) and SAPE (streptavidin-phycoerythrin purchased from Molecular Probes). Was processed. This treatment allows anti-human Fc / biotin to bind to all bound A39R / Fc, and SAPE to bind to anti-human Fc / biotin and fluorescent identification labels on A39R / Fc bound to cells. Cause it to occur. Cells were analyzed for all bound proteins using fluorescence detection flow cytometry. Results show that A39R semaphorin binds well to human NK cells, murine splenic B cells, human PB T cells, human T, B, erythroblasts, lymphoblasts and myeloid progenitors, fibroblasts and epithelial cell lineages It was shown that. Table 1 lists the results of the flow cytometry study. “+” Indicates that binding was detected between the cell surface and A39R. “-” Indicates that no binding was detected between the cell surface and A39R.
[0092]
[Table 1]

[0093]
Example 4
Identification of putative semaphorin receptors
CB23 cells (human umbilical cord blood B cell line) and human PBT T cells that were tested positive for binding to A39R were tested for putative receptor expression, and some receptors as membrane bound molecules, soluble molecules or both. Tested to determine if expressed. In general, the analysis radiolabels CB23 and human PB T cell surfaces, harvests, and treats cell supernatants and lysates with A39R / Fc fusion proteins to precipitate all putative receptors, and Thereafter, visualization of the immunoprecipitate on an electrophoresis gel was included.
[0094]
In particular, the method is first described as described in Benjamin et al., Blood 75: 2017-2023 (1990), [¹²⁵I], approximately 1 × 10⁷Radiolabeling of CB23 or PB T cells. Cultured cell supernatants were collected and clarified by centrifugation at 14,000 rpm for 30 minutes. Cell lysates are incubated for 30 minutes on ice in 1 ml phosphate buffered saline containing 1% Triton-X 100 containing protease inhibitors, phenylmethylsulfonyl fluoride, pepstatin-A, and leupeptin. Was generated by The lysate was clarified by centrifugation at 14,000 rpm for 30 minutes. To precipitate all receptors present in the lysate and / or supernatant, 200 μl of cell supernatant or lysate was incubated with 2 μg of A39R / Fc fusion protein prepared as described in Example 1. Incubation was performed for 1 hour at 4 ° C. with gentle rocking. Similarly, Fc protein control samples were prepared and incubated. Following incubation, protein A sepharose beads (# 17-0780-01, Pharmacia Biotech Inc., Piscataway, NJ) were added to the lysate and supernatant, and the mixture was incubated at 4 ° C. with gentle rocking for 1 hour. The beads were washed thoroughly with PBS 1% Triton-X 100 solution. Bound protein was eluted and analyzed by SDS PAGE. The protein band was visualized by autoradiography and a single approximately 200 kDa band was found to bind to A39R / Fc but not to the control Fc protein. The semaphorin receptor was present in cell lysates and cell supernatants, confirming its expression as a membrane bound protein and as a secreted soluble protein.
Example 5
Isolation and sequencing of semaphorin receptors
Using the A39R / Fc fusion protein prepared as described in Example 1, a human semaphorin receptor polypeptide was isolated and a method for purification of the isolated polypeptide was confirmed. Protease containing CB23 cell pellet with 1 mM, 10 μg / ml APMSF, and 1 mM EDTA each of PMSF, leupeptin, aprotinin, pepstatin A in homogenization buffer (10 mM phosphate, 30 mM NaCl, pH 7.4) The semaphorin receptor was isolated by suspending in the inhibitor solution. The cells were homogenized in dounce and overlaid with a 41% sucrose solution in homogenization buffer and spun off on a Beckman SW-28 rotor at 25,000 rpm, 4 ° C. for 45 minutes. The intermediate phase was collected and diluted with cold homogenization buffer, downsed and spun. The resulting clear membrane pellet was stored at -80 ° C.
[0095]
Membrane pellets prepared from 240 ml packed cells were prepared from 20 mM Tris, 150 mM NaCl, protease inhibitors identified above, 1% Triton X-100 and CaCl.₂MgCl₂, And McCl₂Combined with 100 ml of an aqueous solution of 0.1 mM salt (buffer A). The suspended pellet was downed and spun on a SW-28 rotor at 25,000 rpm, 4 ° C. for 30 minutes. The supernatant was loaded onto a 100 ml wheat germ agglutinin column and eluted with 10 column volumes of buffer A at a rate of 1 ml / min. The protein specifically bound to the column was then eluted with buffer A containing 0.2M N-acetylglucosamine.
[0096]
Fractions that tested positive for protein were pooled and incubated with 100 μg of A39R / Fc fusion protein for 1 hour at 4 ° C. The incubated mixture was passed through a Sepharose column to remove non-specifically bound components and then passed through a 0.5 ml column of protein A / Sepharose solid support. The Protein A / Sepharose solid support was washed with 20 column volumes of PBS containing 1% Triton X-100 and then with PBS to wash off any unbound components. The protein retained on the Protein A / Sepharose column was then eluted in a stepwise fashion with a 0.35 ml fraction of 50 mM citric acid at pH 3.0. Fractions that tested positive for protein were combined and concentrated to 50 μl using a 10 kD MWCO Centricon concentrator. The resulting protein in the concentrated sample was reduced and then alkylated using standard DTT and iodoacetic acid methods. The alkylated protein was then electrophoresed on an 8% gel. Proteins on the gel were visualized with Coomassie G in 50% MeOH with 5% acetic acid and then destained in 50% MeOH.
[0097]
The approximately 200 kD band, located by comparison to protein standards, was excised with a razor and washed overnight in 100 mM ammonium carbonate. Gel slices were fast evaporated to dryness and a 1:10 solution of trypsin in 100 mM ammonium carbonate was added to the dry slide. The slides were incubated for 16 hours at 37 ° C., and then the proteins in the sections were extracted three times with 50% acetonitrile containing 5% formic acid with 30 minutes incubation with each extraction.
[0098]
Trypsin digested peptide fragments were lyophilized, dissolved in 50 μl of 0.1% trifluoroacetic acid, and separated by RP-HPLC on a 500 μid (inner diameter) × 25 cm capillary column packed with C-18 reverse phase packing. The HPLC liquid phase was an acetonitrile / water gradient of 10% after 5 minutes and 85% after 105 minutes. The eluted protein was detected at 215 nm. As each protein eluted, it was collected in separate fractions and N-terminal sequence analysis of the peptides in the fractions was performed on a 494 Procise sequencer according to the manufacturer's instructions.
[0099]
The RP-HPLC fraction obtained as described above was dried in a vacuum centrifuge, and the peptide in the fraction was dissolved in 6 μl of 50% methanol containing 0.5% acetic acid. 2 μl of each peptide solution was loaded onto a nanospray chip (Protein Analysis Company, Ozense, Denmark). Data were obtained on a Finnigan TSQ700 triple quadrupole mass spectrometer (San Jose, CA) equipped with a nanospray source. Mass spectra were obtained with unit resolution. For series mass spectrometry, the first quadrupole was operated with sufficient resolution to pass the 3-4 Da width, and the third quadrupole was operated with unit resolution. The collision gas was supplied at a pressure of 4 mTorr. Methyl esterification was performed using standard esterification methods.
[0100]
In-line mass spectrometric analysis of tryptic peptides provided amino acid sequence information for the isolated portion of the purified protein. Serial mass spectral data were used in computer-assisted screening of non-redundant protein databases and EST databases using a partial SEQUEST algorithm search tool (Eng, JK, et al., J Am Soc. Mass 1994). Peptide query sequence GluGluThrProValPheTyrLys corresponding to amino acids 421-428 of SEQ ID NO: 2 and AsnIleTylIleTyrLeuThrAlaGlyLys corresponding to amino acids 436-445 are EST 248534 (deposited with reference number N78220, reference number N78220) It was identified as containing a peptide sequence that had sex. ThrValLeuPheLeuGlyThrGlyAspGlyGlnLeuLeuLeuLys, corresponding to amino acids 388-401, identified EST R08946 as containing 100% identity to the query.
[0101]
The 100% identity between the portion of EST 248534 and the three peptide fragments of the purified protein strongly suggested that the cDNA contained within EST 248534 represents a portion of the nucleotide sequence for the coding region of the purified protein. The source of the semaphorin receptor cDNA was identified using a phage library screening method and PCR primers based on EST 248534.
[0102]
The oligonucleotide primer had the following nucleotide sequence:

  PCR isolation and amplification methodologies were performed using a panel of human tissue cDNA phage libraries as templates for PCR reactions. The PCR reaction mixture was prepared in a final reaction volume of 30 μl, 1 μl of phage library stock, 0.3 μM final concentration of PCR oligonucleotide primers, 1 × PC2 buffer (Ab Peptides, Inc., St. Louis, Mo.), dATP, dCTP. , DGTP, dTTP (Pharmacia Biotech) 0.2 mM, 16: 1 mixed Klen-Taq / Vent polymerase (Klen-Taq polymerase, Ab Peptides, Inc. and Vent polymerase, New England Biolabs, Beverly, Mass.) 0.2 μl Included. PCR reaction cycles were performed using Strabogene, Robocycler 96, La Jolla, CA, 1 cycle at 98 ° C for 5 minutes; 30 cycles at 98 ° C for 45 seconds; 30 cycles at 68 ° C for 45 seconds; 30 cycles at 72 ° C for 45 seconds Cycle; and one cycle at 72 ° C. for 5 minutes. CDNAs in several libraries were positively identified as containing DNA encoding purified VESPR protein based on the appearance of appropriately sized DNA bands in electrophoretic PCR products.
[0103]
Two of the phage libraries, human foreskin fibroblasts and human dermal fibroblasts, were selected for further analysis. The library was seeded according to established methods and probed with a radiolabeled random primer probe derived from a PCR amplification product using EST 248534 as a template. The PCR conditions used to obtain the amplification products were as described above, and the probe was generated using the Prime-IT II random primer labeling kit from Stratagene, La Jolla, California. About 1 x 10⁶  Using a cpm / ml purified probe, a human foreskin phage library on a nylon membrane filter was prepared in 10 × Denhardts solution, pH 7.5, 50 mM Tris, 0.9M NaCl, 0.01% sodium pyrophosphate. Probed overnight at 63 ° C. in hybridization buffer of 1% sodium dodecyl sulfate and 200 μg / ml denatured fragmented salmon sperm DNA. After probing, the probed membranes were once scanned at 63 ° C. once for 6 × SSC, 0.1% SDS for 20 minutes, once for 2 × SSC, 0.1% SDS for 20 minutes, once for 1 × SSC, 0. Washed with 1% SDS for 20 minutes and once with 0.1 × SSC, 0.1% SDS for 20 minutes. The probed and washed filters were exposed to X-omat AR X-ray film (Eastman-Kodak Corp.) overnight. Four overlapping cDNAs were identified. Overlapping DNA was used with sequenced trypsin digested protein fragments to complete and confirm the VESPR coding sequence as shown in SEQ ID NO: 1 and the amino acid sequence presented in SEQ ID NO: 2.
Example 6
Monoclonal antibody against A39R semaphorin
This example illustrates a method for preparing an antibody against A39R semaphorin. Purified A39R / Fc was prepared as described in Example 1 above. The purified protein was used to generate antibodies against A39R semaphorin as described in US Pat. No. 4,411,993. Briefly, mice were immunized with A39R / Fc at 10 μg at 0, 2 and 6 weeks. The first immunization was performed using Vaxcell, Inc. Of TITERMAX adjuvant and subsequent immunizations were prepared with incomplete Freund's adjuvant (IFA). At 11 weeks, mice were IV boosted with 3-4 μg A39R / Fc in PBS. Three days after IV boost, spleen cells were harvested and fused with an Ag8.653 myeloma fusion partner using 50% aqueous PEG 1500 solution. Hybridoma supernatants were screened for A39R antibody by a dot blot assay for A39R / Fc and inappropriate Fc protein.
Example 7
Northern blot analysis of tissues expressing semaphorin receptors
The following describes a Northern blot experiment performed to identify tissues and cell types that express the VESPR polypeptides of the invention. The results confirm the cell binding results obtained using flow cytometric analysis and A39R / Fc fusion protein.
[0104]
As described in Example 5, as a result of the EST database search, EST (EST 248534), which is considered to be a partial clone of VESPR of SEQ ID NO: 2, was discovered. Riboprobe templates were generated using PCR techniques and oligonucleotide primers based on nucleotides 1-372 of EST 248534. The upstream and downstream primers containing nucleotides 1-372 of EST 248534 had the following sequences:

The underlined portion is the T7 site.
[0105]
Two primers were used and the PCR product was isolated from EST 248534 and amplified for use in generating riboprobes. Using Ambion's MAXIscript SP6 / T7 kit, 3 μl RNAse free water, 2 μl 10 × transcription buffer, 10 mM dATP / dCTP / dGTP 1 μl each, 5 μl 5 ′ / 3 ′ EST 248534 PCR product, 5 μl Amersham [ α³²Riboprobes were generated by combining P] UTP 10 mCi / ml, 2 μl of T7 RNA polymerase at room temperature. The combination was microcentrifuged, briefly spun and incubated at 37 ° C. for 30 minutes. Thereafter, 1 μl of DNAse was added to the mixture and reacted at 37 ° C. for 15 minutes. The reaction product was passed through two column volumes of G-25 packing (Boehringer). 1 μl of riboprobe was counted for 1 minute with a scintillation counter, and cpm / ml was measured.
[0106]
Polyadenylated RNA from various cell lines is fractionated on a 1.2% agarose formaldehyde gel and the RNA is blotted onto a Hybond nylon membrane (Amersham, Arlington Heights, Ill.) To generate a Northern blot did. A standard Northern blot generation method was used as described in Maniatis (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Lab. Press, 1989). Total RNA multiple tissue northern blots were purchased commercially (BioChain Institute, Inc., San Leandro, CA, catalog numbers 021001, 021002, 021003).
[0107]
Northern blots were prepared using 50% formamide hybridization solution (30 ml 20 x SSC, 2 ml 100 x Denhardt's reagent, 1 ml 10 mg / ml denatured fragmented salmon sperm DNA, 50 ml 100% formamide and 20 ml Prehybridization in 10% SDS). Total RNA blots were prehybridized at 42 ° C for 4 hours, and poly A + RNA blots were prehybridized at 63 ° C for 4 hours. Riboprobe⁶  Added to clean hybridization solution (same as prehybridization solution) at a count of cpm / ml. The prehybridization solution was removed from the blot and the hybridization solution and riboprobes were added to the blot. Hybridization proceeded overnight with gentle shaking. Total RNA blots were hybridized at 63 ° C. and poly A + RNA blots were hybridized at 63 ° C.
[0108]
The probed total RNA blot was once at 42 ° C. for 30 minutes in 2 × SSC containing 0.05% SDS and once at 55 ° C. for 30 minutes in 2 × SSC containing 0.05% SDS; 63 ° C. Twice, washed with 0.1 x SSC containing 0.1% SDS for 30 minutes; washed 3 times with 0.1 x SSC containing 0.1% SDS for 30 minutes and then exposed to X-ray film did. The poly A + blot was washed once at 63 ° C. with 2 × SSC solution containing 0.05% SDS for 30 minutes and once with 1 × SSC containing 0.1% SDS for 30 minutes and then X-rayed Exposed to film.
[0109]
The results of exploring the Northern blot and visualization of the resulting X-ray film for positive binding probes confirms that VESPR is expressed in the same cells that showed positive binding in flow cytometry experiments. Hybridizing RNA was detected in MP-1, HFF and CB23 cells. Primary tissues that showed positive RNA included heart, brain, lung, spleen and placenta. No RNA was detected in RAJI cells.
Example 8
Generation of AHV semaphorin Fc fusion protein
The following describes preparing an AHV semaphorin / immunoglobulin fusion protein (AHV sema / Fc). The method involved preparing a DNA construct encoding the fusion protein, transfecting a cell line with the DNA construct, and collecting the supernatant from the transfected cells.
[0110]
DNA encoding AHV-sema is described in Ensser et al. Gen. Vir. 76: 1063-1067, 1995. Using the PCR technique and a synthetic oligonucleotide primer based on the published AHV-sema sequence from Alserafin herpesvirus DNA strain WC11 (Plowright, W. et al., Nature 188: 1167-1169, 1960), AHV -DNA encoding sema amino acids 70-653 was isolated and amplified. A Spe 1 site was introduced by the upstream oligonucleotide primer. A Not 1 site was introduced downstream of the stop codon by a downstream oligonucleotide primer. The general method used to isolate soluble AHV sema is described by Spriggs et al., J. MoI. Virology, 70: 5557 (1996).
[0111]
A restriction fragment containing an immunoglobulin mutein Fc region as described in Goodwin et al., Cell 73: 447-456, 1993 is described in the murine IL-7 signal peptide and US Pat. No. 5,011,912. FLAG^TMLigated into an expression vector (pDC409) containing the octapeptide. The encoded PCR amplified AHV sema DNA is then mutated with the human protein Fc region, murine IL-7 signal peptide and FLAG.^TMTwo-way ligation was performed in an expression vector containing the peptide. The resulting DNA construct was transfected into the monkey kidney cell line CV-1 / EBNA (with co-transfection of pSV3neo). After 7 days in culture with medium containing 0.5% low immunoglobulin bovine serum, 0.2% azide solution was added to the supernatant and the supernatant was filtered through a 0.22 μm filter. Subsequently, approximately 1 liter of culture supernatant was passed through a BioCad Protein A HPLC protein purification system using a 4.6 x 100 mm Protein A column (POROS 20A from PerSeptive Biosystems) at 10 ml / min. The Protein A column binds to the Fc portion of the fusion protein in the supernatant, immobilizes the fusion protein, and allows other components in the supernatant to pass through the column. The column was washed with 30 ml PBS solution and the bound fusion protein was eluted from the HPLC column with citric acid adjusted to pH 3.0. The eluted purified fusion protein was neutralized with 1M HEPES solution at pH 7.4 at the time of elution.
Example 9
Expression of recombinant semaphorin receptor
With the semaphorin receptor (VESPR) amino acid sequence of the protein purified as described in Example 5 and the information and radiolabeled probe obtained from the EST database search, also as described in Example 5. The cDNA obtained using the hybridization methodology of is used to generate cDNA and to transfect cells with the cDNA to allow expression of the recombinant VESPR polypeptide.
[0112]
The cDNA in the DC409 expression vector derived from pDC406 is transfected into CV1 / EBNA cells using standard techniques (McMahon et al., EMBO J. 10: 2821, 1991). More specifically, CV1 EBNA cells were placed 2 x 10 per 10 cm plate in 10 ml Dulbecco's minimal essential medium (medium) supplemented with 10% fetal calf serum.⁶Spread by cell density. Cells are allowed to attach overnight at 37 ° C. The medium is replaced with 1.5 ml of medium containing a DNA mixture containing 66.7 μM chloroquine and 5 μg of cDNA encoding VESPR. Medium containing 175 μl and 25 μl DEAE dextran is added to the cells. Cells and cDNA are incubated for 5 hours at 37 ° C. The cDNA mixture is removed and cells are shocked with 1 ml of fresh medium containing 10% DMSO for 2.5 minutes. The medium is replaced with fresh medium and the cells are allowed to grow for at least 3 days.
[0113]
To recover the soluble form of VESPR, the supernatant containing the soluble form is collected and the VESPR protein is recovered using HPLC or affinity chromatography techniques. To recover the membrane-bound form of VESPR, the transfected cells are harvested, fixed with 1% paraformaldehyde, washed and used in their intact form.
Example 10
VESPR binding studies
To examine the binding properties of the receptor polypeptides of the invention, cells expressing the membrane-bound VESPR extracellular domain were expressed by Goodwin et al., Cell 73: 447-456 (1993) and Spriggs et al., J Virol 70: 5557 ( 1996)) was performed by subjecting it to a slide binding assay as described.
[0114]
A pDC409 expression vector derived from pDC406 (McMahon et al., EMBO J. 10: 2821, 1991) but with a single Bgl 2 was selected for the cloning method. VESPR cDNA encoding amino acids 19-1100 was subcloned into the pDC409 expression vector through the Sal 1 (5 ') and Not 1 (3') sites to form a DNA construct.
[0115]
CV-1 / EBNA cells were transfected via DEAE / dextran with 2 μg of VESPR cDNA (encoding amino acids 19-1100) in pDC409 (Giri et al., EMBO J. 13: 2822, 1994). Transfected cells were cultured for 3 days and CV-1 / EBNA cell monolayers were incubated with 1 μg / ml of A39R / Fc, AHV sema / Fc, or control Fc protein. Thereafter, the incubation cells are washed, and¹²⁵Incubated with I-labeled mouse anti-human IgG (Jackson Immunoresearch, West Grove, PA). After extensive washing, the cells were fixed, dipped in a photographic emulsion and developed as described in Gearing et al., EMBO J 8: 3667-3676 (1989). Positive binding was determined by the presence of exposed or dark silver grains covering cells expressing VESPR bound to Fc protein.
Example 11
Flow cytometry and inhibitory binding studies
The following describes flow cytometric analysis of CB23 cells for binding to A39R / Fc fusion protein (Example 1) and AHV sema / Fc fusion protein (Example 9). Also described below is a study aimed at measuring the inhibition of AHV sema and A39R binding at an excess of A39R / polyHis fusion protein prepared as described in Example 2. .
[0116]
Flow cytometry analysis starts with about 1 x 10⁶CB23 cells were incubated in FACS buffer containing 3% normal goat serum and 3% normal rabbit serum for 30 minutes on ice to block non-specific binding. A39R / Fc, AHV sema / Fc and a portion of control Fc protein were added at various concentrations and incubation was continued for 30 minutes. Cells were washed and then incubated with phycoerythrin-conjugated Fc specific anti-human IgG in FACS buffer. Cells were washed and analyzed on a FACScan, Becton Dickinson, Bedford, Massachusetts. The results showed positive binding of AHV semaphorin and A39R semaphorin.
[0117]
Binding inhibition studies are about 1 x 10⁶CB23 cells were incubated in FACS buffer for 30 minutes on ice. A39R / polyHis and control His protein were added to different samples at various concentrations and incubation was continued for another 30 minutes. Thereafter, A39R / Fc or AHV Sema / Fc was added to the incubated cells at various concentrations and the incubation was continued for another 30 minutes. Cells were washed and then incubated with phycoerythrin-conjugated Fc specific anti-human IgG in FACS buffer. Cells were washed again and analyzed on a FACScan. The results demonstrated that using A39R / polyHis, A39R and AHV sema were completely inhibited, but not heterologous His-containing proteins.
Example 12
Human B cell aggregation with A39R semaphorin
To examine the human B cell response to A39R semaphorin, human tonsil B cells were purified as described in Spriggs et al., J Exp Med 176: 1543 (1992). A39R / polyHis fusion protein was prepared as described in Example 2. A solution of A39R / polyHis fusion protein is prepared such that the final A39R concentration is 1 μg / ml and the A39R / polyHis fusion protein solution is about 10⁵In vitro cultures of purified B cells were incubated. Incubation continued for about 24 hours resulting in cell aggregation. Prior to adding the fusion protein to the culture, cell aggregation was blocked when a 10-fold molar excess of monoclonal antibody against A39R, prepared as described in Example 6, was added to the fusion protein preparation. Furthermore, aggregation was blocked when A39R semaphorin was heat inactivated prior to addition to the culture.
[0118]
This study confirms that VESPR is expressed on B cells and that the interaction between A39R and VESPR results in B cell aggregation. B cell aggregation indicates B cell activation. Activated B cells are known to secrete cytokines, produce antibodies, or become antigen presenting cells.
Example 13
Mouse dendritic cell and macrophage aggregation with A39R semaphorin
To examine dendritic cell and macrophage responses to A39R, mouse cell cultures were contacted with A39R semaphorin and the effects of the combination were observed. Murine dendritic cell cultures containing macrophages were obtained by immunizing mice with Flt-3 and isolating and purifying cells as described in Maraskovsky et al., J Exp Med 184: 1953 (1996). It was.
[0119]
Briefly, female C57B1 / 6 mice were injected once daily with a solution of 10 μg Flt3L and 1 μg mouse serum albumin in 100 μl PBS for 9-10 consecutive days. NH after immunization₂Single cell suspensions of the spleen were prepared by disrupting spleen tissue between ground glass slides and depleting red blood cells in the presence of Cl. Residual cells were incubated with mAbs against Thy-1, B220, NK1.1, and TER119 and then incubated with 10% rabbit complement. The incubated cells were then washed and the remaining mAb coated cells were removed using anti-immunoglobulin (Ig) coated magnetic beads. Residual enriched cells were cultured or sorted for diverse cell populations.
[0120]
Cells selected for sorting were stained with anti-CD11c and anti-CD11b and sorted for C and D / E populations as described by Maraskovsky et al., J Exp Med 184: 1953-1962, 1996.
[0121]
A39R / polyHis fusion protein was prepared as described in Example 2. The A39R / polyHis fusion protein solution was about 10 at a final A39R concentration of 1 μg / ml.⁵Incubated with in vitro cultures of categorized or depleted mouse cells. Within 4-6 hours, the cells began to aggregate. Aggregation is blocked when a 10-fold molar excess of a monoclonal antibody to A39R, prepared as described in Example 6, is added to the A39R / polyHis fusion protein preparation prior to addition of the fusion protein to the mouse cell culture. It was done.
[0122]
This study confirms that VESPR is expressed on dendritic cells and macrophages, and that the interaction between A39R and VESPR results in dendritic cell and macrophage aggregation.
Example 14
A39R semaphorin upregulates CD69 activating antigen
To examine the effect of A39R semaphorin on cultured dendritic cells, mice were injected daily with Flt3-L preparation for 9 days. Mouse dendritic cells were harvested and then cultured for 5 days in medium containing 10% FBS and 20 ng / ml GM-CSF.
[0123]
On day 5, 1 μg / ml of A39R / polyHis fusion protein was added to the culture. On day 6, cells were stained with diagnostic antibody. According to the results of the diagnostic antibody staining experiment, CD11c⁺, CD11b⁺It was shown that the amount of CD69 activating antigen expressed by cells (dendritic cells) was increased, thus demonstrating that the interaction of A39R semaphorin and its receptor up-regulates CD69 expression.
[0124]
When the fusion protein was heat inactivated, the fusion protein had no effect on the CD69 antigen. The typical change in average fluorescence intensity between unstained and stained cells was between approximately 500 to 2500 channels. Again, these results show a significant effect of the interaction between A39R semaphorin and its membrane-bound receptor on the regulation of CD69 activation antigen, a transient and early expression marker of cell activation. Prove it.
Example 15
Evaluation of the effect of A39R on IL-12 production
To study the role of A39R in the production of IL-12 from mouse spleen cells, mice were immunized with flt3-L and dendritic cells were generated, harvested and purified as described in Example 13. did.
[0125]
Approx. 5 x 10⁵Cells / 0.5 ml of purified unsorted dendritic cells were incubated in modified DMEM medium in the presence of another (1 × 10⁶500 ng / ml): 20 ng / ml muGM-CSF (Immunex, Seattle, WA), 20 ng / ml γ-IFN (Genzyme, Boston, Mass.), 10 μg / ml SAC (CalBiochem, La Jolla, CA). Each cell preparation was further treated with 1 μg / ml A39R / polyHis fusion protein alone or in combination with 1 μg / ml or 0.1 μg / ml muCD40L trimer (Immunex, Seattle, WA). Cultivation is humidified at 37 ° C and 10% CO in air.₂Incubated for 16-18 hours. Following incubation, the viability of each group of cultured cells was measured and the supernatants were collected and assayed for muIL12 (P70) using an ELISA assay kit (Genzyme, Boston, Mass.). MuIL12 levels were calculated with reference to a standard curve constructed with recombinant cytokines.
[0126]
ELISA studies, in particular, demonstrated that A39R interacts with its receptor and synergizes with interferon and SAC in the production of IL-12 from unsorted mouse dendritic cells. This in vivo IL-12 induction promotes natural killer cell activation and gamma interferon production and contributes to upregulate gamma interferon sensitive cytokines.
Example 16
Testing the effect of A39R on the regulation of MHC class II and CD86 on monocytes
The following experiments describe upregulation of MHC class II and CD86 by interaction of A39R with its membrane-bound receptor. Peripheral blood from healthy donors was diluted 1: 1 with low endotoxin PBS at pH 7.4 and room temperature. Subsequently, 35 ml of diluted blood was layered on 15 ml of Isolymph (Gallard and Schlesinger Industries, Inc., Carl Place, NY) and centrifuged at 2200 rpm for 25 minutes at room temperature. The plasma layer was saved. The PBMC layer was collected and washed 3 times to remove Isolymph. Washed PBMC were resuspended in X-Vivo 15 serum free medium (BioWhittaker, Walkersville, MD) and added to the T175 flask. The flask was previously coated with 2% gelatin (Sigma, St. Louis, MO) and pretreated with a stored plasma layer for 30 minutes. PBMC at 37 ° C, 5% CO₂For 90 minutes and then gently rinsed 3 times with a 10 ml wash of low endotoxin PBS. Adherent monocytic cells were harvested by incubating the cells in enzyme-free dissociation buffer (Gibco, BRL) and washing the cells many times in PBS. Monocytes are centrifuged at 2500 rpm for 5 minutes, counted, and 5 x 10 in 1 ml.⁵Cells / well were arranged in 24-well plates. The culture was 95% pure.
[0127]
Purified monocyte cells were cultured for 7-9 days in the presence of 20 ng / ml GM-CSF and 100 ng / ml IL-4 in order to differentiate the cells into a more dendritic cell-like phenotype. On days 7-9, cultures were treated with 1 μg / ml A39R / polyHis or control polyHis containing protein and the next day cells and supernatants were harvested for analysis.
[0128]
In flow cytometry experiments to examine monocyte-derived dendritic cell surface markers, cells were stained with conjugated mAbs directed against specific proteins. Staining showed that in most peripheral blood donors tested, A39R treatment down-regulated CD86 and MHC class II expression on these cells. Since CD86 and MHC class II molecules are markers of enhanced antigen presentation by dendritic cells, their down-regulation suggests an immunosuppressive effect of the interaction between A39R and its receptor on this cell population Is done.
Example 17
CD54 upward control
The following describes the effect of interaction between A39R semaphorin and its receptor on purified monocytes, and more particularly the effect of CD54 expression on monocytes after incubation with semaphorin. Freshly isolated monocytes from peripheral blood donors were purified as described in Example 16 except that they were placed in overnight culture in the presence of A39R / polyHis or control protein.
[0129]
After overnight culture, flow cytometry was performed using mAbs directed against cultured cells and monocyte-specific cell surface markers. In all donors tested, the level of CD54 surface expression was enhanced in the presence of A39R, but not in the presence of heat-inactivated A39R. Similarly, CD54 surface expression was not enhanced in cultures containing control protein.
[0130]
CD54, also known as ICAM-1, is an adhesion molecule and its increased expression is considered to indicate cell activation. These data indicate that it is possible to activate newly isolated human monocytes by promoting the interaction of A39R with its receptor.
Example 18
Cytokine induction from newly isolated human monocytes
Freshly isolated human monocytes were purified as described in Example 16 and cultured as described in Example 17. After overnight incubation with A39R / polyHis, monocyte supernatants were examined for the presence of pro-inflammatory cytokines. In all tested donors, IL-6 and IL-8 were induced by A39R protein. Heat inactivated A39R and control protein did not induce IL-6 or IL-8. Furthermore, cytokine production was blocked by including a mAb directed against A39R.
[0131]
The results of this experiment demonstrate that A39R, or a homologue of this protein, may induce cytokine production by newly isolated monocytes through interaction with its receptor. Conveniently, a soluble form of VESPR can be used to react with A39R or a homologue thereof and inhibit the pro-inflammatory activity of monocytes.
Example 19
Monocyte aggregation research
To examine the response of human monocytes to the interaction of semaphorin with its receptors on monocytes, monocytes were purified as described in Example 17 and A39R as described in Example 2. / PolyHis fusion protein was prepared. The fusion protein and purified cultured monocytes were incubated. Incubation continued for 20 hours resulting in monocyte aggregation. Results demonstrated in Example 17 suggested that the observed monocyte aggregation occurred as a result of CD54 upregulation. However, other factors may also contribute to aggregation.
[0132]
This study confirms that the semaphorin receptor of the present invention is expressed on monocytes and that the interaction between A39R and VESPR results in monocyte aggregation. As with B cells, monocyte aggregation indicates activation of monocytes.
Example 20
Monoclonal antibody against VESPR
This example illustrates a method for preparing an antibody against a VESPR polypeptide. Purified VESPR polypeptide is prepared as described in Example 10. The purified protein is used to generate antibodies against VESPR as described in US Pat. No. 4,411,993. Briefly, mice are immunized with 10 μg with VESPR at 0, 2 and 6 weeks. The first immunization was performed using Vaxcell, Inc. Of TITERMAX adjuvant and subsequent immunization is prepared with incomplete Freund's adjuvant (IFA). At 11 weeks, mice are IV boosted with 3-4 μg VESPR in PBS. Three days after IV boost, spleen cells are harvested and fused with an Ag8.653 myeloma fusion partner using 50% aqueous PEG 1500 solution. Hybridoma supernatants are screened for VESPR antibodies by a dot blot assay for VESPR and inappropriate Fc protein.
[Sequence Listing]

Claims (17)

A polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and capable of binding to semaphorins. below:
Fragments of sequences of (b) (a); ( a) to no amino acid x ₁ of SEQ ID NO: 2 944, where x ₁ is Ru 1 or 35 der;
A polypeptide comprising an amino acid sequence selected from the group consisting of: wherein the polypeptide is capable of binding to semaphorins. A polypeptide capable of binding to semaphorins comprising the amino acid sequence of amino acids x ₁ to 944 of SEQ ID NO: 2, wherein x ₁ is 1 or 35. DNA encoding a polypeptide that can bind to semaphorins, comprising the following (a) a nucleic acid sequence of SEQ ID NO: 1;
(B) DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2;
(C) DNA encoding a polypeptide consisting of the amino acid sequence of amino acids x ₁ to 944 of SEQ ID NO: 2, wherein x ₁ is 1 or 35;
(D) DNA that is completely complementary to DNA capable of hybridizing to the DNA of (a) under stringent conditions;
(E) DNA that degenerates to DNA of (a)-(d) by degeneracy of the genetic code; and
(F) DNA completely complementary to the DNA of (a)-(e);
The DNA selected from the group consisting of: DNA encoding a polypeptide capable of binding to semaphorins,
(A) DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2;
(B) DNA encoding a polypeptide consisting of an amino acid sequence of amino acids x ₁ to 944 of SEQ ID NO: 2, wherein x ₁ is 1 or 35;
(C) DNA that degenerates to DNA of (a) or (b) by degeneracy of the genetic code; and
(D) DNA that is completely complementary to the DNA of (a)- (c)
The DNA selected from the group consisting of: A DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. A DNA encoding a polypeptide that can bind to semaphorins consisting of the amino acid sequence of amino acids x ₁ to 944 of SEQ ID NO: 2, wherein x ₁ is 1 or 35. DNA encoding a fragment of the amino acid sequence of amino acids x ₁ to 944 of SEQ ID NO: 2, wherein x ₁ is 1 or 35, wherein said fragment is capable of binding to semaphorins, DNA. A fusion protein capable of binding to semaphorins,
The amino acid sequence of amino acids x ₁ to 944 of SEQ ID NO: 2, wherein x ₁ is 1 or 35; and
The amino acid sequence of a peptide that facilitates purification of the fusion protein, a signal or leader peptide, and / or a peptide that oligomerizes the fusion protein;
The fusion protein comprising: 10. The fusion protein of claim 9 , wherein the peptide is selected from the group consisting of an antibody Fc region, [alpha] -factor leader, polyHis tag, antigenic identifying peptide, and FLAG <(R)> tag. A fusion protein capable of binding to semaphorins,
The amino acid sequence of amino acids x ₁ to 944 of SEQ ID NO: 2 or the amino acid sequence thereof, wherein x ₁ is 1 or 35, and wherein the fragment is capable of binding to semaphorins; and of the fusion protein The amino acid sequence of a peptide that facilitates purification, a signal or leader peptide, and / or a peptide that oligomerizes the fusion protein;
The fusion protein comprising: 12. The fusion protein of claim 11 , wherein the peptide is selected from the group consisting of an Fc region of an antibody, an α-factor leader, a polyHis tag, an antigenic identifying peptide, and a FLAG® tag. A DNA encoding the fusion protein according to any one of claims 9-12 . A recombinant expression vector comprising the DNA of any one of claims 4-8 or 13 . A host cell transformed with the expression vector of claim 14 . 16. A method for preparing a polypeptide comprising culturing the host cell of claim 15 under conditions that promote expression of the polypeptide. An antibody immunoreactive with the polypeptide of any one of claims 1-3 .