JP4552076B2 - IL-9 receptor variant useful for the treatment and diagnosis of atopic allergy including asthma and related diseases - Google Patents

Abstract

This invention relates to the diagnosis, treatment and methods for discovery of new therapeutics for atopic asthma and related disorders based on variants of Asthma Associated Factor (2). One embodiment of the invention is a variant of AAF2, wherein codon 173 is deleted resulting in the loss of glutamine 173 from the mature protein precursor. This single amino acid deletion results in a non-functional AAF2 protein and therefore the presence of this phenotype should be associated with less evidence of atopic asthma. Correspondingly, the lack of susceptibility to an asthmatic, atopic phenotype is characterized by the loss of glutamine at codon 173. The invention includes isolated DNA molecules which are variants of the wild type sequence as well as the proteins encoded by such DNA and the use of such DNA molecules and expressed protein in the diagnosis and treatment of atopic asthma.

Description

ゲノムDNAは、生物の細胞中に存在するすべてのDNA配列を含む。これは、メッセンジャーRNA（「mRNA」）に「転写」される。相補的DNA（「cDNA」）は、mRNAの逆転写により作成される、mRNAの相補的なコピーである。ゲノムDNAと異なり、mRNAとcDNAは両方とも、DNAのタンパク質コード領域またはポリペプチドコード領域（いわゆる「エキソン」）のみを含有する。ゲノムDNAはまた、「イントロン」を含有し、これはタンパク質をコードしない。
実際、真核生物遺伝子は、それによりコードされるタンパク質に対して断続的であり、イントロンにより中断されたエキソンからなる。RNAに転写された後、イントロンはスプライシングにより除去されて、成熟メッセンジャーRNA（mRNA）が形成される。エキソンの間のスプライス点は、典型的にはスプライシングプロセスのシグナルとして作用する共通配列により決定される。スプライシングは、一次RNA転写産物からのイントロンの削除と、切断されたイントロンのいずれかの側にある残りのRNAの末端の結合もしくは融合からなる。イントロンの有無、イントロンの組成、および遺伝子当たりのイントロンの数は、同じ種の株の間でも異なり、同じ基本的な機能遺伝子を有する種の間でも異なることがある。ほとんどの場合にイントロンは必須ではなく害はないと考えられているが、この分類は必ずしも絶対的ではない。例えば１つの遺伝子のイントロンは、別のエキソンであることもある。ある場合には、スプライシングの代替的なまたは異なるパターンが、DNAの同じ１つのストレッチから、異なるタンパク質を形成することがある。実際、イントロンの構造的特徴と基礎的スプライシング機構は、異なる種類のイントロンの分類の基礎となる。
エキソンについて、これらは、別個のドメインまたはモチーフ、例えばタンパク質の機能性ドメイン、折り畳みドメイン、もしくは構造的エレメント；または短いポリペプチド配列、例えば逆ターン、ループ、グリコシル化シグナルおよび他のシグナル配列、または非構造ポリペプチド、リンカー領域、に対応する。本組合せ法のエキソンモジュールは、天然に存在するエキソン配列または突然変異（例えば、点突然変異、末端切断、融合）を受けている天然に存在するエキソン配列に対応する核酸配列を含有してもよい。
DNAの操作にもどると、DNAは、ある既知の部位でDNAを切断する「制限酵素」およびDNAをつなぐDNAリガーゼを使用して、切断、スプライス、および他の操作をすることができる。このような技術は当業者に公知であり、例えばSambrookら、Molecular Cloning:A Laboratory Manual,第２版、Cold Spring Harbor Laboratory Press（1985）、またはAusubelら、Current Protocols in Molecular Biology,John Wiley & Sons,Inc.（1994）に記載されている。
特定のサイズおよび配列のDNAは、次に「レプリコン」（これは、プラスミド、コスミド，またはウイルスのような、それ自身の制御下で複製ができる遺伝成分である）中に挿入することができる。「組換えベクター」または「発現ベクター」は、DNAセグメントが挿入されるレプリコンであり、DNAの発現（すなわち、DNAによりコードされるタンパク質の産生）を可能にする。発現ベクターは、実験室で作製されるか、他の実験室から入手できるか、または市販品を購入することができる。
組換えベクター（当該分野において種々の名前で知られている）は、「形質転換」として一般的に知られているプロセスにより宿主中に導入される。形質転換は、任意の方法（感染、直接摂取、形質導入、Ｆ−接合、微量注入法、または電気穿孔法）により、宿主細胞中に外来DNAセグメントを移動することを意味する。
単細胞宿主細胞（組換え宿主細胞、細胞および細胞培養物として種々の名前で知られている）は、細菌、酵母、昆虫細胞、植物細胞、哺乳動物細胞、およびヒト細胞を含む。特に好適な実施態様において、宿主細胞は，大腸菌（E.coli）、シュードモナス（Pseudomonas）、バシラス（Bacillus），ストレプトミセス（Streptomyces），酵母、CHO、R1-1、B-W、LH、COS-J、COS-7、BSC1、BSC40、BMT10、およびS69細胞を含む。酵母細胞は特に、サッカロミセス（Saccharomyces），ピキア（Pichia）、カンジダ（Candida）、ハンゼヌラ（Hansenula）、およびトルロプシス（Torulopsis）を含む。
当業者は認識できるように、宿主細胞によるDNAセグメントの発現は、適切な制御配列または成分が必要である。制御配列は、使用される宿主細胞により変化するが、例えば原核生物では、プロモーター、リボゾーム結合部位、および／または転写停止部位がある。真核生物では、このような制御配列には、プロモーターおよび／または転写停止部位がある。当業者は認識できるように、これらの制御配列を注意深く選択し配置することにより、ポリペプチドの発現を増強、すなわち標準的なレベルより上昇させることができる。
他の実施態様において、使用されるプロモーターには、ヒトサイトメガロウイルス（CMV）プロモーター、テトラサイクリン誘導性プロモーター、サルウイルス（SV40）プロモーター、モロニーマウス白血病ウイルス長い末端反復配列（LTR）プロモーター、糖質コルチコイド誘導性マウス乳癌ウイルス（MMTV）プロモーター、ヘルペスチミジンキナーゼプロモーター、マウスおよびヒトアクチンプロモーター、HTLV1およびHIV IL-9 5’フランキング領域、ヒトおよびマウスIL-9受容体5’フランキング領域、細菌tacプロモーターおよびキイロショウジョウバエ（Drosophila）熱ショックタンパク質足場結合領域（SAR）エンハンサー成分がある。
DNAは、ペプチド、オリゴペプチド、およびタンパク質のようなある長さのポリペプチドとして発現される。ポリペプチドはまた、グリコシル化、アセチル化、リン酸化などの翻訳的修飾も含む。
本発明に関係のある他の分子生物学的方法は、「連鎖解析」である。連鎖解析は、形質または疾患と相関する染色体または染色体領域を同定するための分析方法である⁴⁷。染色体は、その上に遺伝子が組み立てられる遺伝の基本的単位である。遺伝子以外に、当業者は、染色体上に「DNAマーカー」を証明している。DNAマーカーは、その同定と配列決定が容易に行なうことができる、DNAの既知の配列である。連鎖解析法は、疾患遺伝子（例えば喘息の感受性に関係する遺伝子）を特定の染色体にマッピングすることに応用されている^47,48。
本出願人らは、上記および下記のすべての文献を参照により本明細書に組み込む。
本明細書の説明および開示された発明の実施から、本発明の他の実施態様は当業者には明らかであろう。明細書および実施例は単に例示のためであり、本発明の範囲は請求の範囲によってのみ示されるものである。
背景情報は提供したので、本出願人らは、ここで本発明の好適な面を説明する。
実施例１
IL-9受容体転写産物多型の同定
Philadelphia,Pennsylvania地域からの52人の集団を、喘息とアトピーに関してランダムに確認した。総血清IgEを、酵素結合免疫吸着アッセイ（ELISA、Genzyme、Cambridge、Massachusetts）により測定した。
ヒトIL-9受容体cDNAの構造型を評価するために、これらの52人の無関係のドナーからPBMCを単離し、PHAとPMAの存在下で培養した（実施例４に記載）。本出願人らの実験室の以前のデータは、分裂促進物質による刺激後６日目に１次培養物中のIL-9受容体メッセージの発現の動力学を証明した。従って本出願人らは、細胞を６日間培養し、この時点で細胞を採取し，実施例５に記載のようにそのRNAとDNAを単離した。
RNAを逆転写し、実施例５に記載のように完全長IL-9受容体cDNAに特異的なプライマーを使用してPCRにより増幅した。各個人からの増幅産物をTA PCRクローニングベクターにクローン化し、予測されるインサートを含有する10クローン（消化とゲル電気泳動により測定した）を完全に配列決定し、構造または配列の変化について解析した。
上記スクリーニングから７個の主要な変異体を同定した。これらのcDNAは、コドン173の欠失、エキソン４の欠失、エキソン５の２つの別の欠失、エキソン８の欠失、および成熟タンパク質のコドン344でARGからHISへの変異を有する完全長cDNAを示す。これらの遺伝子バックグランドのそれぞれに追加の変異体が存在する。Arg対立遺伝子は、8 Ser/4 Asn繰り返しと7 Ser/4 Asn繰り返しに関連し、His対立遺伝子は、9 Ser/4 Asn繰り返し、9 Ser/3 Asn繰り返し、および10 Ser/2 Asnに関連する。これらの変異体のすべてを、図１に記載する。
変異体を、クローン化cDNAの発現を指令するCMVプロモーターとその後のSV40ポリアデニル化シグナルを含有する真核生物発現ベクターpCEP4（Clontech）中にクローン化した。このベクターはまた、ベクターを含有し、おそらく、クローン化したcDNAをCMVプロモーターの制御下で発現する真核生物細胞の選択に使用される、ヒグロマイシンＢ耐性遺伝子を含有する。組換えプラスミドを、配列により解析し、正しいcDNAインサートを含有するプラスミドを、真核生物受容体細胞（例えば、実施例３に記載のシリアンハムスター繊維芽細胞TK-ts13、ヒト神経膠芽細胞腫T98G、ヒト骨髄白血病株TF-1、およびマウス骨髄前駆体細胞系32D）にトランスフェクトした。増殖とアポトーシスにおける、IL-9リガンドに対する応答として、機能を生物学的に評価した（実施例７と10）。
ARGおよびHIS変異体を含有する完全長IL-9受容体cDNAを行なった実験は、TK-ts13ハムスター繊維芽細胞またはヒトT98G神経膠芽細胞腫細胞である。細胞をトランスフェクトし、48時間後ヒト特異的カルボキシ末端抗体を使用して、ウェスタンブロットとin situ染色により解析した（Santa Cruz）（実施例８）。in situ解析は、ハムスターとヒト系の両方で，両方の型の受容体が発現されるらしいことを証明した（図11と図12）。興味深いことに、ヒトとハムスター系で両方の型のウェスタンブロットは同等レベルで発現され、TS1細胞系中でARGとHIS受容体型との間には移動パターンに差があり（図12）、これは、グリコシル化、リン酸化などの翻訳後修飾の差を示唆する。この生化学的な差は、機能の変化が表現型の変化を引き起こす機構かも知れない。
種々の置換の頻度を、一般的集団中の各変異体の普及率の、偏りのない推定値として使用した。アンケートにより遺伝子型を表現型と比較した。アレルギーと喘息の診断は、アンケートを再検討して、医師が行なった。Arg344対立遺伝子についてホモ接合性である個人は、ヘテロ接合性またはホモ接合性His344と比較した時、アレルギーと喘息の証拠を示す可能性が有意に低かった（図９）。
実施例２
IL-9受容体遺伝子のゲノム解析
アレルギーおよび／または喘息の人のゲノム解析を行うために、Ｘ／Ｙ偽常染色体領域上に位置する真のIL-9受容体遺伝子についてPCR特異的プライマーを作成し、かつ染色体９，10、16、および18上に位置する高度に保存されたIL-9受容体偽遺伝子を排除するように，以下の方策を設計した。まず、IL-9受容体遺伝子の２つの公表された偽遺伝子とゲノム配列の間で、配列アライメントを行なった。次に，真の遺伝子と偽遺伝子の間の分岐領域でプライマーを設計し、次にCoreill DNA Repository（Camden,NJ）から入手した単一の染色体特異的ハイブリッドを使用して、PCRにより解析した。プライマーが、ＸおよびＹハイブリッドから正しいサイズの生成物のみを産生するなら、これらをしっかりした増幅について最適化した。いくつかの場合に、分岐領域に向けられたプライマーはXY特異的ではなく、従って本出願人らは、IL-9受容体遺伝子配列に比較して偽遺伝子に対してミスマッチの数を多くするために、特定のプライマー中の追加の塩基の変異を導入した。表１は、XY特異的増幅についてのプライマーの配列と最適アニーリング温度を含む。XY増幅についてこれらのプライマーの特異性を、図13に示す。

これらのプライマーは、アトピー性喘息および関連疾患に対する感受性と耐性の診断に使用可能な、これらの遺伝子中のDNA配列の変異を解析するための新規の方法を示す。
この技術を使用して、IL-9受容体遺伝子中の配列の変異を検出するために、本出願人らの集団中の人についてDNA配列解析により、各エキソンを調べた⁴⁴。解析を繰り返して、配列多型をアーチファクトから区別した。受容体変異体とアレルギー性表現型との関連を図９に示す。受容体対立遺伝子の配列を図１〜８に示す。
実施例３
IL-9受容体発現とリガンド結合アッセイ
市販の技術を使用してサプライヤー（Pierce）の勧めに従って、精製した組換えIL-9および構造または機能がIL-9に似ている可能性のある化合物を、高比活性になるように蛍光標識した。ヒトMo7eとマウス32D細胞を増殖させ、37℃で、それぞれ10％（容量／容量）ウシ胎児血清、50mM ２−メルカプトエタノール、0.55mM L-アルギニン、0.24mM L-アスパラギン、および1.25mM L-グルタミンを補足したダルベッコ改変イーグル培地0.8ml、または10％（容量／容量）ウシ胎児血清、50mM ２−メルカプトエタノール、0.55mM L-アルギニン、0.24mM L-アスパラギン，および1.25mM L-グルタミンを補足したRPMIに再懸濁した。TF1.1（ヒトIL-9受容体を欠失したTF1細胞）、T98G、TK^-、およびマウス32D細胞（実施例７と10）を、後述のようにそのまま、またはヒトIL-9受容体構築物でトランスフェクション後に使用した。完全長または末端切断型のIL-9受容体の１つを含有するプラスミドDNAを、pCEP4プラスミド（Clontech）中にクローン化し、Qiagenカラム（Qiagen）を用いて遠心分離により精製した。電気穿孔法の直前に0.4cmのキュベット中の細胞にプラスミドDNA（50μg）を加えた。２重電気パルス（750Ｖ／74オーム／40マイクロファラドと100Ｖ／74オーム／2100マイクロファラド）後に、直ちに細胞を、IL-9を補足した新鮮な培地に希釈した。
ヒグロマイシンＢ（400μg/ml〜1.6mg/ml）で１４日間選択した後に、安定なトランスフェクション細胞が得られた。ヒグロマイシン耐性クローンを、実施例８に記載のようにウェスタンブロットとin situ染色によりIL-9受容体の発現について分析した。
蛍光顕微鏡でリアルタイムに、細胞受容体の結合を直接視覚化する。対照細胞（トランスフェクトしてない）と既知のIL-9受容体変異体のそれぞれでトランスフェクトした細胞で、結合とインターナリゼーションを経時的に追跡する。特異的結合を証明するために、過剰の非標識リガンドまたはブロッキング抗体を並行実験で使用する。
HAジタグを有するかまたは有さない、アミノ酸44〜270を含む可溶性IL-9受容体をまた、異なる型のヒト標識組換えIL-9とともにインキュベートする。異なる量のFLAGタグ付き（IBI）リガンドを0.5μgの可溶性受容体とともに、PBS中で室温で30分間インキュベートする。１μgの抗HA抗体または抗FLAGモノクローナル抗体（IBI）とともに、EBCバッファー（50mMトリス、ｐＨ7.5；0.1M NaCl；0.5％ NP40）を加え（300μl），氷上で１時間インキュベートする。各試料に40μlのプロテインＡセファロース溶液を加え、４℃で１時間混合した。試料を11,000×Ｇで１分間遠心分離し、ペレットを500μlのEBCで４回洗浄した。ペレットを26μlの２×SDSバッファーに溶解し、４分間沸騰させ、18％SDSポリアクリルアミドゲルで電気泳動した。ウェスタンブロットを、抗IL-9受容体抗体（Santa Cruz Inc.）またはFLAGタグ付きrhIL-9に対する抗FLAGモノクローナル抗体（IBI）で釣り上げた。治療薬としての候補物質を、リガンド結合の拮抗作用を測定することにより評価する。受容体発現を図11と14に示す。
実施例４
細胞の単離と培養
製造業者（Pharmacia Biotech,AB Uppsala Sweden）に従ってエンドトキシン試験を行なったFicoll-Paque PLUSを使用して密度勾配遠心分離により、健常ドナーからヒト末梢血単核細胞（PBMC）を単離した。同一遺伝子のヒト血清または熱不活性化FBSを最終濃度10％になるように補足したRPMI-1640（Bethesda Research Labs（BRL）,Bethesda,MD）７ml中で、PBMC（５×10⁶）、マウス脾臓細胞（５×10⁶）、または５×10⁶個のMo7e細胞を培養した。細胞を、刺激せずに、またはPMA ５μg/ml／PHA ５μg/ml、もしくはPHA ５μg/mlとrhIL2 50ng/ml（R & D Systems,Minneapolis,MN）で刺激して、37℃で24時間培養した。
実施例５
PCR産物のDNAとRNA単離、rtPCR、クローニング、および配列決定
Nicolaides and Stoeckert⁴⁶が記載したように、培養したPBMCからマイトジェンで６日間刺激後、細胞質RNAとゲノムDNAを抽出した。各供給源から１μgのRNAを，70℃で＞10分間変性し、次に2.5単位のSuperscriptII逆転写酵素（GIBCO,BRL）、１U/lのRNAseインヒビター、2.5mMオリゴd（T）16プライマー、１mMずつのdATP、dCTP、dGTP、dTTP、50mM KCl、10mMトリス塩酸（pH7.0）、25mM MgCl₂を使用して、37℃で１時間cDNAに逆転写（V+）した。偽逆転写反応物を陰性対照として使用した。
逆転写反応物の20分の１を、6.7mM MgCl₂、16.6mM（NH₄）₂SO₄、67mMトリス塩酸（pH8.8）、10mM ２−メルカプトエタノール、６％ DMSO、1.25mMずつのdNTP、2.5ＵのAmplitaq DNAポリメラーゼ、およびヒトcDNA IL-9エキソン２（フォワード5’-GCT GGA CCT TGG AGA GTG-3’）（配列番号１）とエキソン９（リバース5’-GTC TCA GAC AAG GGC TCC AG-3’）（配列番号２）に相当する300ngずつのオリゴヌクレオチドを、含有するPCR（50μl）中で使用した。反応混合物を以下のPCR条件に付した：95℃で120秒、次に94℃で30秒、58℃で90秒、72℃で90秒を３５サイクル。最後に、反応混合物を伸長のために、72℃で15分を１サイクルを行なった。
hIL-9受容体cDNAを示すPCR産物を、1.5％アガロースゲルでゲル電気泳動に付し、臭化エチジウム染色を用いて視覚化した。疑似逆転写反応の産物（ここでRNAの代わりにH₂Oを使用した）を、すべての実験で陰性対照増幅として使用した。
PCR産物を、TAクローニングベクター（Invitrogen,San Diego,CA）中にサブクローニングした。ヒトcDNAの増幅により、1614塩基対の産物が得られた。hIL-9受容体cDNAインサートを含有するプラスミドを、従来法により単離した（Sambrookら、（1989）Molecular Cloning:A Laboratory Manual,第２版、Cold Spring Harbor Laboratory Press,New York）。増幅後，各インサートを含みかつこれを囲むDNA配列を、PCR誘導性またはクローニング誘導性のエラーの測定のために、自動シーケンサー（ABI 377,Perkin Elmer）で、蛍光ジデオキシターミネーターを使用して、配列決定（Sangerら、1977,Proc.Natl.Acad.Sci.USA 74:5463）により解析した。クローニングおよび／またはポリメラーゼ誘導性の配列エラーの無いhIL-9受容体cDNAインサートを、発現ベクターにサブクローニングした。
実施例６
in vitroでのIL-9受容体構築物のクローニングと発現
hIL-9受容体を、エピソーム真核細胞発現ベクターpCEP4（Clontech）中にサブクローニングした。標準的方法を使用してCMVプロモーターに対してセンス配向で、pCEP4ポリリンカー中にBamH1-XhoI断片として，インサートをクローン化した（図10）。記載したように、構築物を細胞宿主中で発現させた。
実施例７
（Mo7e、32D、TF1.1、TK ^- ts13、およびT98G）を使用する細胞アッセイ
変異体IL-9受容体の機能の評価と、アトピー性喘息の治療に有効な化合物のスクリーニングのために、細胞系を使用した。IL-9により誘発される抗アポトーシスまたはベースライン増殖応答に拮抗する能力について、化合物を試験した。ある細胞系でベースライン抗アポトーシスまたは増殖応答がいったん確立されたら、３重の３回反復アッセイでの応答の統計的に有意な低下を、拮抗作用の証拠であると考えた。直接の観察、トリパンブルー染色（当業者に公知の技術）、または酸性ホスファターゼ活性の低下により、細胞傷害性から真の拮抗応答を区別した。他の増殖性物質（例えば、インターロイキン３またはインターロイキン４）に対して活性が証明されるかどうかを評価して、各化合物について拮抗作用の特異性を評価した。
適切な培地中で使用するために、組換えIL-9または構造もしくは機能がIL-9に似ている可能性のある化合物を精製し、調製した。推定アゴニストおよびアンタゴニストを、水、食塩水、またはDMSOと水で調製した。細胞はそのまま使用したか、または実施例３に記載のようにIL-9受容体構築物でトランスフェクトした後に使用した。増殖因子を24時間枯渇させた後に、細胞を可変量の精製IL-9または構造もしくは機能がIL-9に似ている可能性のある化合物を加えてまたは加えないでインキュベートした。
細胞数の指標として細胞内に存在する酸性ホスファターゼの量を測定する、Abacus Cell Proliferation Kit（Clontech,Palo Alto,CA）を使用して、細胞増殖を評価した。基質p-ニトロフェニルホスフェート（pNPP）を酸性ホスファターゼによりp-ニトロフェノールに変換し、これを酵素濃度の指標として測定した。各ウェルにpNPPを加え、37℃で１時間インキュベートした。次に１Ｎの水酸化ナトリウムを加えて、酵素反応を停止させ、p-ニトロフェノールの量を、Dynatech 2000プレートリーダー（Dynatech Laboratories,Chantilly,VA）を使用して410nmで定量した。細胞数と吸光度を比較する標準曲線を使用して、アッセイの直線範囲を決定した。吸光度測定値がアッセイの直線範囲内にある時のみ、測定結果を使用した。簡単に説明すると、リガンドを含むかまたは含まない平底マイクロタイタープレート（150または200μlウェル）中の４重の細胞試料を用いて、37℃で72〜96時間アッセイを行なった。存在する細胞の数の尺度として酸性ホスファターゼを使用した。すべての実験は、少なくとも２回繰り返した。
アポトーシスの初期マーカーである細胞外ホスファチジルセリンを認識することによりデキサメタゾン誘導性アポトーシスを測定するAnnexin Vキットを使用してサプライヤー（Clontech）が記載するように、アポトーシスを測定した。アポトーシス細胞数は、Annexin V染色細胞のパーセントとして、蛍光顕微鏡によりスコア化した。
Mo7e系はヒト巨核芽球細胞系であり、RPMI 1640（GIBCO/BRL,Gaithersburg,MD），20％ウシ胎児血清（Hyclone）および10ng/ml IL-3（R & D Systems,Minneapolis,MN）中で培養した。T98G系は、RPMI 1640（GIBCO/BRL）中で増殖されるヒト神経膠芽細胞腫細胞系である。ハムスター繊維芽細胞TK-ts13系も、マウス骨髄腫前駆体系であるマウス32D細胞系とともに使用し、いずれも10％ウシ胎児血清（Hyclone）を含有するRPMI 1640（GIBCO/BRL）中で培養し、さらに32D細胞系では１ng/mlのIL-3を使用した。TF1.1は，IL-2受容体ガンマサブユニット（ウェスタンブロットとrtPCRにより確認された）を発現することが知られているヒト骨髄白血病系であるが、その先祖（TF1）と比較すると、rtPCR、免疫染色、およびウェスタンブロット分析により，すでにIL-9受容体は持っていない。TF1.1は、RPMI 1640（GIBCO/BRL）と10％ウシ胎児血清（Hyclone）中で培養した。すべての細胞系は、IL-9を含めて複数のサイトカインに応答する。細胞系に栄養を与え，再度72時間毎に２×10⁵細胞/mlで再接種した。
細胞を2000rpmで10分間遠心分離し、0.5％ウシ血清アルブミン（GIBCO/BRL,Gaithersburg,MD）とインスリン−トランスフェリン−セレン（ITS）補助因子（GIBCO/BRL,Gaithersburg,MD）を含むRPMI 1640中に再懸濁した。血球計で細胞を数え、１×10⁵細胞/mlに希釈し、96ウエルのマイクロタイタープレートにまいた。各ウェルは、0.15または0.2mlを含有し、細胞により10,000〜50,000細胞／ウェルの最終濃度を与えた。Mo7e細胞を，50ng/mlの幹細胞因子（SCF）（R & D Systems,Minneapolis,MN）単独、50ng/ml SCFプラス50ng/ml IL-3（R & D Systems,Minneapolis,MN）、または50ng/ml SCFプラス50ng/ml IL-9で刺激した。細胞と基本培地のみを含有する対照を含めた。試験化合物（すなわち、組換えIL-9タンパク質、ペプチド、小分子）の連続希釈物を、３重測定で各試験条件に加えた。IL-9受容体でトランスフェクトしなかったTF1.1細胞を、応答と非特異的細胞傷害性の独立した対照として使用した。培養物を５％ CO₂中で37℃で72〜96時間インキュベートした。
実施例８
トランスフェクトされた細胞系中の外因性IL-9受容体のin situおよびウェスタン分析
IL-9受容体のin situ染色は以下のように行なった。細胞をカバーグラスの上で24時間増殖させ、次に接着性の細胞を含有するカバーグラスを、カルシウムとマグネシウムを含有するリン酸緩衝化生理食塩水（PBS）（Gibco/BRL）で２回洗浄した。IL-9受容体の細胞内染色のために、細胞を、４％パラホルムアルデヒド／PBSプラス0.1％トリトン-X中で、室温で15分間固定してから、抗ヒトIL-9受容体抗体で処理した。細胞外染色のために、細胞を固定前に抗体で処理した。次に細胞をPBSで２回洗浄し、dH₂O中の7.5％BSAにより室温で30分ブロックした。次に、PBSで洗浄した細胞を、１％BSA/PBS中の抗ヒトIL-9受容体（IL-9受容体のカルボキシ末端に対するポリクローナル抗体）の10μg/ml溶液と共に、室温で１時間インキュベートした。PBSで細胞を３回洗浄し、次に１％BSA/PBS中の抗ウサギローダミン結合抗体の10μg/ml溶液中、室温で30分間インキュベートした。次に細胞をPBSで３回洗浄して、１μg/mlのDAPIを使用して室温で１分間カウンター染色した。dH₂O中で細胞を３回洗浄し、顕微鏡スライドに固定し、蛍光顕微鏡により分析した。トランスフェクトしたCOS7細胞についての結果を、図14に示す。
0.5％溶解バッファー（トリス50mM,NaCl 150mM,NP40）、１mM DTTおよびプロテアーゼインヒビター中の細胞抽出物の直接溶解から得られたタンパク質溶解物について、ウェスタンブロットを行い、５分間沸騰させた。試料を、トリス−グリシンランニングバッファー中の４〜20％トリス−グリシンSDSゲル（Novex）で電気泳動した。次に、Trans Blot II装置（Bio Rad）を使用して、電気ブロットにより、タンパク質をニトロセルロースに移した。移動後、膜を，TBS-T（（20mMトリス、137mM NaCl、pH7.6）プラス0.05％ツイーン20）プラス５％ブロット（blotto）中、室温で１時間ブロックした。次に、TBS-T中のIL-9受容体のカルボキシ末端に対するポリクローナル抗体（１μg/ml）を使用して、ブロットを１時間探した。次にTBS-T中でブロットを10分間３回洗浄し、TBS-T中の第２抗ウサギ西洋ワサビペルオキシダーゼ結合抗体（１：10,000）を使用して30分間探した。上記のようにブロットを洗浄し、化学発光基質であるルミノール／エンハンサー溶液（Pierce）と共に室温で５分間インキュベートし、次に１〜60秒間フィルムに露光した。図11と12を参照されたい。
実施例９
真のIL-9Rゲノム増幅法
IL-9Rは，ヒトゲノムの他の遺伝子座（第９、第10、第16、第18染色体）に４つの高度に相同性（＞90％ヌクレオチド同一性）のプロセシングされていない偽遺伝子を有するため、標準的プライマーデザインを使用する真のIL-9R（XYq偽常染色体領域中に位置する生物学的機能性タンパク質をコードする遺伝子）の特異的増幅は不可能であった。これらの他の遺伝子の高度の同一性のために、標準的プライマーデザインを使用するゲノムPCR増幅により、すべての遺伝子が同時増幅され、従って真の遺伝子の配列解析が不明確になった。真のIL-9R構造は、本出願に記載の喘息または癌のような他の疾患（Renauldら、Oncogene,9:1327-1332,1994;Grussら、Cancer Res.,52:1026-1031,1992）に対する素因に関係しうるので、その構造を研究するために、特異的アンプリマーを以下のように設計した：
IL-9R偽遺伝子と真の遺伝子の配列をMac Vectorソフトウェアを使用してアラインメントした。次に、真の遺伝子と偽遺伝子の間で分岐した領域について、各エキソンの周りのイントロン配列を調べた。次にこれらの領域に対してプライマーを設計し、これを使用して、各ヒト染色体を含有するヒト／齧歯類ハイブリッドDNAをPCR増幅した。生成物を３％アガロースゲルで泳動し、４つの偽遺伝子の増幅の無い真のIL-9R増幅について分析した。アニーリング温度とバッファー条件（DMSO含量５〜10％）を変えて、特異的PCR増幅条件も最適化した。まだ偽遺伝子の増幅が存在した場合は、プライマー配列中にヌクレオチド変異を入れて、真の遺伝子と比較して偽遺伝子からのより大きな分岐を生じさせた。プライマー配列を実施例２に示し、その特異性を図13に示す。
実施例10
細胞増殖アッセイとサイトカイン刺激
種々の型のヒIL-9受容体を発現するTS1細胞の増殖応答を調べるために、細胞をPBSで洗浄し、D-MEM、10％ウシ胎児血清中に再懸濁した。10³細胞/ウェルを、96ウェルマイクロプレート中に３回反復で接種し、適宜、組換えヒトIL-9またはマウスIL-9（R & D Systems,Minneapolis,MN）を最終濃度５ng/mlで加えた。酸性ホスファターゼアッセイを使用して７日後に、細胞増殖を評価した。簡単に説明すると、0.1Ｍ酢酸ナトリウム（pH5.5）,0.1％トリトンX-100および10mM p-ニトロフェニルホスフェート（Sigma 104ホスファターゼ基質）を含有するバッファー50μlを、ウェルに加えた。プレートを室温で１〜0.5時間インキュベートし、10μl／ウェルの１Ｎ水酸化ナトリウムで反応を停止させ、Dynatech Model MR600で410nmで吸光度を読んだ。サイトカイン刺激後のシグナル伝達カスケードのタンパク質のチロシンリン酸化を解析するために、種々の型のヒトIL-9受容体を発現するTS1細胞をPBSで洗浄し、D-MEM,0.5％ウシ血清アルブミンに再懸濁し、37℃で６時間インキュベートした。続いて、20×10⁶細胞をヒトIL-9またはマウスIL-9（100ng/ml）で５分間処理し，直ちに冷PBSで洗浄した。実施例11に記載のように細胞をRIPAバッファーで溶解した。
実施例11
免疫沈降、免疫ブロッティングおよび抗体
典型的には20〜50×10⁶細胞を、１mlのRIPAバッファー（１％NP40、0.5％デオキシコール酸ナトリウム、0.1％SDS、１mM PMSF、50mMフッ化ナトリウム、１nMオルトバナジン酸ナトリウム、および１×「完全」プロテアーゼインヒビター混合物を含有するPBS、Cat.No.1697498 Boehringer Mannheim）で溶解し、氷上で45分間インキュベートした。溶解物をEppendorf微量遠心分離機中で20分間遠心分離し、上清を回収し、新しいチューブに移した。免疫沈降のために、１〜５μgの抗体を溶解物に加え、４℃で一晩インキュベートした。20mlのプロテインA+Gアガロース結合ビーズを２時間加え、次にRIPAバッファーを使用して４回洗浄した。ビーズをLaemmliバッファー中に再懸濁し、３分間沸騰してから電気泳動した。タンパク質をImmobilion-P膜（Millipore）に移し、西洋ワサビペルオキシダーゼ結合２次抗体を使用して化学発光検出アッセイ（Pierce）により検出した。マウスおよびヒトIL-9受容体の特異的抗体（sc698），マウスJak1、Irs1、Irs2、Stat1、Stat2、Stat3、Stat4、Stat5、およびホスホチロシン（PY）は、Santa Cruz（Santa Cruz,CA）から購入した。抗Jak3およびモノクローナル抗ヒトIL-9受容体MAB290は、それぞれUpstate BiotechnologyとR & D Systemsから購入した。図15は、ヒトIL-9受容体の異なる変異体による、Jakファミリーのメンバーの活性化を証明する。
実施例12
IL-9受容体ゲノム多型の同定
既に記載されているように（NicolaidesとStoeckert,Biotechniques 8:154-156,1990）、志願者ドナーのPBMCからゲノムDNAを単離した。配列番号14と配列番号17のプライマーを使用するPCRにより、ヒトIL-9R遺伝子のイントロン５の配列解析を行ない、分子サイズ約1243塩基対の産物を得た。既に記載されているバッファー（Nicholaidesら、Genomics 30:195-206,1995）で、94℃で30秒、62℃で1.5分、72℃で1.5分を35サイクル行って増幅した。次に産物を精製し、標準的配列プロトコールを使用して配列決定した。複数の個体のイントロン５からの配列を調べると、エキソン６の配列の-213ヌクレオチド上流にヌクレオチド変化があり、このためチミジン（公表されている配列）がシトシンヌクレオチドに変化していた。この変異の例を図17に示す。
本発明を種々の具体的な材料、方法および例を参照して説明し例示したが、本発明は、この目的のために選択された材料の特定の組合せや方法に限定されるものではないことを理解されたい。当業者が理解できるように、このような細部の多くの変更が可能である。
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14.Hamid G,Azzawi M,Ying Sら．Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma（喘息からの粘膜気管支生検材料におけるインターロイキン-5のmRNAの発現）．J Clin Invest 1991;87:1541-1546.
15.Djukanovic R,Roche WR,Wilson JWら．Mucosal inflammation in asthma（喘息における粘膜炎症）． Am Rev Respir Dis 1990;142:434-57.
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17.Robinson DS,Hamid Q,Ying Sら．Prednisolone treatment in asthma is associated with modulation of bronchoalveolar lavage cell interleukin-4,interleukin-5,and interferon-cytokine gene expression（喘息のプレドニゾロン治療は気管支肺胞洗浄細胞のインターロイキン-4、インターロイキン-5およびインターフェロン-サイトカイン遺伝子発現のモジュレーションと関連する）． Am Rev Respir Dis 1993;148:401-406.
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24.Hultner,L,Moeller,J,Schmitt,E,Jager,G,Reisbach,G,Ring,J.Dormer,P.Thiol-sensitive mast cell lines derived from mouse bone marrow respond to a mast cell growth-enhancing activity different from both IL-3 and IL-4（マウス骨髄由来のチオール感受性マスト細胞系はIL-3およびIL-4と異なるマスト細胞増殖促進活性に応答する）. J Immunol 1989;142:3440-3446.
25.Dugas,B,Renauld,J-C,Pene,J,Bonnefoy,J,Peti-Frere,C,Braquet,P,Bousquet,J,Van Snick,J,Mencia-Huerta,JM.Interleukin-9 potentiates the interleukin-4-induced immunoglobulin（IgG,IgM and IgE）production by normal human B lymphocytes（インターロイキン-9は正常ヒトBリンパ球によるインターロイキン-4-誘導免疫グロブリン（IgG,IgM,IgE）産生を増強する）． Eur J Immunol 1993;23:1687-1692.
26.Petit-Frere,C,Dugas,B,Braquet,P,Mencia-Huerta,JM.Interleukin-9 potentiates the interleukin-4-induced IgE and IgG1 release from murine B lymphocytes（インターロイキン-9はマウスBリンパ球からのインターロイキン-4-誘導IgEおよびIgG1放出を増強する）．lmmunology 1993;79:146-151.
27.Behnke,JM,Wahid,FN,Grencis,RK,Else,KJ,Ben-Smith,AW,Goyal,PK.Immunological relationships during primary infection with Heligmosomoides polygyrus（Nematospiroides dubius）:downregulation of speciftc cytokine secretion（IL-9 and IL-10）correlates with poor mastocytosis and chronic survival of adult worms（Heligmosomoides polygyrus（Nematospiroides dubius）による一次感染中の免疫学的関係:特定のサイトカイン分泌（IL-9およびIL-10）のダウンレギュレーションは低い肥満細胞症および成虫の慢性的生存と相関する）． Parasite Immunol 1993;15:415-421.
28.Gessner,A,Blum,H,Rollinghoff,M.Differential regulation of IL-9 expression after infection with Leischmania major in susceptible and resistant mice（感受性および耐性マウスにおけるLeischmania major感染後のIL-9発現の示差的調節）． Immunobiology 1993;189:419-435.
29.Renauld J-C,Druez C,Kermouni Aら．Expression cloning of the murine and human interleukin 9 receptor cDNAs（マウスおよびヒト・インターロイキン-9受容体cDNAの発現クローニング）． Proc Natl Acad Sci 89:5690-5694（1992）.
30.Chang M-S,Engel G,Benedict Cら．Isolation and characterization of the Human interleukin-9 receptor gene（ヒト・インターロイキン-9受容体遺伝子の単離および特性決定）． Blood 83:3199-3205（1994）.
31.Renauld J-C,Goethals A,Houssiau Fら.Human P40/IL-9.Expression in activated CD4+ T cells,Genomic Organization,and Comparison with the Mouse Gene（ヒトP40/IL-9.活性化CD4+ T細胞での発現、ゲノム構成、およびマウス遺伝子との比較）．J Immunol 144:4235-4241（1990）.
32.Kelleher K,Bean K,Clark SCら．Human interleukin-9:genomic sequence,chromosomal location,and sequences essential for its expression in human T-cell leukemia virus（HTLV-1-transformed human T cells）（ヒト・インターロイキン-9:ゲノム配列、染色体位置、およびヒトT細胞白血病ウイルス（HTLV-1形質転換ヒトT細胞）におけるその発現に不可欠な配列）．Blood 77:1436-1441（1991）.
33.Houssiau FA,Schandene L,Stevens Mら．A cascade of cytokines is responsible for IL-9 expression in human T cells.Involvement of IL-2,IL-4,and IL-10（サイトカインのカスケードはヒトT細胞におけるIL-9発現に関与する。IL-2、IL-4およびIL-10の関与）． J of Immunol.154:2624-2630（1995）.
34.Miyazawa K,Hendrie PC,Kim Y-Jら．Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel factor to stimulate proliferation of the human factor-dependent cell line,Mo7e（組換えヒト・インターロイキン-9はタンパク質チロシンリン酸化を引き起こし、ヒト因子依存性細胞系Mo7eの増殖を刺激するためにSl因子と相乗的に作用する）．Blood 80:1685-1692（1992）.
35.Yin T,Tsang M L-S,Yang Y-C.JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes（JAK1キナーゼはインターロイキン-4受容体および4PS/インスリン受容体基質-1様タンパク質と複合体を形成し、Tリンパ球内でインターロイキン-4およびインターロイキン-9により活性化される）． J Biol Chem 269:26614-26617（1994）.
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37.Chu JW,and Sharom FJ.Glycophorin A interacts with interleukin-2 and inhibits interleukin-2-dependent T-lymphocyte proliferation（グリコホリンAはインターロイキン-2と相互作用し、インターロイキン-2依存性Tリンパ球増殖を阻害する）．Cell Immunol 145:223-239（1992）.
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48.Meyers DA,Postma DS,Panhuysen CIMら．Evidence for a locus regulating total serum IgE levels mapping to chromosome 5（総血清IgEレベルを調節する遺伝子座が第5染色体にマップされる証拠）． Genomics 1994;23:464 470.41.Reference to related applications
This application is related to US Provisional Application No. 60 / 032,224, filed Dec. 2, 1996, which is incorporated herein by reference. This application also includes U.S. Patent Application Nos. 08 / 697,419, 08 / 697,360, 08 / 697,473, 08 / 697,472, 08 / 697,471, 08 / 702,105, 08 / 702,110, 08 / 702,168, and 08 / 697,440 (all of which were filed on August 23, 1996) and 08/874, 503 (which was filed on June 13, 1997) This is incorporated herein by reference.
Field of Invention
The present invention describes the biological changes of the IL-9 receptor (Asthma Associated Factor 2) (SEQ ID NO: 1) and these variants, asthma, atopic allergy, and related diseases. Correlate with susceptibility. The present invention also teaches how to utilize these IL-9 receptor sequence variants for diagnosis of susceptibility or resistance to asthma and atopic allergy. Furthermore, methods for using IL-9 receptor variants in the development of asthma drugs that depend on modulation of IL-9 activity are described.
Background of the Invention
Inflammation is a complex process in which the body's defense system fights foreign substances. Although fights against foreign substances are necessary for the survival of the body, some defense systems inappropriately respond as dangerous to harmless foreign substances, so that Injury the tissue.
Atopic allergy is a disease in which the genetic background dominates the response to environmental stimuli. This disease is generally characterized by an increased ability of lymphocytes to produce IgE antibodies in response to ubiquitous antigens. Activation of the immune system by these antigens also causes their ingestion, permeation through the skin, or allergic inflammation that occurs after inhalation. When this immune activation occurs, followed by lung inflammation, the disease is broadly characterized as asthma. Certain cells are important for the inflammatory response in the respiratory tract, including T cells and antigen presenting cells, B cells that produce IgE, and mast cells / basophils that store inflammatory mediators and bind to IgE There are spheres, eosinophils that release additional mediators. These inflammatory cells accumulate in the field of allergic inflammation and the harmful products they release contribute to the tissue destruction associated with the disease.
Asthma is generally defined as an inflammatory disease of the airways, but clinical symptoms arise from intermittent airflow blockages. This is a chronic disabling disease that seems to have increased morbidity and severity¹. It is estimated that 30-40% of the population has atopic allergies, and 15% of children and 5% of adults have asthma.¹. In other words, a huge burden is placed on medical resources in the treatment of these diseases.
Both diagnosis and treatment of asthma and related diseases are problematic¹. In particular, assessment of inflamed lungs is often difficult and often the cause of inflammation cannot be determined. Although atopic asthma is an environmental genetic disease, knowledge about specific mutant genes has only recently been discovered. The detection methods and their role, diagnosis and prognosis of these genetic variants in inflammation have not yet been determined. What is needed in the art is the development of a technique that facilitates the diagnosis of atopic asthma that is specifically associated with mutations in genes involved in susceptibility / resistance to atopic disease.
Modern therapies have an inherent set of drawbacks. Β-agonists, the main therapeutic agents, reduce symptoms, ie transiently improve lung function, but do not affect basal inflammation, so lung tissue is still at risk. In addition, continued use of β-agonists results in desensitization, reducing efficacy and safety². Anti-inflammatory steroids, substances that can reduce basal inflammation, have a unique set of side effects ranging from immunosuppression to bone loss².
Alternative treatment strategies are being evaluated due to problems arising from conventional treatment^38-39. Glycophorin A³⁷, Cyclosporine³⁸And the nonapeptide fragment of IL-2³⁶All inhibit interleukin-2-dependent T lymphocyte proliferation²⁸. But these are known to have many other actions². For example, cyclosporine has been used as an immunosuppressant after organ transplantation. These drugs are an alternative to steroids in the treatment of asthma patients.^36-39These may inhibit interleukin-2-dependent T lymphocyte proliferation and may inhibit immune functions that appear to be very important in connection with homeostasis. What is needed in the art is to facilitate the development of therapeutics specifically designed to treat the causes of atopic asthma but not its symptoms. These therapies are the most likely ways to avoid the toxicity associated with non-specific treatments. This therapeutic is downstream of immune function (eg, IL-2 mediated T lymphocyte activation required for the development of asthma), explains the incidental nature of the disease, and provides a pathway to explain its close association with allergies Selectively target. If there is a common biological change in the pathway and the individual showing the change is not immune compromised or ill except for the symptoms of atopic asthma, the pathway should be an appropriate target for asthma treatment. Nature proves.
Due to the difficulties associated with the diagnosis and treatment of atopic allergies, including asthma, the complex pathophysiology of these diseases has been studied in detail. Although these diseases are difficult to define because they consist of many factors and take many types, some common features have been found in asthmatic patients. Examples of such features include abnormal skin test responses to allergen antigenic stimulation, pulmonary eosinophilia, bronchial hyperresponsiveness (BHR), bronchodilator reversibility, and airflow blockage^3-10. These expressions of traits associated with asthma are studied quantitatively or qualitatively.
In many cases, elevated IgE levels are associated with increased BHR, bronchoconstrictor response to various stimuli^4,6,8,9. BHR is thought to reflect the presence of airway inflammation^6,8Is considered a risk factor for asthma^11-12. BHR is associated with bronchial inflammation, including eosinophil infiltration into the lung, and allergic predisposition in asthmatic patients^6,8,13-18.
Hereditary components for atopic asthma have been documented in many studies^4,10. However, these diseases involve age and sex, as well as many environmental factors such as allergens, viral infections, and pollutants.^19-21Family studies are difficult to interpret. Furthermore, since there are no biochemical defects known to be associated with susceptibility to these diseases, mutant genes and their abnormal gene products can only be recognized by the abnormal phenotype they produce.
The functions of IL-9 and IL-9 receptors (IL-9 pathway) are expanding from those originally recognized. Although the IL-9 pathway acts as a stimulator of T cell proliferation, this cytokine is also known to mediate proliferation of erythroid progenitor cells, B cells, mast cells, and fetal thymocytes^22,23. IL-9 pathway synergizes with IL-3 to cause mast cell activation and proliferation^{twenty four}. The IL-9 pathway also enhances IL-4 induced production of IgE, IgG and IgM by normal human B lymphocytes^{twenty five}Enhances IL-4-induced release of IgE and IgG by mouse B lymphocytes²⁶. The role of the IL-9 pathway in mucosal inflammatory responses to parasitic infections has also been demonstrated^27,28.
However, it is not known how the sequence of IL-9 receptor correlates with atopic asthma and bronchial hyperresponsiveness. IL-9 is known to bind to specific receptors expressed on the surface of target cells^23,29,30. This receptor actually consists of two protein chains. One protein chain is known as the IL-9 receptor and binds specifically to IL-9. Another protein chain is shared with the IL-2 receptor^{twenty three}. In addition, the cDNA encoding the human IL-9 receptor has been cloned and sequenced^23,29,30. This cDNA encodes a 522 amino acid protein with high homology to the mouse IL-9 receptor. The extracellular region of this receptor is highly conserved, with 67% homology between mouse and human proteins. The cytoplasmic region of the receptor is not very conserved. The human cytoplasmic domain is much larger than the corresponding region of the mouse receptor^{twenty three}.
The IL-9 receptor gene has also been characterized³⁰. It exists as a single copy in the mouse genome and contains 9 exons and 8 introns³⁰. The human genome contains at least four IL-9 receptor pseudogenes. The human IL-9 receptor gene is mapped to the 320 kb subtelomeric region of sex chromosomes X and Y^{twenty three}.
Despite these studies, no variant of the IL-9 receptor gene has been found. Therefore, there is a specific need for genetic information on atopic allergies, asthma, bronchial hyperresponsiveness, and elucidation of the role of IL-9 receptors in the pathogenesis of these diseases. This information can be used to diagnose atopic allergies and related diseases, using methods to identify genetic variants of this gene associated with these diseases. Furthermore, there is a need for methods of employing IL-9 receptor mutants for the development of therapeutics to treat these diseases.
Summary of invention
Applicants have discovered natural variants of the human IL-9 receptor (also known as asthma-related factor 2 or AAF2) and have associated these variants with the pathology of asthma and related diseases. These discoveries have developed a method for discovering diagnostic and therapeutic agents for atopic asthma. In addition, Applicants have shown that the IL-9 receptor is responsible for many antigen-induced responses in mice (including bronchial hyperresponsiveness, increased eosinophils and cell numbers in bronchial lavage fluid, and increased serum total IgE). To make it very important. These findings are typical of atopic asthma and associated allergic inflammation.
In addition, Applicants have found that a nucleic acid mutation from G to A exists at position 1273 of the cDNA (SEQ ID NO: 2), which is predicted from arginine to histidine at codon 344 of the human IL-9 receptor precursor protein. Cause amino acid substitutions. When arginine residues are present in both alleles of an individual, this is associated with less atopic asthma. That is, Applicants confirmed the presence of a non-asthmatic phenotype characterized by the codon 344 arginine present in both individuals in both IL-9 receptor gene products. As a further important result, Applicants have confirmed the presence of susceptibility to the asthmatic, atopic phenotype characterized by histidine at codon 344. Thus, the present invention encompasses purified and isolated DNA molecules having such sequences, as well as proteins encoded by this DNA.
Applicants have also confirmed the presence of a splice variant of IL-9R (SEQ ID NO: 3) lacking the glutamine residue at position 173 of the IL-9R precursor protein (FIG. 5). Furthermore, Applicants have demonstrated that this mutant cannot transcribe signals via the Jak-Stat pathway (FIG. 15) and that it cannot induce cell proliferation upon stimulation with IL-9 (FIG. 16). Thus, individuals with this allele will be less susceptible to atopic asthma and related diseases.
The present applicants further confirmed that there is a mutant of IL-9R genomic DNA in which thymine residue nt-213 (exon 6 to 213 nt upstream) of intron 5 is converted to a cytosine nucleotide. . Such mutations can increase the frequency of splice variants that remove glutamine residues at the start of exon 6.
Further, Applicants have discovered a variant of IL-9R (SEQ ID NO: 4) lacking exon 8, which causes mutations in the reading frame of exon 9 and the immature stop codon. Such mutants interfere with signaling in the Jak-Stat pathway, and thus individuals with this allele will also be less susceptible to atopic asthma and related diseases.
The biological activity of IL-9 is due to its binding to the IL-9 receptor and subsequent propagation of regulatory signals in specific cells, and thus IL-9 function is dependent on IL-9 antagonist and IL-9 or its Can be hampered by interaction with receptors. Down-regulation, i.e., loss of function controlled by IL-9, is accomplished in a number of ways. Administering an antagonist capable of blocking IL-9 binding to the receptor is one important method and such antagonists are within the scope of the claimed invention. An example is the administration of a polypeptide product encoded by a naturally occurring soluble DNA sequence of the IL-9 receptor, which encodes a polypeptide consisting of exons 2 and 3 (SEQ ID NO: 5). . The other two variants are soluble forms of IL-9R (which consists of exons 2, 3 and 4 and in some cases four amino acids from different reading frames of exon 5 (SEQ ID NO:32) (FIG. 6), and in other cases 27 amino acids of different reading frames of exon 5 (SEQ ID NO:33) (Including FIG. 7).
Methods for identifying agonists and antagonists of the IL-9 receptor pathway can be identified by assessing receptor-ligand interactions as described in detail in the literature. These methods can be adapted to high-throughput automated assays that facilitate chemical screening and identification of potential therapeutic agents. Agonists are recognized by identifying specific interactions with the IL-9 receptor. The lack of binding to the labeled putative ligand using a 100-1000 fold excess of unlabeled ligand is generally accepted as evidence of specific receptor binding. Many labels and detection schemes can be used in these experiments. Evidence for antagonists is also the addition of increasing concentrations of test compounds to known ligands and receptors with no binding.
The mutant receptor information provides a means for generating expression vectors that can be used to generate soluble receptors for receptor binding assays. Mutagenesis of these soluble receptors can be used to determine which amino acids are very important for binding to the ligand and which amino acids assist the structure-based design of the antagonist. it can.
Cells lacking the human IL-9 receptor can be transiently or stably transfected with expression vectors containing mutant receptors and used to assay IL-9 pathway activity. it can. Both of these activities may be cell growth or apoptosis prevention that is attributed to the IL-9 pathway. These cells can be used to identify receptor agonists and antagonists as described above.
The above methods are various effective methods that utilize mutant forms of IL-9 receptor to develop therapeutic agents for atopic asthma and other related diseases.
A number of techniques have been described that can be used to diagnose atopic asthma that recognize single nucleotide variants in the IL-9 receptor, including DNA sequencing, restriction fragment length polymorphism (RFLP) There are allele-specific oligonucleotide analysis (ASO), ligation chain reaction (LCR), chemical cleavage, and single-strand conformation polymorphism analysis (SSCP). The use of these techniques as well as one or more other techniques described in the literature can be used to detect one or more mutations in the IL-9 receptor gene or mRNA transcript, which are Those skilled in the art will readily understand that they are within the scope of the invention.
Additional techniques (including ELISA, immunoprecipitation, Western methods, and immunoblotting methods) may be used to detect amino acid variants of the IL-9 receptor. That is, polyclonal or monoclonal antibodies that specifically recognize various types of structures of the IL-9 receptor are also within the scope of the present invention, to explain susceptibility or resistance to atopic asthma and related diseases. It is a useful diagnostic method.
The above methods represent various effective methods for diagnosing atopic asthma and other related diseases.
Thus, Applicants have provided methods of using IL-9 receptors to identify antagonists that can modulate the interaction of IL-9 with its receptors. More particularly, Applicants assay IL-9 receptor function to identify compounds or agents that are administered in an amount sufficient to down-regulate IL-9 pathway expression or function. Provide a method.
Since the Applicants have identified the role of the IL-9 pathway in atopic allergy, bronchial hyperresponsiveness and asthma, they also provide methods for diagnosis of susceptibility and resistance to atopic allergy, asthma, and related diseases.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description of the invention, illustrate the principles of the invention.
[Brief description of the drawings]
FIG. 1: Schematic representation of human IL receptor cDNA. Squares indicate exons 2-9 including the coding region (scale is relative size and is shown as a dashed line except for the 3 'untranslated portion of exon 9). The transmembrane region is encoded by exon 7, the intracellular domain is encoded by exons 8 and 9, and the extracellular domain is encoded by exons 2-6. Arrows indicate polymorphisms or aberrant splices that affect exon subsequences; a) deletion of the first 5 or 29 nucleotides of exon 5; b) deletion of the first 3 nucleotides of exon 6 (codon 173) C) arg / gly polymorphism at codon 310; d) arg / his polymorphism at codon 344; e) polymorphism of codon 410 + n consisting of 8 or 9 serines; *) exon 3, 4 or 8 polymorphism; Complete deletion.
Figure 2: cDNA with translation of wild-type IL-9R precursor protein with Arg allele at codon 344 (nucleotides 1272-1274) and 8 Ser / 4 Asn repeats starting at codon 410 (nucleotides 1470-1472). An array.
FIG. 3: Translated cDNA sequence of IL-9R precursor protein with His allele at codon 344 (nucleotides 1272-1274) and 9 Ser / 4 Asn repeats starting at codon 410 (nucleotides 1470-1472).
FIG. 4: Translated cDNA sequence of the IL-9R precursor protein with a deletion of exon 8 causing a frameshift in exon 9 and the production of 11 non-wild type amino acids and immature stop codons.
FIG. 5: Translated cDNA sequence of IL-9R precursor protein with a glutamine deletion at codon 173.
FIG. 6: Translated cDNA sequence of IL-9R precursor protein with an alternative splice in exon 5 that causes the production of an immature stop codon and 27 non-wild type amino acids.
FIG. 7: Translated cDNA sequence of IL-9R precursor protein with an alternative splice in exon 5 that causes the production of an immature stop codon and four non-wild type amino acids.
FIG. 8: Translated cDNA sequence of the IL-9R precursor protein with a deletion of exon 4, producing a stop codon as the first codon of exon 5.
FIG. 9: Table showing the relationship between IL-9 receptor genotype and atopic allergy. Arg / Arg individuals are homozygous for the Arg allele with 8 Ser / 4 Asn repeats. Arg / His individuals are in exon 9, Arg allele with 8 Ser / 4 Asn repeats, 9 Ser / 4 Asn repeats, 9 Ser / 3 Asn repeats, and 10 Ser / 2 Asn repeats. Heterozygous for the His allele with His / His individuals are homozygous for the His allele in Exon 9 with 9 Ser / 4 Asn repeats, 9 Ser / 3 Asn repeats, and 10 Ser / 2 Asn repeats. Arg / Arg individuals are protected from atopic allergies. Arg / His and His / His individuals are susceptible to atopic allergy (P = 0.002).
Figure 10: Map of IL-9 receptor expression construct.
Figure 11: TK^-Western blot of recombinant IL-9 receptor protein using a C-terminal antibody probe in a transfected cell line (left: Arg allele with 8 Ser / 4 Asn repeats; right: 9 Ser / 4 Asn repeats His alleles).
Figure 12: Human ILs in TS1 cells showing differences in mobility between Arg344 mutant (GR8) with 8 Ser / 4 Asn repeats and His344 mutant (GH9) with 9 Ser / 4 Asn repeats -9 receptor mutant expression. There is a mobility shift that demonstrates the difference in post-translational modification of these two mutant forms of the IL-9 receptor.
Figure 13: XY specific amplimer for specific amplification of IL-9 receptor gene. Pseudogenes on chromosomes 9, 10, 16, or 18 are not amplified by PCR. (M represents mouse DNA, H represents human DNA, and C represents hamster DNA).
FIG. 14: Immunoreactivity of anti-human IL-9 receptor neutralizing antibody with wild type and delta-Q receptors. Panel A): COS7 cells were treated with LXSN vector alone (A and B), wild type IL-9R (C and D), wild type IL-9R with 9 Ser residues starting at codon 410 (E and F). ), Δ-Q 173 mutant (G and H), and Δ-Q 173 (I and J) with 9 Ser residues starting at codon 410 and transiently transfected as described Example 8: Incubate sequentially with MAB290 and anti-mouse IgG Texas Red binding antibody (B, D, F, H, and J). DAPI staining (A, C, E, G, and I) is performed to visualize all cells in the photographic field. Panel B): Same as A), but cells were first fixed / permeabilized, then incubated with a C-terminal specific antibody (sc698) and then with an anti-rabbit IgG Texas Red binding antibody. Horizontal bar = 10 microns.
Figure 15: Activation of Jak, Stat, and Irs family members of different variants of the human IL-9 receptor. TS1 cells expressing GH9, ΔQGR8, or ΔQGH9 are deprived of nutrients for 6 hours and then treated with no cytokines (-), with mouse IL-9 (m), or with human IL-9 (h) for 5 minutes did. Cell extracts were immunoprecipitated with various antibodies specific for different members of the Jak, Stat and Irs families. The immunoblot was first reacted with an anti-phosphotyrosine antibody to detect only tyrosine-phosphorylated proteins, then stripped and re-fished with the same antibodies used to immunoprecipitate each protein. GH9, ΔQGR8, and ΔQGH9 are as shown in FIG.
Figure 16: Proliferation of TS1 cells expressing different types of human IL-9 receptor. Cells were inoculated into a 96-well plate (1000 / well) so that each cell could be measured in quadruplicate, and treated with no cytokine, mouse IL-9 or human IL-9 (5 ng / ml). Seven days later, a colorimetric assay was performed to determine the number of cells and the ratio of treated / untreated cells (control%) was calculated to assess growth rate. LxSN = cells transfected with empty vector; GR8 is wild type IL-9R; GH9 is a His344 variant with 9 Ser residues starting at codon 410; ΔQGR8 and ΔQGH9 are wild type and His344, respectively ΔQ173 mutant on + 9-Ser background.
FIG. 17: Genomic DNA sequence of IL-9R intron 5 having a mutation in the nucleic acid 213 nt upstream from exon 6 (wherein T residue is changed to C residue as indicated by the arrow).
Detailed Description of the Invention
Applicants have elucidated the IL-9 pathway and compositions that affect this treatment used in the development of therapeutics, diagnostics, and methods for identifying drugs to prevent or treat atopic asthma and related diseases This solved the needs in the field.
Asthma includes airway inflammatory disorders with reversible airflow blockage. By atopic allergy is meant atopy and related diseases, including asthma, bronchial hyperresponsiveness (BHR), rhinitis, urticaria, intestinal allergic inflammatory diseases, and various types of eczema. Atopy is a hypersensitivity to environmental allergens and is expressed as an increase in serum total IgE or abnormal skin test response to allergens compared to controls. BHR means bronchial hyperresponsiveness, which is an increase in bronchoconstriction response to various stimuli.
By analyzing the DNA of individuals exhibiting atopic allergies and asthma-related diseases, Applicants have identified polymorphisms in the IL-9 receptor (IL-9R) gene that can be correlated with asthma expression. The IL-9 receptor gene (also known as asthma-related factor 2 or AAF2) is an interleukin-9 receptor that is a cytokine receptor associated with a variety of functions including regulation of the human myeloid and lymphatic systems Means the gene locus. The human IL-9 receptor gene of the present invention is found in the telomere region at the next end of the XY chromosome.
Applicants refer to a polymorphism as a change in a specific DNA sequence (called a “locus”) from a normal sequence. In general, two or more alleles containing a locus are identified by those skilled in the art, and a locus is defined as polymorphic when at least a common allele is present at a frequency of 1% or more To do.
IL-9R has four highly homologous (> 90% nucleotide identity) non-processing pseudogenes at other loci (chromosomes 9, 10, 16, 18) of the human genome It is impossible to specifically amplify true IL-9R (a gene encoding a biologically functional protein located in the XYq pseudoautosomal region) using standard primer designs there were. Because of the high identity of these other genes, genomic PCR amplification using a standard primer design causes simultaneous amplification of all genes, making true gene sequence analysis unclear. The true IL-9R structure is found in other diseases such as asthma or cancer described in this application (Renauld et al., Oncogene, 9: 1327-1332, 1994; Gruss et al., Cancer Res., 52: 1026-1031, 1992 In order to study the true IL-9R structure, Applicants designed a specific amplimer. This specific primer was found to be reliable for IL-9R amplification without the amplification of four pseudogenes. The primer sequences are shown in Example 2 and their specificity is demonstrated in FIG.
Applicants also amplified the entire coding region of IL-9 receptor cDNA by RT-PCR using RNA extracted from PBMC (peripheral blood mononuclear cells) purified from 50 donors. FIG. 1 shows the most frequent mutations found in the 50 people analyzed. Exons 3, 4, 5, 6 and 8 were affected by aberrant splicing events in samples where full-length cDNA could also be cloned. Some transcripts show a complete deletion of exon 3, which causes a frameshift and generates a stop codon after 79 unrelated residues. In the case of exon 4 deletion also, a frameshift occurred and the first codon in exon 5 was converted to a stop codon. In some other cDNAs, exon 5 showed a partial deletion of the first 5 or 29 nucleotides, both of which caused a frameshift and formed an early stop codon in exon 5. Thus, in all cases, the putative truncated protein will lack most of the extracellular domain and all of the transmembrane and cytoplasmic domains. If secreted, these forms may function as soluble receptors. Finally, the first three nucleotides of exon 6 corresponding to codon 173 are often removed by splicing, resulting in a deletion of glutamine at this codon position and no other mutations in the remaining protein sequence. . This splice variant is probably related to the variant (SEQ ID NO: 24) found in intron 5 of genomic DNA that increases the frequency of splice variants (Figure 17 and Example 12).
Applicants have also found allelic variations limited to the coding sequence of exon 9. Polymorphisms associated with codons 310 and 410 have already been disclosed^29,30(Kermouni, A. et al., Genomics, 371-382 (1995)). Codon 310 encodes arginine or glycine, respectively, depending on whether the first nucleotide of the codon is adenine or guanidine. Codon 410 (referred to as “410 + n” from this codon) begins a sequence of 8 or 9 AGC trinucleotide repeats that will be translated into 8 or 9 serines, respectively.
Applicants have found a new polymorphism at codon 344. Here the second nucleotide is either adenine or guanidine and the two possible residues encoded by this codon are histidine or arginine, respectively. In addition, a correspondence is observed between codons 344 and 410 + n, arginine at codon 344 is always present with 8 serines at codon 410 + n, and histidine at codon 344 is found with 9 serines. The human IL-9 receptor cDNA originally cloned from the human megakaryoblastic leukemia cell line Mo7e shows 9 serines at codon 410 + n and, unlike our clone, arginine at codon 344. Show²⁹. Another megakaryoblastic leukemia cell line, UT-7, has been reported to have the same arginine / 9-serine allele³⁰. Applicants have cloned 16 cDNAs from the Mo7e cell line and found that 6 clones have 8 serines at codon 410 + n and arginine at codon 344. The remaining 10 clones showed published sequences. Applicants have also genotyped human acute myeloid leukemia cell line KG-1 and found it to be a histidine / 9-serine homozygote.
RNA corresponding to these DNA molecules is isolated using standard methods in the art such as, for example, Sambrook et al., Molecular Clonig: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1985). . Applicants mean that isolated means that DNA is free of at least a portion of contaminants that are bound to nucleic acids or polypeptides present in the natural environment.
The present invention also includes proteins encoded by these nucleotide sequences. The invention also includes fragments of this molecule. Applicants mean that a fragment is a portion of a nucleic acid sequence that maintains the function of the entire sequence. As is known in the art, fragments are obtained from deletions, additions, substitutions and / or modifications.
The source of the IL-9 receptor variant of the present invention is human. Alternatively, DNA or a fragment thereof may be synthesized by a method known in the art. Also, using genetic engineering techniques to create DNA by any accepted genetic engineering technique, cloning the DNA into an expression vehicle, and transfecting the cells that express the compound with the vehicle to produce the compound. Can do. See, for example, the method described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1985).
Mutant IL-9 receptor sequences that may be associated with atopic allergy and asthma-like phenotypes, and evidence of other sequences that may be associated with the absence of asthma-like phenotypes diagnose susceptibility to atopic asthma and related diseases Provide a method. Certain mutants can produce soluble receptors that can be used to treat these diseases.
The receptor is a soluble or membrane-bound component that recognizes and binds to a molecule, and the IL-9 receptor of the present invention is a component that recognizes and binds to IL-9. The function of the IL-9 receptor consists of binding to IL-9 or an IL-9-like molecule and propagating its regulatory signals in specific cells^29,30,34,35. When human IL-9 binds to the human IL-9 receptor, it causes phosphorylation of the IL-9 receptor itself and activation of proteins in the Jak-Stat pathway (Jak1, Stat1, Stat3, Stat5, and Irs2) (Demoulin, JB. Et al., Molecular and Cellular Biology, p. 4710-4716, Sept. 1996). Applicants examined whether IL-9R or its mutants showed a bias in the activation of these proteins and extended this analysis to Jak3 and Irs1. All proteins in the pathway including Jak3 and Irs1 were confirmed to be phosphorylated by IL-9R activation. It was also confirmed that IL-9R mutants with mutations at codons 310, 344 and 410 + n provide the same upregulation as wild type IL-9R. Accordingly, one aspect of the present invention is a therapeutic agent for the treatment of atopic asthma that inhibits interactions in the Jak-Stat pathway.
Unlike the wild type receptor and other tested mutants, the ΔQ173 mutant was unable to activate any protein in the Jak-Stat pathway (FIG. 15). Furthermore, the ΔQ173 mutant was unable to support cell growth by IL-9 stimulation (FIG. 16). Therefore, individuals expressing the ΔQ173 mutant are unlikely to be susceptible to atopic asthma and related diseases. Accordingly, one aspect of the invention is a therapeutic agent that increases the expression of the ΔQ173 splice variant for the treatment of atopic asthma and related diseases.
One diagnostic embodiment is the recognition of mutations in the DNA sequence of the IL-9 receptor gene or transcript. One method uses a nucleic acid molecule (also known as a probe) having a sequence complementary to the IL-9 receptor of the present invention under sufficient hybridization conditions, as will be appreciated by those skilled in the art. It is to be. In one embodiment, the nucleic acid molecule will specifically bind to the codon of mature IL-9 receptor protein Arg344, or His344, and in another embodiment, will bind to both Arg344 and His344. In yet another embodiment, it will bind to the codon of Gln173. These methods may be used to recognize other variants of the IL-9 receptor. Another method for recognizing DNA sequence variations associated with these diseases is direct DNA sequence analysis by multiple methods known in the art.⁴⁴. Another embodiment is the detection of DNA sequence mutations in the IL-9 receptor gene associated with these diseases^40-44. These include the polymerase chain reaction, restriction fragment length polymorphism (RFLP) analysis, and single strand conformation analysis. In a preferred embodiment, the Applicants specifically describe a method for recognizing polymorphisms in the IL-9 receptor associated with the His344 and Arg344 alleles at the gene level using ASO PCR. provide. In other embodiments, a binding chain reaction can be used to distinguish these alleles of the IL-9 receptor gene.
The invention also includes methods for the identification of antagonists of IL-9 and its receptors. An antagonist is a compound that itself lacks pharmacological activity but causes an effect by interfering with the action of an agonist. To identify antagonists of the invention, one may test for competitive binding with known agonists or down-regulation of IL-9-like function as described herein and in the cited references.^2,22-35.
Specific measurements to screen for drugs useful for treating atopic allergy based on the known role of IL-9 receptors for T lymphocyte proliferation, IgE synthesis, and release from mast cells Law is used^29,30,33-35. Another assay relates to the ability of the human IL-9 receptor to specifically induce rapid and transient tyrosine phosphorylation of multiple proteins in Mo7e cells³⁴. Since this response is dependent on IL-9 receptor expression and activation, this is a convenient method or assay for the characterization of potentially useful compounds. Tyrosine phosphorylation of Stat3 transcription factor appears to be specifically associated with IL-9 receptor function³⁵This response represents a simple method or assay for characterization of compounds within the scope of the present invention. Yet another method for characterizing IL-9 receptor function is transfected with a human receptor that can be used to assess human IL-9 function using cell proliferation assays. Includes the use of known mouse TS1 clones²⁹. These methods can be used to identify IL-9 receptor antagonists.
In a further embodiment, the invention encompasses downregulation of IL-9 expression or function by administering a soluble IL-9 receptor molecule that binds to IL-9. Applicants and Renalud et al.²⁹Demonstrated the existence of a soluble form of the IL-9 receptor. This molecule can be used to block the binding of IL-9 to cell-bound receptors and acts as an antagonist of IL-9. Soluble receptors are used to bind cytokines or other ligands and control their function⁴⁵. A soluble receptor is a type of membrane-bound receptor that exists in solution or outside the membrane. Soluble receptors may be present because there are no molecular segments normally associated with the membrane. This segment is commonly referred to in the art as the transmembrane domain of a gene, or the membrane-bound segment of a protein. That is, in one embodiment of the invention, the soluble receptor is a fragment or analog of a membrane bound receptor.
Applicants have identified three splice variants of the human IL-9 receptor that cause the production of proteins that can act as soluble receptors. One splice variant deleted exon 4, which introduced a frameshift that became a stop codon as the first codon of exon 5. This variant produces an approximately 45 residue peptide containing an epitope reactive with an antibody that blocks IL-9 / IL-9R interaction. The other two variants have a deletion in exon 5, which creates an immature stop codon early in the exon, but in these cases there will be no deletion of exon 4. These variants will produce a protein of about 100 residues that also contains the epitope recognized by the blocking antibody.
Soluble IL-9 receptor may be used to screen drug candidates including antagonists useful for treating atopic asthma and related diseases. For example, screening of peptides and single chain antibodies using phage display could be facilitated using soluble receptors. Phage that bind to the soluble receptor can be isolated and the molecule can be identified by affinity capture of the receptor and bound phage. In addition, screening for compounds for agents useful in the treatment of atopic asthma and related diseases can incorporate ligands that bind in the absence of soluble receptors and antagonists. Because these molecules are in the vicinity, detection of ligand-receptor interactions occurs. Antagonists are recognized by blocking these interactions.
The present invention further includes pharmaceutical compositions comprising a compound of the present invention in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Martin, E.W., Remington's Pharmaceutical Sciences, which is specifically incorporated herein by reference.
The compounds used in the treatment methods of the present invention may be administered systemically or locally, taking into account the condition being treated, the need for site-specific treatment, the amount of drug administered, and the like. May be.
Topical administration may be used. Any common topical formulation (eg, solution, suspension, gel, ointment, salve, etc.) may be used. The preparation of such topical formulations is described in detail in the field of pharmaceutical formulations, as exemplified, for example, by Remington's Pharmaceutical Science, 17th Edition, Mack Publishing Company, Easton, Pa. For topical administration, these compounds can also be administered as powders or sprays (especially in aerosol form). In a preferred embodiment, the compounds of the invention may be administered by inhalation. For inhalation therapy, the compound may be a solution useful for administration in a dosage calibrated inhaler or in a form suitable for a dry powder inhaler.
The active ingredient is administered in a pharmaceutical composition suitable for systemic administration. As is known, if administered systemically, the drug may be prepared for oral administration as a powder, pill, tablet, etc., or as a syrup or elixir. For intravenous, intraperitoneal or intralesional administration, the compounds are prepared as solutions or suspensions that can be administered by injection. In some cases, it is useful to prepare these compounds as suppositories, or as long-term release formulations for subcutaneous sedimentation, or in formulations for intramuscular injection.
An effective amount is that amount that down-regulates a function regulated by the IL-9 receptor. The specific effective amount will vary from symptom to symptom, and in some cases will vary with the severity of the condition being treated and the patient's sensitivity to the treatment. Thus, a particular effective amount is optimally determined at that time and place by routine experimentation. However, in the treatment of asthma and related diseases of the present invention, a formulation containing 0.001-5% by weight, preferably about 0.01-1%, will usually be a therapeutically effective amount. When administered systemically, an amount of 0.01-100 mg / kg body weight / day, but preferably about 0.1-10 mg / kg, will often affect the outcome of treatment.
Applicants also provide a method of screening for compounds that down-regulate functions regulated by the IL-9 receptor. Whether the function expressed by the IL-9 receptor is down-regulated may be confirmed using standard methods in the art^29,30,34,35. In a specific embodiment, Applicants provide a method for identifying compounds with functions comparable to IL-9. In one embodiment, IL-9 receptor function is assessed in vitro. As known to those skilled in the art, activation of the human IL-9 receptor specifically induces rapid and transient tyrosine phosphorylation of multiple proteins in cells responsive to IL-9. Tyrosine phosphorylation of Stat3 transcription factor appears to be specifically associated with the action of the IL-9 pathway. Another way to characterize the function of IL-9 and IL-9-like molecules relies on “stable expression” of the IL-9 receptor in the mouse TS1 or TF1 clone, which usually Not expressed. These transfectants can be used to assess human IL-9 receptor function using cell proliferation assays.²⁹.
The invention also encompasses a convenient screening assay for saturating and specific ligand binding based on cell lines expressing IL-9 receptor variants.^23,29. IL-9 receptor is a broad range of cell types (K562, C8166-45, KG-1, B cells, T cells, mast cells transfected with human IL-9 receptor, HL60, HL60-clone 5, human IL TS1 transfected with -9 receptor, 32D transfected with human IL-9 receptor, neutrophils, megakaryocytes (UT-7 cells)³⁰The human megakaryoblastic leukemia cell line Mo7e³⁴, TF1²⁹, Macrophages, eosinophils, fetal thymocytes, human kidney cell line 293³⁰, And mouse 32D and embryonic hippocampal progenitor cell lines^23,29,30Expressed).
In practicing the present invention, conventional terms and techniques of molecular biology, pharmacy, immunology, and biochemistry are used, which are within the ordinary skill of the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, or Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
We will provide the following basic background information. The body's genetic material or DNA is lined up on 46 chromosomes, each containing two arms connected by a centromere. Each chromosome is divided into segments called p or q. The symbol p indicates the shorter chromosomal arm when measured from the centromere to the nearest telomere. The longer arm of the chromosome is indicated by the symbol q. The position on the chromosome is indicated by the chromosome number (ie chromosome 5) and the coordinates of the p or q region (ie q31-q33). The body also has sex chromosomes X and Y. During meiosis, the X and Y chromosomes exchange DNA sequence information in a region known as the pseudoautosomal region.
DNA (deoxyribonucleic acid) consists of two complementary strands of nucleotides, which contain four different base compounds, adenine (A), thymine (T), cytosine (C), and guanine (G). A on one strand binds to T on the other strand, C on one strand binds to G on the other strand to form complementary “base pairs”, with each pair in one strand Has one base.
A contiguous group of three nucleotides (“codon”) encodes one amino acid. For example, the three nucleotide CAG encodes the amino acid glutamine. The 20 naturally occurring amino acids and their one letter codes are as follows:
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Asparagine or
Aspartic acid Asx B
Cysteine Cys C
Glutamine Gln Q
Glutamate Glu E
Glutamine or
Glutamic acid Glx Z
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valin Val V
Amino acids constitute proteins. Amino acids may be hydrophilic (ie, exhibit affinity for water) or hydrophobic (ie, dislike water). That is, amino acids represented by G, A, V, L, I, P, F, Y, W, C, and M are hydrophobic, and S, Q, K, R, H, D, E, N, The amino acids denoted by and T are hydrophilic. In general, the hydrophilic or hydrophobic nature of amino acids affects the folding of the peptide chain and thus affects the three-dimensional structure of the protein.
DNA is related to proteins as follows:

Genomic DNA includes all DNA sequences present in the cells of an organism. This is “transcribed” into messenger RNA (“mRNA”). Complementary DNA (“cDNA”) is a complementary copy of mRNA made by reverse transcription of mRNA. Unlike genomic DNA, both mRNA and cDNA contain only the protein coding region or the polypeptide coding region of DNA (so-called “exons”). Genomic DNA also contains “introns”, which do not encode proteins.
Indeed, eukaryotic genes consist of exons interrupted by introns that are intermittent to the protein encoded thereby. After being transcribed into RNA, introns are removed by splicing to form mature messenger RNA (mRNA). The splice point between exons is typically determined by a consensus sequence that acts as a signal for the splicing process. Splicing consists of the deletion of an intron from the primary RNA transcript and the binding or fusion of the remaining RNA ends on either side of the cleaved intron. The presence or absence of introns, the composition of introns, and the number of introns per gene vary between strains of the same species and can also vary between species having the same basic functional gene. In most cases introns are not essential and are considered harmless, but this classification is not necessarily absolute. For example, an intron of one gene may be another exon. In some cases, alternative or different patterns of splicing may form different proteins from the same single stretch of DNA. In fact, intron structural features and basic splicing mechanisms are the basis for the classification of different types of introns.
For exons, these are distinct domains or motifs such as protein functional domains, folding domains, or structural elements; or short polypeptide sequences such as reverse turns, loops, glycosylation signals and other signal sequences, or non- Corresponds to structural polypeptide, linker region. The exon module of the combination method may contain a nucleic acid sequence corresponding to a naturally occurring exon sequence that has undergone a naturally occurring exon sequence or mutation (eg, point mutation, truncation, fusion). .
Returning to manipulation of DNA, DNA can be cleaved, spliced, and otherwise manipulated using a “restriction enzyme” that cleaves DNA at some known site and a DNA ligase that joins the DNA. Such techniques are known to those skilled in the art, for example Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1985), or Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons. , Inc. (1994).
DNA of a particular size and sequence can then be inserted into a “replicon”, which is a genetic component that can replicate under its own control, such as a plasmid, cosmid, or virus. A “recombinant vector” or “expression vector” is a replicon into which a DNA segment is inserted, allowing expression of the DNA (ie, production of a protein encoded by the DNA). Expression vectors can be made in the laboratory, obtained from other laboratories, or purchased commercially.
Recombinant vectors (known in the art under various names) are introduced into a host by a process commonly known as “transformation”. Transformation means the transfer of foreign DNA segments into the host cell by any method (infection, direct uptake, transduction, F-mating, microinjection, or electroporation).
Unicellular host cells (known under various names as recombinant host cells, cells and cell cultures) include bacteria, yeast, insect cells, plant cells, mammalian cells, and human cells. In a particularly preferred embodiment, the host cell comprises E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, R1-1, BW, LH, COS-J, Includes COS-7, BSC1, BSC40, BMT10, and S69 cells. Yeast cells include, among others, Saccharomyces, Pichia, Candida, Hansenula, and Torulopsis.
As one skilled in the art will recognize, expression of a DNA segment by a host cell requires appropriate regulatory sequences or components. The control sequences vary depending on the host cell used, but for example in prokaryotes there are promoters, ribosome binding sites and / or transcription termination sites. In eukaryotes, such control sequences include a promoter and / or a transcription termination site. As one skilled in the art will recognize, careful selection and placement of these control sequences can enhance the expression of the polypeptide, ie, raise it above standard levels.
In other embodiments, the promoter used includes human cytomegalovirus (CMV) promoter, tetracycline inducible promoter, simian virus (SV40) promoter, Moloney murine leukemia virus long terminal repeat (LTR) promoter, glucocorticoid Inducible mouse mammary tumor virus (MMTV) promoter, herpes thymidine kinase promoter, mouse and human actin promoter, HTLV1 and HIV IL-9 5 'flanking region, human and mouse IL-9 receptor 5' flanking region, bacterial tac promoter And Drosophila heat shock protein scaffold-binding domain (SAR) enhancer component.
DNA is expressed as a length of polypeptide such as peptides, oligopeptides, and proteins. Polypeptides also include translational modifications such as glycosylation, acetylation, phosphorylation and the like.
Another molecular biological method relevant to the present invention is “linkage analysis”. Linkage analysis is an analytical method for identifying chromosomes or chromosomal regions that correlate with traits or diseases⁴⁷. A chromosome is the basic unit of inheritance on which genes are assembled. In addition to genes, those skilled in the art have proven “DNA markers” on chromosomes. A DNA marker is a known sequence of DNA that can be easily identified and sequenced. Linkage analysis has been applied to mapping disease genes (eg, genes related to asthma susceptibility) to specific chromosomes.^47,48.
Applicants incorporate all of the above and below references herein by reference.
From the description herein and the practice of the disclosed invention, other embodiments of the invention will be apparent to those skilled in the art. The specification and examples are illustrative only and the scope of the invention is indicated only by the claims.
Since background information has been provided, Applicants will now describe preferred aspects of the invention.
Example 1
Identification of IL-9 receptor transcript polymorphism
A population of 52 people from the Philadelphia, Pennsylvania region was randomly identified for asthma and atopy. Total serum IgE was measured by enzyme-linked immunosorbent assay (ELISA, Genzyme, Cambridge, Massachusetts).
To assess the structural type of human IL-9 receptor cDNA, PBMCs were isolated from these 52 unrelated donors and cultured in the presence of PHA and PMA (described in Example 4). Our previous laboratory data demonstrated the kinetics of IL-9 receptor message expression in primary cultures 6 days after stimulation with mitogens. Applicants therefore cultured the cells for 6 days, at which point the cells were harvested and their RNA and DNA were isolated as described in Example 5.
RNA was reverse transcribed and amplified by PCR using primers specific for full-length IL-9 receptor cDNA as described in Example 5. Amplified products from each individual were cloned into a TA PCR cloning vector and 10 clones (measured by digestion and gel electrophoresis) containing the expected insert were fully sequenced and analyzed for structural or sequence changes.
Seven major mutants were identified from the above screen. These cDNAs are full length with a deletion of codon 173, a deletion of exon 4, two other deletions of exon 5, a deletion of exon 8, and an ARG to HIS mutation at codon 344 of the mature protein. cDNA is shown. There are additional mutants in each of these genetic backgrounds. The Arg allele is associated with 8 Ser / 4 Asn repeats and 7 Ser / 4 Asn repeats, and the His allele is associated with 9 Ser / 4 Asn repeats, 9 Ser / 3 Asn repeats, and 10 Ser / 2 Asn repeats . All of these variants are described in FIG.
Mutants were cloned into the eukaryotic expression vector pCEP4 (Clontech) containing the CMV promoter directing the expression of the cloned cDNA followed by the SV40 polyadenylation signal. This vector also contains a hygromycin B resistance gene, which is used to select eukaryotic cells that contain the vector and possibly express the cloned cDNA under the control of the CMV promoter. The recombinant plasmid was analyzed by sequence and the plasmid containing the correct cDNA insert was transformed into a eukaryotic receptor cell (eg, Syrian hamster fibroblast TK-ts13 as described in Example 3, human glioblastoma T98G , Human myeloid leukemia strain TF-1, and mouse bone marrow precursor cell line 32D). Function was assessed biologically as a response to IL-9 ligand in proliferation and apoptosis (Examples 7 and 10).
Experiments performed with full-length IL-9 receptor cDNA containing ARG and HIS variants are TK-ts13 hamster fibroblasts or human T98G glioblastoma cells. Cells were transfected and analyzed 48 hours later by Western blot and in situ staining using human specific carboxy terminal antibodies (Santa Cruz) (Example 8). In situ analysis demonstrated that both types of receptors appear to be expressed in both hamster and human systems (Figures 11 and 12). Interestingly, both types of Western blots are expressed at equivalent levels in human and hamster lines, and there is a difference in migration patterns between ARG and HIS receptor types in the TS1 cell line (Figure 12). Suggests differences in post-translational modifications such as glycosylation, phosphorylation, etc. This biochemical difference may be the mechanism by which changes in function cause phenotypic changes.
Various replacement frequencies were used as unbiased estimates of the prevalence of each variant in the general population. The questionnaire compared genotypes with phenotypes. Allergies and asthma were diagnosed by doctors after reviewing the questionnaire. Individuals who are homozygous for the Arg344 allele were significantly less likely to show evidence of allergy and asthma when compared to heterozygous or homozygous His344 (FIG. 9).
Example 2
Genome analysis of IL-9 receptor gene
To perform genome analysis of allergic and / or asthmatic individuals, PCR-specific primers are generated for the true IL-9 receptor gene located on the X / Y pseudoautosomal region and chromosomes 9, 10, 16 The following strategy was designed to eliminate the highly conserved IL-9 receptor pseudogene located on and 18th. First, a sequence alignment was performed between two published pseudogenes of the IL-9 receptor gene and the genomic sequence. Next, primers were designed at the branch region between the true gene and the pseudogene, and then analyzed by PCR using a single chromosome-specific hybrid obtained from Coreill DNA Repository (Camden, NJ). If the primers produced only the correct size product from the X and Y hybrids, they were optimized for robust amplification. In some cases, primers directed to the branch region are not XY-specific, so Applicants have increased the number of mismatches to the pseudogene compared to the IL-9 receptor gene sequence. In addition, additional base mutations in specific primers were introduced. Table 1 contains the primer sequences and optimal annealing temperatures for XY specific amplification. The specificity of these primers for XY amplification is shown in FIG.

These primers represent a novel method for analyzing DNA sequence variations in these genes that can be used to diagnose susceptibility and resistance to atopic asthma and related diseases.
Using this technique, each exon was examined by DNA sequence analysis for people in our population to detect sequence variations in the IL-9 receptor gene.⁴⁴. The analysis was repeated to distinguish sequence polymorphisms from artifacts. The relationship between receptor variants and allergic phenotypes is shown in FIG. The sequence of the receptor allele is shown in FIGS.
Example 3
IL-9 receptor expression and ligand binding assay
Fluorescent labeling of purified recombinant IL-9 and compounds that may be similar in structure or function to IL-9 with high specific activity, as recommended by the supplier (Pierce) using commercially available technology did. Growing human Mo7e and mouse 32D cells at 37 ° C, 10% (volume / volume) fetal calf serum, 50 mM 2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mM L-asparagine, and 1.25 mM L-glutamine, respectively Dulbecco's modified Eagle's medium supplemented with 0.8 ml or RPMI supplemented with 10% (volume / volume) fetal calf serum, 50 mM 2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mM L-asparagine, and 1.25 mM L-glutamine Resuspended. TF1.1 (TF1 cells lacking human IL-9 receptor), T98G, TK^-And mouse 32D cells (Examples 7 and 10) were used as such or after transfection with human IL-9 receptor constructs as described below. Plasmid DNA containing one of the full-length or truncated IL-9 receptors was cloned into the pCEP4 plasmid (Clontech) and purified by centrifugation using a Qiagen column (Qiagen). Immediately prior to electroporation, plasmid DNA (50 μg) was added to the cells in a 0.4 cm cuvette. Immediately after double electric pulses (750 V / 74 ohms / 40 microfarads and 100 V / 74 ohms / 2100 microfarads), the cells were diluted in fresh medium supplemented with IL-9.
Stable transfected cells were obtained after 14 days of selection with hygromycin B (400 μg / ml to 1.6 mg / ml). Hygromycin resistant clones were analyzed for IL-9 receptor expression by Western blot and in situ staining as described in Example 8.
Visualize cell receptor binding directly in real time with a fluorescence microscope. Binding and internalization are followed over time in control cells (untransfected) and cells transfected with each of the known IL-9 receptor variants. To demonstrate specific binding, excess unlabeled ligand or blocking antibody is used in parallel experiments.
Soluble IL-9 receptor comprising amino acids 44-270, with or without HA ditag, is also incubated with different types of human labeled recombinant IL-9. Different amounts of FLAG-tagged (IBI) ligand are incubated with 0.5 μg of soluble receptor in PBS for 30 minutes at room temperature. EBC buffer (50 mM Tris, pH 7.5; 0.1 M NaCl; 0.5% NP40) is added (300 μl) together with 1 μg of anti-HA antibody or anti-FLAG monoclonal antibody (IBI) and incubated on ice for 1 hour. 40 μl of Protein A Sepharose solution was added to each sample and mixed at 4 ° C. for 1 hour. The sample was centrifuged at 11,000 × G for 1 minute and the pellet was washed 4 times with 500 μl EBC. The pellet was dissolved in 26 μl of 2 × SDS buffer, boiled for 4 minutes and electrophoresed on an 18% SDS polyacrylamide gel. Western blots were picked up with anti-IL-9 receptor antibody (Santa Cruz Inc.) or anti-FLAG monoclonal antibody (IBI) against FLAG-tagged rhIL-9. Candidate substances as therapeutic agents are evaluated by measuring antagonism of ligand binding. Receptor expression is shown in FIGS.
Example 4
Cell isolation and culture
Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by density gradient centrifugation using Ficoll-Paque PLUS, which was endotoxin tested according to the manufacturer (Pharmacia Biotech, AB Uppsala Sweden). In 7 ml of RPMI-1640 (Bethesda Research Labs (BRL), Bethesda, MD) supplemented with human serum of the same gene or heat-inactivated FBS to a final concentration of 10%, PBMC (5 × 10 5⁶), Mouse spleen cells (5 × 10⁶) Or 5 × 10⁶Individual Mo7e cells were cultured. Cells are stimulated without stimulation or with PMA 5 μg / ml / PHA 5 μg / ml, or PHA 5 μg / ml and rhIL2 50 ng / ml (R & D Systems, Minneapolis, Minn.) And cultured at 37 ° C. for 24 hours did.
Example 5
DNA and RNA isolation, rtPCR, cloning, and sequencing of PCR products
Nicolaides and Stoeckert⁴⁶As described above, cytoplasmic RNA and genomic DNA were extracted from cultured PBMC after stimulation with mitogen for 6 days. 1 μg RNA from each source was denatured at 70 ° C. for> 10 minutes, then 2.5 units Superscript II reverse transcriptase (GIBCO, BRL), 1 U / l RNAse inhibitor, 2.5 mM oligo d (T) 16 primer, 1 mM each of dATP, dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCl (pH 7.0), 25 mM MgCl₂Was reverse transcribed (V +) into cDNA for 1 hour at 37 ° C. A mock reverse transcription reaction was used as a negative control.
1/20 of the reverse transcription reaction product, 6.7 mM MgCl₂, 16.6mM (NH_Four)₂SO_Four, 67 mM Tris-HCl (pH 8.8), 10 mM 2-mercaptoethanol, 6% DMSO, 1.25 mM each dNTP, 2.5 U Amplitaq DNA polymerase, and human cDNA IL-9 exon 2 (forward 5'-GCT GGA CCT TGG PCR (50 μl) containing 300 ng oligonucleotides corresponding to AGA GTG-3 ′) (SEQ ID NO: 1) and exon 9 (reverse 5′-GTC TCA GAC AAG GGC TCC AG-3 ′) (SEQ ID NO: 2) ) Used in. The reaction mixture was subjected to the following PCR conditions: 35 cycles of 95 ° C for 120 seconds, then 94 ° C for 30 seconds, 58 ° C for 90 seconds, 72 ° C for 90 seconds. Finally, one cycle of 15 minutes at 72 ° C. was performed for extension of the reaction mixture.
PCR products showing hIL-9 receptor cDNA were subjected to gel electrophoresis on a 1.5% agarose gel and visualized using ethidium bromide staining. Pseudo reverse transcription reaction product (where H instead of RNA₂O was used) as a negative control amplification in all experiments.
The PCR product was subcloned into a TA cloning vector (Invitrogen, San Diego, Calif.). Amplification of human cDNA resulted in a 1614 base pair product. A plasmid containing the hIL-9 receptor cDNA insert was isolated by conventional methods (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). After amplification, the DNA sequence containing and surrounding each insert is sequenced using a fluorescent dideoxy terminator with an automated sequencer (ABI 377, Perkin Elmer) for the measurement of PCR- or cloning-induced errors. Analysis by determination (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463). HIL-9 receptor cDNA inserts without cloning and / or polymerase-induced sequence errors were subcloned into expression vectors.
Example 6
Cloning and expression of IL-9 receptor constructs in vitro
The hIL-9 receptor was subcloned into the episomal eukaryotic expression vector pCEP4 (Clontech). The insert was cloned as a BamH1-XhoI fragment in the pCEP4 polylinker in a sense orientation relative to the CMV promoter using standard methods (FIG. 10). The construct was expressed in the cellular host as described.
Example 7
(Mo7e, 32D, TF1.1, TK ^- Cell assay using ts13, and T98G)
Cell lines were used to evaluate the function of mutant IL-9 receptors and to screen for compounds effective in the treatment of atopic asthma. Compounds were tested for their ability to antagonize IL-9-induced anti-apoptotic or baseline proliferative responses. Once a baseline anti-apoptotic or proliferative response was established in a cell line, a statistically significant decrease in response in a triple triplicate assay was considered evidence of antagonism. The true antagonistic response was distinguished from cytotoxicity by direct observation, trypan blue staining (technique known to those skilled in the art), or reduced acid phosphatase activity. Each compound was evaluated for specificity of antagonism by assessing whether activity was demonstrated against other proliferative substances (eg, interleukin 3 or interleukin 4).
Recombinant IL-9 or compounds that may resemble IL-9 in structure or function were purified and prepared for use in appropriate media. Putative agonists and antagonists were prepared in water, saline, or DMSO and water. Cells were used as such or after transfection with IL-9 receptor construct as described in Example 3. After 24 hours of depletion of growth factors, cells were incubated with or without variable amounts of purified IL-9 or compounds that might resemble IL-9 in structure or function.
Cell proliferation was assessed using the Abacus Cell Proliferation Kit (Clontech, Palo Alto, Calif.), Which measures the amount of acid phosphatase present in the cells as an indicator of cell number. The substrate p-nitrophenyl phosphate (pNPP) was converted to p-nitrophenol by acid phosphatase and measured as an indicator of enzyme concentration. PNPP was added to each well and incubated at 37 ° C. for 1 hour. 1N sodium hydroxide was then added to stop the enzyme reaction and the amount of p-nitrophenol was quantified at 410 nm using a Dynatech 2000 plate reader (Dynatech Laboratories, Chantilly, VA). A standard curve comparing cell number and absorbance was used to determine the linear range of the assay. Measurement results were used only when the absorbance measurements were within the linear range of the assay. Briefly, assays were performed at 37 ° C. for 72-96 hours using quadruple cell samples in flat bottom microtiter plates (150 or 200 μl wells) with or without ligand. Acid phosphatase was used as a measure of the number of cells present. All experiments were repeated at least twice.
Apoptosis was measured as described by the supplier (Clontech) using the Annexin V kit, which measures dexamethasone-induced apoptosis by recognizing extracellular phosphatidylserine, an early marker of apoptosis. Apoptotic cell numbers were scored by fluorescence microscopy as a percentage of Annexin V stained cells.
Mo7e is a human megakaryoblastic cell line in RPMI 1640 (GIBCO / BRL, Gaithersburg, MD), 20% fetal calf serum (Hyclone) and 10 ng / ml IL-3 (R & D Systems, Minneapolis, MN) In culture. The T98G line is a human glioblastoma cell line grown in RPMI 1640 (GIBCO / BRL). The hamster fibroblast TK-ts13 line is also used with the mouse 32D cell line, which is the mouse myeloma progenitor line, and both are cultured in RPMI 1640 (GIBCO / BRL) containing 10% fetal calf serum (Hyclone), In addition, 32 ng cell line used 1 ng / ml IL-3. TF1.1 is a human myeloid leukemia line known to express the IL-2 receptor gamma subunit (confirmed by Western blot and rtPCR), but compared to its ancestor (TF1), rtPCR No IL-9 receptor already present by immunostaining and Western blot analysis. TF1.1 was cultured in RPMI 1640 (GIBCO / BRL) and 10% fetal calf serum (Hyclone). All cell lines respond to multiple cytokines including IL-9. Nourish the cell line and again 2 × 10 every 72 hours^FiveRe-inoculated with cells / ml.
Cells are centrifuged at 2000 rpm for 10 minutes in RPMI 1640 containing 0.5% bovine serum albumin (GIBCO / BRL, Gaithersburg, MD) and insulin-transferrin-selenium (ITS) cofactor (GIBCO / BRL, Gaithersburg, MD) Resuspended. Count cells with a hemocytometer, 1 × 10^FiveDilute to cells / ml and seed into 96-well microtiter plates. Each well contained 0.15 or 0.2 ml and gave a final concentration of 10,000-50,000 cells / well with cells. Mo7e cells are treated with 50 ng / ml stem cell factor (SCF) (R & D Systems, Minneapolis, MN) alone, 50 ng / ml SCF plus 50 ng / ml IL-3 (R & D Systems, Minneapolis, MN), or 50 ng / ml Stimulation with ml SCF plus 50 ng / ml IL-9. A control containing only cells and basal medium was included. Serial dilutions of test compounds (ie recombinant IL-9 protein, peptide, small molecule) were added to each test condition in triplicate. TF1.1 cells that were not transfected with IL-9 receptor were used as independent controls for response and non-specific cytotoxicity. 5% CO in culture₂Incubated for 72-96 hours at 37 ° C.
Example 8
In situ and western analysis of exogenous IL-9 receptor in transfected cell lines
In situ staining of IL-9 receptor was performed as follows. Cells are grown on coverslips for 24 hours, then coverslips containing adherent cells are washed twice with phosphate buffered saline (PBS) containing calcium and magnesium (Gibco / BRL) did. For intracellular staining of IL-9 receptor, cells were fixed in 4% paraformaldehyde / PBS plus 0.1% Triton-X for 15 minutes at room temperature and then treated with anti-human IL-9 receptor antibody did. For extracellular staining, cells were treated with antibody before fixation. The cells are then washed twice with PBS and dH₂Blocked with 7.5% BSA in O for 30 minutes at room temperature. The cells washed with PBS were then incubated with a 10 μg / ml solution of anti-human IL-9 receptor (polyclonal antibody against the carboxy terminus of IL-9 receptor) in 1% BSA / PBS for 1 hour at room temperature. . Cells were washed three times with PBS and then incubated for 30 minutes at room temperature in a 10 μg / ml solution of anti-rabbit rhodamine-conjugated antibody in 1% BSA / PBS. Cells were then washed 3 times with PBS and counterstained for 1 minute at room temperature using 1 μg / ml DAPI. dH₂Cells were washed 3 times in O, fixed on a microscope slide and analyzed by fluorescence microscopy. The results for transfected COS7 cells are shown in FIG.
Protein lysates from direct lysis of cell extracts in 0.5% lysis buffer (Tris 50 mM, NaCl 150 mM, NP40), 1 mM DTT and protease inhibitors were Western blotted and boiled for 5 minutes. Samples were electrophoresed on 4-20% Tris-Glycine SDS gel (Novex) in Tris-Glycine running buffer. The protein was then transferred to nitrocellulose by electroblotting using a Trans Blot II instrument (Bio Rad). After transfer, membranes were blocked in TBS-T ((20 mM Tris, 137 mM NaCl, pH 7.6) plus 0.05% Tween 20) plus 5% blot (blotto) for 1 hour at room temperature. The blot was then searched for 1 hour using a polyclonal antibody (1 μg / ml) against the carboxy terminus of the IL-9 receptor in TBS-T. The blot was then washed 3 times for 10 minutes in TBS-T and looked for 30 minutes using a second anti-rabbit horseradish peroxidase conjugated antibody (1: 10,000) in TBS-T. Blots were washed as above, incubated with the chemiluminescent substrate luminol / enhancer solution (Pierce) for 5 minutes at room temperature, and then exposed to film for 1-60 seconds. See Figures 11 and 12.
Example 9
True IL-9R genome amplification method
IL-9R has four highly homologous (> 90% nucleotide identity) unprocessed pseudogenes at other loci (chromosomes 9, 10, 16, and 18) of the human genome. Specific amplification of true IL-9R (a gene encoding a biologically functional protein located in the XYq pseudoautosomal region) using standard primer designs was not possible. Because of the high degree of identity of these other genes, genomic PCR amplification using a standard primer design resulted in simultaneous amplification of all genes, thus obscure true gene sequence analysis. The true IL-9R structure is found in other diseases such as asthma or cancer described in this application (Renauld et al., Oncogene, 9: 1327-1332, 1994; Gruss et al., Cancer Res., 52: 1026-1031, 1992 In order to study its structure, a specific amplimer was designed as follows:
The sequences of IL-9R pseudogene and true gene were aligned using Mac Vector software. Next, the intron sequence around each exon was examined for the region branched between the true gene and the pseudogene. Primers were then designed for these regions and used to PCR amplify human / rodent hybrid DNA containing each human chromosome. The product was run on a 3% agarose gel and analyzed for true IL-9R amplification without amplification of 4 pseudogenes. Specific PCR amplification conditions were also optimized by changing the annealing temperature and buffer conditions (DMSO content 5-10%). If there was still pseudogene amplification, a nucleotide mutation was introduced into the primer sequence, resulting in a larger branch from the pseudogene compared to the true gene. Primer sequences are shown in Example 2 and their specificity is shown in FIG.
Example 10
Cell proliferation assay and cytokine stimulation
To examine the proliferative response of TS1 cells expressing various types of hi-IL-9 receptor, the cells were washed with PBS and resuspended in D-MEM, 10% fetal calf serum. Ten^ThreeCells / well were seeded in triplicate in 96-well microplates and recombinant human IL-9 or mouse IL-9 (R & D Systems, Minneapolis, Minn.) Was added at a final concentration of 5 ng / ml as appropriate. . Cell growth was assessed after 7 days using an acid phosphatase assay. Briefly, 50 μl of buffer containing 0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100 and 10 mM p-nitrophenyl phosphate (Sigma 104 phosphatase substrate) was added to the wells. Plates were incubated for 1-0.5 hours at room temperature, quenched with 10 μl / well of 1N sodium hydroxide, and absorbance read at 410 nm on a Dynatech Model MR600. To analyze tyrosine phosphorylation of proteins in the signal transduction cascade following cytokine stimulation, TS1 cells expressing various types of human IL-9 receptors were washed with PBS and added to D-MEM, 0.5% bovine serum albumin. Resuspend and incubate at 37 ° C. for 6 hours. Next, 20 × 10⁶Cells were treated with human IL-9 or mouse IL-9 (100 ng / ml) for 5 minutes and immediately washed with cold PBS. Cells were lysed with RIPA buffer as described in Example 11.
Example 11
Immunoprecipitation, immunoblotting and antibodies
Typically 20-50x10⁶Cells were washed with 1 ml RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 50 mM sodium fluoride, 1 nM sodium orthovanadate, and PBS containing 1 × “complete” protease inhibitor mixture. No. 1697498 Boehringer Mannheim) and incubated on ice for 45 minutes. The lysate was centrifuged for 20 minutes in an Eppendorf microcentrifuge and the supernatant was collected and transferred to a new tube. For immunoprecipitation, 1-5 μg of antibody was added to the lysate and incubated overnight at 4 ° C. 20 ml of protein A + G agarose-bound beads were added for 2 hours and then washed 4 times using RIPA buffer. The beads were resuspended in Laemmli buffer, boiled for 3 minutes and then electrophoresed. The protein was transferred to an Immobilion-P membrane (Millipore) and detected by a chemiluminescent detection assay (Pierce) using a horseradish peroxidase-conjugated secondary antibody. Mouse and human IL-9 receptor specific antibodies (sc698), mouse Jak1, Irs1, Irs2, Stat1, Stat2, Stat3, Stat4, Stat5, and phosphotyrosine (PY) are purchased from Santa Cruz (Santa Cruz, CA) did. Anti-Jak3 and monoclonal anti-human IL-9 receptor MAB290 were purchased from Upstate Biotechnology and R & D Systems, respectively. FIG. 15 demonstrates the activation of Jak family members by different variants of the human IL-9 receptor.
Example 12
Identification of IL-9 receptor genomic polymorphism
Genomic DNA was isolated from volunteer donor PBMC as previously described (Nicolaides and Stoeckert, Biotechniques 8: 154-156, 1990). Sequence analysis of intron 5 of the human IL-9R gene was performed by PCR using the primers of SEQ ID NO: 14 and SEQ ID NO: 17, and a product having a molecular size of about 1243 base pairs was obtained. Amplification was carried out with 35 cycles of the buffer already described (Nicholaides et al., Genomics 30: 195-206, 1995) at 94 ° C for 30 seconds, 62 ° C for 1.5 minutes, and 72 ° C for 1.5 minutes. The product was then purified and sequenced using standard sequencing protocols. Examination of the sequence from intron 5 of multiple individuals revealed that there was a nucleotide change upstream of -213 nucleotides in the sequence of exon 6, and thus thymidine (the published sequence) was changed to a cytosine nucleotide. An example of this mutation is shown in FIG.
Although the invention has been described and illustrated with reference to various specific materials, methods and examples, the invention is not limited to the specific combinations and methods of materials selected for this purpose. I want you to understand. Many variations of such details are possible as will be appreciated by those skilled in the art.
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Claims (1)

A method of detecting susceptibility to asthma or related diseases in a human subject comprising determining the presence or absence of a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 4 in a sample, wherein existence of the nucleic acid molecules exhibit a lower susceptibility than for asthma or related disease.

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