JP3778922B2 - Intercellular adhesion molecule and its binding ligand - Google Patents

Description

上記等式中の入れた細胞の数はインキュベートしていない細胞と完全培地のみを含有するコントロールチューブ中のmｌ当りの細胞数である。上記等式中の遊離細胞の数は試験チューブからのmｌ当りの非凝集細胞の数に等しい。上記の手順は“Rothlein, R.等、J. Exper. Med.１６３：１１３２−１１４９（１９８６）”によって開示されている。
実施例３
ＬＦＡ−１依存性細胞凝集
実施例２で記載した定量的凝集アッセイをエプスタイン−バールウイルス形質転換細胞系ＪＹを用いて行った。マイクロタイタープレート中の培地にＰＭＡを加えて、細胞凝集を観察した。時間経過ビデオ記録計はマイクロタイターウェル底部のＪＹ細胞が動いており活性な膜波動および仮足運動を示していた。隣接細胞の仮足間の接触はしばしば細胞−細胞粘着をもたらした。粘着が持続した場合、細胞接触領域は尾脚（uropod）に移動した。接触は激しい細胞運動および反対方向への細胞の引っ張り合いにも依らず維持できた。PMA処理および未処理細胞間の主な違いは１度形成された上記接触の安定性において現われていた。ＰＭＡにおいては、細胞密集が出現し、その周りに粘着した追加の細胞として粘度の成長があった。
粘着を測定する第２の手段としては、実施例２で記載した定量的アッセイを用いた。細胞懸濁液を２時間、２００rpmで振り、ヘモサイトメーターに移し、凝集物中に含まれない細胞を計数した。ＰＭＡの不存在では、４２％（ＳＤ＝２０％、Ｎ＝６）のＪＹ細胞が２時間後、凝集物中にあったが、５０μｇ／mｌのＰＨＡと共に同じ条件下でインキュベートしたＪＹ細胞は凝集物中に８７％（ＳＤ＝８％、Ｎ＝６）の細胞を有していた。凝集の動力学的検討はＰＭＡがすべての試験時間で凝集速度および強度を促進していることを示した（第３図）。
実施例４
抗ＬＦＡ−１モノクローナル抗体を用いての細胞の凝集抑制
ＰＭＡ誘起細胞凝集への抗ＬＦＡ−１モノクローナル抗体の効果を試験するために、そのような抗体を実施例２の定量凝集アッセイに従ってインキュベートした細胞に加えた。このモノクローナル抗体はＰＭＡの存在または不存在下のいずれにおいても細胞凝集の形成を抑制していることが判った。ＬＦＡ−１のアルファー鎖に対するモノクローナル抗体のF（ab′）₂およびFab′フラグメントは双方とも細胞凝集を抑制できた。一方、本質的に１００％の細胞が抗ＬＦＡ−１抗体の不存在下では凝集を形成し、抗体を加えたときは２０％以下の細胞が凝集物中に見い出された。この実験の結果はRothlein, R.等により開示された〔J. Exper. Med. １６３：１１３２−１１４９（１９８６）〕。
実施例５
細胞凝集はＬＦＡ−１レセプターを必要とする：
ＥＢＶ形質転換リンパ芽球細胞を実施例１で記載した方法により患者から調製した。この細胞をＬＦＡ−１を認識し得るモノクローナル抗体に対してスクリーニングし、細胞がＬＦＡ−１欠損であることを見い出した。
実施例２で記載した定量凝集アッセイを上記ＬＦＡ−１欠損細胞を用いて行った。この細胞はＰＭＡの存在においても自発的に凝集しなかった。
実施例６
ＩＣＡＭ−１の発見
実施例５のＬＦＡ−１欠損細胞をカルボキシフルオレスセインジアセテートで標識した〔Patarroyo, M.等、Cell. Immunol.６３：237−２４８（１９８１）〕。標識細胞を同原またはＪＹ細胞と１：１０の比で混合し凝集物中のフルオレスセイン標識細胞の割合を“Rothlein, R.等、J. Exper. Med.１６３：１１３２−１１４９（１９８６）”の方法に従って測定した。ＬＦＡ−１欠損細胞はＬＦＡ−１発現性細胞と共凝集し得ることを見い出した（第４図）。
ＬＦＡ−１が凝集形成またはその維持にのみに重要であるかどうかを決定するために、ＬＦＡ−１に結合し得る抗体を上記の前以って形成させた凝集に加えた。抗体の添加は上記の前以って形成させた凝集を強く分裂させることが判った。時間経過ビデオ記録計は前以って形成させた凝集へのモノクローナル抗体の添加が２時間以内で分裂を生じ始めることを示した（第１表）。ＬＦＡ−１に対するモノクローナル抗体の添加後、凝集中の個々の細胞の促足運動および形状変化は変らないまま続いた。個々の細胞は凝集物の周囲から次第に解離し、８時間までに細胞は殆んど分散した。ビデオ時間経過によれば、ＬＦＡ−１モノクローナル抗体による前以って形成させた凝集の分裂は時間を逆のぼって行ったＬＦＡ−１モノクローナル抗体の不存在下での凝集過程と等価であった。

実施例７
ＬＦＡ−１依存性凝集における２価イオンの必要性
細胞毒性Ｔ細胞とターゲット間のＬＦＡ−１依存性粘着はマグネシウムの存在を必要とする〔Martz, E.,J. Cell., Boll. ８４：５８４、５９８（１９８０）〕、ＰＭＡ誘起ＪＹ細胞凝集を２価カチオン依存性について試験した。ＪＹ細胞はカルシウムまたはマグネシウムイオンを含まない培地中では凝集しなかった（実施例２のアッセイを用いて）。２価マグネシウムの添加は０．３ｍＭ程の低い濃度で凝集を行った。カルシウムイオン単独の添加は殆んど効果がなかった。しかしながら、カルシウムイオンはマグネシウムイオンのＰＭＡ誘起凝集を補佐する能力を増強することが判った。１．２５ｍＭのカルシウムイオンを培地に加えたときは、０．０２ｍＭ程の低いマグネシウムイオン濃度で凝集を補佐することが判った。これらのデータは細胞のＬＦＡ−１依存性凝集がマグネシウムイオンを必要とすること、およびカルシウムイオンがそれ自体は不十分であるけれどもマグネシウムイオンと共に凝集を増大させることを示している。
実施例８
抗ＩＣＡＭ−１モノクローナル抗体を発現し得るハイブリドーマ細胞の単離
ＩＣＡＭ−１に結合し得るモノクローナル抗体を“Rothlein, R.等、J. Immunol. １３７：1270−１２７４（１９８６）”の方法に従って単離した。該文献は参考として本明細書に引用する。即ち、３匹のＢＡＬＢ／ＣマウスをＬＦＡ−１欠損患者からのＥＢＶ形質転換末梢血単核細胞で腹腔内免疫した（Springer, T. A.等、J. Exper. Med.１６０：１９０１（１９８４））。１mｌＲＰＭＬ１６４０培地中約１０⁷個の細胞を各免疫に用いた。免疫はひ臓細胞をマウスから取り出す前の４５日、２９日および４日で行い所望のハイブリドーマ細胞系を産生させた。ひ臓細胞の取り出し前３日に、マウスに追加の０．１５mｌ培地中１０⁷細胞を投与した（静注）。
上記の動物からの単離ひ臓細胞をＰ３×７３Ag８．６５３ミエローマ細胞と４：１の比率で“Galfre, G.等、Nature２６６：５５０（1977）”のプロトコールに従って融合させた。得られたハイブリドーマ細胞の各アリコートを９６ウェルマイクロタイタープレートに入れた。各ハイブリドーマ上清を凝集抑制についてスクリーニングし、１つの抑制ハイブリドーマ（６００以上のすべてのウェル試験のうちから）をクローニングし限定稀釈によりサブクローニングした。このサブクローンはＲＰ１／１．１．１．と表示した（以後“ＲＰ１／１”と表示する）。
モノクローナル抗体ＲＰ１／１はＬＦＡ−１発現性細胞系ＪＹのＰＭＡ刺激凝集を一貫して抑制することを見い出した。ＲＰ１／１モノクローナル抗体はいくつかのＬＦＡ−１αまたはβサブユニットに対するモノクローナル抗体よりも等価かわずかに小さく凝集を抑制した。これに対し、コントロールのＨＬＡに対するモノクローナル抗体（これはＪＹ細胞上に多量に発現する）は凝集を抑制しなかった。モノクローナル抗体ＲＰ１／１と結合した抗原は細胞間粘着分子−１（ＩＣＡＭ−１）と定義する。
実施例９
ＩＣＡＭ−１を特性決定するための抗ＩＣＡＭ−１モノクローナル抗体の使用
ＩＣＡＭ−１の性質を限定するために、特にＩＣＡＭ−１がＬＦＡ−１と異なるかどうかを決定するために、細胞たん白質をモノクローナル抗体ＲＰ１／１を用いて免疫沈降させた。免疫沈降は“Rothlein, R.等の方法に従って行った〔J. Immunol.１３７：１２７０−１２７４（1986）〕。ＪＹ細胞を新たに１ｍＭフェニルメチルスルホニルフルオライド、０．２単位／mｌのトリプシンインヒビターアプロチニンを加えた１％トリトンＸ−１００、０．１４ｍのNaCｌ、１０ｍＭのトリス、pH８．０（溶解バッファー）中で５×１０⁷細胞／mｌで２０分間、４℃で溶解させた。溶解物を１０，０００ｘｇで１０分間遠心しＣＮＢｒ活性化グリシン抑制セファローズＣｌ−４Ｂの５０％懸濁液５０mｌで１時間、４℃で前処理した。１mｌの溶解物をセファローズＣｌ−４Ｂに結合させたモノクローナル抗体（１mg／mｌ）の５０％懸濁液の２０μｌで４℃で一夜免疫沈降させた〔Springer, T. A.等、J. Exper. Med.１６０：１９０１（１９８４）〕。セファローズ結合モノクローナル抗体は“March, S.等、の方法に従って炭酸塩バッファー中のセファローズＣｌ−４ＢのＣＮＢｒ活性化法を用いて調製した〔Anal.Biochem.６０：１４９（１９７４）〕。洗浄した免疫沈降物をＳＤＳ−ＰＡＧＥおよび銀染色に“Morrissey, J. H. Anal. Biochem. １１７：３０７（１９８１）”の手順に従って供した。
ＳＤＳサンプルバッファー〔ＨＤ、M. K.等、J. Biol. Chem.２５８：６３６（１９８３）〕による１００℃でのたん白質溶出後、各サンプルを半分に分けて還元（第５Ａ図）または非還元（第５Ｂ図）の各条件下で電気泳動（ＳＤＳ−８％ＰＡＧＥ）に供した。分子量５０Kdおよび２５Kdを有するバンドはモノクローナル抗体セファローズからの免疫グロブリンの長鎖および短鎖に相当した（第５Ａ図、レーン３）。２５〜５０Kd分子量範囲の可変量の他のバンドも観察したが、ヘアリー白血病細胞からの沈降物中では見られず、この白血病細胞は９０Kd分子量バンドのみを与えた。ＬＦＡ−１の１７７Kdαサブユニットおよび９５Kdβサブユニットは還元（第５Ａ図、レーン２）および非還元（第５Ｂ図、レーン２）の両条件下でＩＣＡＭ−１とは異なって浸透した。
ＰＨＡ−リンパ芽球凝集に対してのモノクローナル抗体ＲＰ１／１の効果を測定するために、実施例２で記載した定量凝集アッセイを用いた。即ち、Ｔ細胞芽球細胞をＰＨＡで４日間刺激し、十分に洗浄し、次いで１Ｌ−２状態調節培地の存在下に６日間培養した。ＰＨＡはこの６日間培養で取り込まれ凝集アッセイに寄与しなかった。異なるＴ細胞芽球調製物による３種のアッセイにおいて、ＩＣＡＭ−１モノクローナル抗体は一貫して凝集を抑制した（第２表）。

ＬＦＡ−１モノクローナル抗体はＩＣＡＭ−１モノクローナル抗体よりも一貫して抑制的であり、一方ＨＬＡ−Ａ、ＢおよびＬＦＡ−３モノクローナル抗体は効果なしであった。これらの結果は試験したモノクローナル抗体のうち、ＬＦＡ−１またはＩＣＡＭ−１に結合し得るモノクローナル抗体のみが細胞粘着を抑制し得ることを示している。
実施例１０
ＩＣＡＭ−１に対するモノクローナル抗体の調製
免疫
Balb／ｃマウスをＲＰＭＩ培地中の２×１０⁷ＪＹ細胞０．５mｌで融合前の１０３日および２４日で腹腔内（i.p.）免疫した。融合前４日および３日に、マウスを０．５mｌのＲＰＭ１培地中のＰＭＡ分化Ｕ９３７細胞でi.p.免疫した。
Ｕ９３７細胞の分化
Ｕ９３７細胞（ＡＴＣＣ ＣＲＬ−１５９３）を１０％のウシ胎児血清、１％グルタミンおよび５０mｌの２ng／mｌホルボール−１２−ミリステートアセテート（ＰＭＡ）含有ジエタマイシン（完全培地）を含むＲＰＭＩ中５×１０⁵／mｌで滅菌ポリプロピレン容器中でインキュベートすることによって分化した。このインキュベーションの第３日で、¹/₂容量の培地を除去し新しいＰＭＨ含有完全培地で置き換えた。４日目で、細胞を取り出し、洗浄して免疫用に調製した。
融合
免疫マウスからのひ臓細胞をGalfre等に従ってＰ３×６３Ag８．６５３ミエローマ細胞と４：１の比で融合させた〔Nature２６６：５５０（１９７７）〕。融合後、細胞を９６ウェル平底マイクロタイタープレートに１０⁵ひ臓細胞／ウェルで入れた。
抗ＩＣＡＭ−１陽性細胞の選択
１週間後、５０μｌの上清を凝集用細胞系としてＪＹおよびＳＫＷ−３の両方を用いて実施例２の定量凝集アッセイでスクリーニングした。ＪＹ細胞凝集を抑制するがＳＫＷ−３凝集は抑制しない上清からの細胞を選択し限定稀釈を用いて２回クローニングした。
この実験により、抗ＩＣＡＭ−１モノクローナル抗体を産生する３種のハイブリドーマ系の同定およびクローニングができた。これらのハイブリドーマ系により産生した抗体は、それぞれ、IgG_2a、IgG_2bおよびIgMであった。IgG_2a抗ICAM−１抗体を産生するハイブリドーマ細胞系は表示Ｒ６′５′Ｄ６′Ｅ９′Ｂ２とした。この好ましいハイブリドーマ細胞系により産生した抗体はＲ６′５′Ｄ６′Ｅ９′Ｂ２と表示した（以下、“Ｒ６−５−Ｄ６”と称する）。
実施例１１
ＩＣＡＭ−１の発現と規格化
ＩＣＡＭ−１発現を測定するために、ラジオイムノアッセイを行った。このアッセイにおいては、精製ＲＰ１／１をイオドゲンを用いて１０μCi／μｇの特定の活性にヨー素化した。内皮細胞を９６ウェルプレート中で増殖させ各実験で述べたようにして処理した。各プレートを直接氷上ではなく冷室に０．５〜１時間置くことによって４℃に冷却した。各単一層を冷完全培地で３回洗浄し次いで４℃で３０分間¹²⁵Ｉ ＲＰ１／１でインキュベートした。次いで、単一層を完全培地で３回洗浄した。結合¹²⁵Ｉを０．１Ｎ NaOHを用いて放出し計数した。¹²⁵Ｉ ＲＰ１／１の特異活性を未標識ＲＰ１／１を用いて調整して本試験において用いる抗原密度範囲に亘って直線状シグナルを得た。非特異結合を１，０００倍過剰の未標識ＲＰ１／１の存在下で測定し総結合から差引き特異性結合を得た。
上述のラジオイムノアッセイを用いて測定したＩＣＡＭ発現はヒトヘソ帯静脈内皮細胞（HUVEC）およびヒト伏状静脈内皮細胞（ＨＳＶＥＣ）上で１Ｌ−１、ＴＮＦ−、ＬＰＳおよびＩＮＦガンマーにより増大する（第３表）。伏状静脈内皮細胞は本試験においては成人組織由来の培養大静脈内皮細胞中のヘソ帯静脈内皮細胞からの結果を確認するために用いた。ＩＣＡＭ−１の基礎的発現はヘソ帯静脈内皮細胞よりも伏状静脈内皮細胞上で２倍高い。ヘソ帯静脈内皮細胞の組換えＩＬ−１アルファ、ＩＬ−１ベータ、およびＴＮＦへの露出はＩＣＡＭ発現を１０〜２０倍増大させる。ＩＬ−１アルファ、ＴＮＦおよびＬＰＳは最大効力インデューサーであり、ＩＬ−１は重量基準および応答の飽和濃度では効力はそれより小さい（第３表）。１００ng／mｌでのＩＬ−１ベータはＩＣＡＭ−１発現をＨＵＶＥＣ上で９倍、ＨＳＶＥＣ上で７．３倍増大させ、半−最高増加は１５ng／mｌで起った。５０ng／mｌのｒＴＮＦはＩＣＡＭ−１発現をＨＵＶＥＣ上で１６倍、ＨＳＶＥＣ上で１１倍増大させ、半−最高効果は０．５ng／mｌであった。インターフェロンガンマーはＩＣＡＭ−１発現において１０，０００Ｕ／mｌでＨＵＶＥＣ上で５．２倍またはＨＳＶＥＣ上で３．５倍の有意の増大を示した。１０μｇ／mｌでのＬＰＳの効果はｒＴＮＦの効果と強さにおいて同じようであった。これら媒介物の対の組合せはＩＣＡＭ−１発現において追加の効果または追加の効果よりもわずかに小さい効果をもたらした（第３表）。ｒＴＭＦとｒＩＬ−１ベータまたはｒＩＦＮガンマーとの交差滴定は次善または最善の濃度での効果において共働作用を示さなかった。
ＬＰＳは培地中でときどき見い出される基準で内皮細胞上でのＩＣＡＭ−１発現を増大させたので、基礎的ＩＣＡＭ−１発現がＬＰＳに基づき得る可能性を試験した。いくつかの血清バッチを試験したとき、低エンドトキシン血清が２５％までの低ＩＣＡＭ−１基礎発現を与えることを見い出した。ここで示した結果はすべて低エンドトキシン血清中で増殖させた内皮細胞のものであった。しかしながら、ＬＰＳ中和性抗生物質ポリミキシンＢの１０μｇ／mｌでの添加はＩＣＡＭ１発現をわずかに追加の２５％に減少させた（第３表）。ＩＬ−１またはＴＮＦによる処理時のＩＣＡＭ−１発現の増大はこれらの調製物中の低エンドトキシン値と一致させた１０μｇ／mｌポリミキシンＢの存在によっては効果はなかった（第３表）。

ＨＶＥＣおよびＨＳＶＥＣ−ＨＵＶＥＣまたはＨＳＶＥＣ上のＩＣＡＭ−１発現の上方規格化（upregulation）は９６ウェルプレートに１：３で融合性単一層から種付し融合状に増殖させた。次いで細胞を各物質または培地で１６時間処理しＲＩＡを方法的に行った。すべての数値は４回繰返しで行った。
実施例１２
ＩＣＡＭ−１のインターロイキン１およびガンマーインターフェロン誘起の動力学
皮ふ繊維芽細胞上でのＩＣＡＭ−１発現へのインターロイキン−１およびガンマーインターフェロン効果の動力学をDustin, M. L.等の¹²⁵Ｉヤギ抗マウスIgG結合アッセイを用いて測定した〔J. Immunol. １３７：２４５−２５４（1986）；該文献は参考として本明細書に引用する。〕この結合アッセイを行うために、ヒト皮ふ繊維芽球を９６ウェルマイクロタイタープレート中で２〜８×１０⁴細胞／ウェル（０．３２cm²）に増殖させた。細胞を実施例１で記載したようにして補充したＲＰＭＩ１６４０培地で２回洗浄した。細胞をさらにハンクス平衡塩溶液（ＨＢＳＳ）、１０ｍＭのＨＥＰＥＳ、０．０５％ NaN₃および１０％の熱不活化ウシ胎児血清で１回洗浄した。この結合用バッファーでの洗浄は４℃で行った。各ウェルに上記の結合用バッファー５０μｌおよびＸ６３およびＷ６／３２による適当なハイブリドーマ上清５０mｌを、それぞれ陰性および陽性コントロールとして加えた。３０分間、４℃でのインキュベーション後、ウェルを２回結合用バッファーで洗浄し、第２の抗体¹²⁵Ｉ−ヤギ抗マウスIgGを１００μｌ中５０nCiで加えた。¹²⁵Ｉ−ヤギ抗マウス抗体はイオドゲン（Pierce社）を用いてFraker, P. J.等の方法に従って調製した〔Biochem. Biophys. Res. Commun.８０：８４９（１９７８）〕。４℃で３０分後、ウェルを２回２００μｌの結合用バッファーで洗浄し、細胞層を１００μｌの０．１Ｎ NaOHを加えることによって可溶化した。この処理および１００μｌ洗浄はベックマン５５００ガンマーカウンター中で計数した。分当り結合の特異的カウントを〔モノクローナル抗体によるcpm〕−〔Ｘ６３によるcpm〕として計算した。特異的試剤による誘起を含むすべての工程は４回繰返しで行った。
ＩＣＡＭ−１誘起における半減期２時間を有するインターロイキン１の効果は半減期３．７５時間を有するガンマーインターフェロンの効果よりも急速であった（第６図）。ＩＣＡＭの静止レベルに戻る時間は細胞サイクルまたは細胞増殖によっているようであった。静止状態細胞においては、インターロイキン１およびガンマーインターフェロン効果は２〜３日安定であり、一方対数期培養においては、ＩＣＡＭ−１発現はこれら誘起剤の除去後２日で基本線に近い。
組換えマウスおよびヒトインターロイキン１によるＩＣＡＭ−１誘起および組換えガンマーインターフェロンによるＩＣＡＭ−１誘起のドーズレスポンス曲線を第７図に示す。ガンマーインターフェロンおよびインターロイキン１は１ng／mｌで殆んど同一の効果を有する同じような濃度依存性を有することが判った。ヒトおよびマウス組換えインターロイキン１も同様な曲線を有するが、ＩＣＡＭ−１発現の誘起においてヒトインターロイキン１製剤よりもかなり低い効果を示している。
シクロヘキシミド（たん白質合成のインヒビター）およびアクチノマイシンＤ（ｍＲＮＡ合成のインヒビター）は繊維芽球上でのＩＣＡＭ−１発現におけるインターロイキン１およびガンマーインターフェロンの効果を壊滅させる（第４表）。さらにまた、ツニカマイシン（Ｎ結合グリコシル化のインヒビター）のみはインターロイキン１効果を４３％まで抑制した。これらの結果はたん白質およびｍＲＮＡ合成がＩＣＡＭ−１発現におけるインターロイキン１およびガンマーインターフェロン誘起増大において必要であるがＮ−結合グリコシル化の方はそれを必要としないことを示している。

実施例１３
ＩＣＡＭ−１の組織分布
組織化学試験をヒト器官の凍結組織上で行い胸線、リンパ節、腸、皮ふ、じん臓および肝臓中のＩＣＡＭ−１の分布を測定した。そのような分析を行うために、正常ヒト組織の凍結組織切片（４μｍ厚さ）をアセトン中で１０分間固定しモノクローナル抗体、ＲＲ１／１でCerf−Bensussan, N.等により開示されたアビジン−ビオチンコンプレックス法（Vector Laboratories社、バーリンゲーム、ＣＡ）を用いたイムノパーオキシダーゼ法により染色した〔J. Immunol. １３０：2615（１９８３）〕。抗体とのインキュベーション後、切片をビオチン化ウマ抗マウスIgGおよびアビチン−ビオチン化パーオキシダーゼ複合体で順次インキュベートした。各切片は最終的には３−アミノ−９−エチル−カルバゾール（アルドリッチケミカル社、ミルウォーキー、ＷＩ）を含有する溶液に浸して呈色反応を行った。次いで、各切片を４％ホルムアルデヒド中で５分間固定させてヘマトキシリンで対向染色（counterstain）させた。コントロール（対照）はＲＲ１／１抗体の代りに無関係のモノクローナル抗体でインキュベートした切片を含んでいた。
ＩＣＡＭ−１は主要組織親和性コンプレックス（ＭＨＣ）クラスII抗原の分布と殆んど同じ分布を有していることが判った。すべての組織中の血管（大および小共に）の殆んどはＩＣＡＭ−１抗体による内皮細胞染色を示した。管内皮染色はじん臓、肝臓および正常皮ふ中の血管に較べてリンパ節、へん桃腺およびパイヤー班中の小胞間（パラコーチカル）領域でより強い。肝臓においては、染色は殆んど洞様毛細血管ライニング細胞に限定され、門静脈および動脈の殆んどをライニングしているヘパトサイトおよび内皮細胞は染色されなかった。
胸線ずい質においては、大細胞の拡散染色および樹木染色模様がみられた。皮質においては、染色像は巣状であり主として樹木状であった。胸腺細胞は染色されてなかった。末梢リンパ球組織においては、２次リンパ濾胞の胚中心細胞は強く染色していた。あるリンパ濾胞においては、染色像は殆んど樹木状であり、リンパ球の認識し得る染色はなかった。マントル領域の細胞のかすかな染色も観察された。さらに、細胞質拡大（指間小網細胞）を有する樹木状細胞および小胞内またはパラコーチカル領域のリンパ球の小数はＩＣＡＭ−１結合性モノクローナル抗体で染色していた。
細胞様マクロファージはリンパ節および小腸の薄膜プロプリア内で染色されていた。試験した器官の殆んどのストローマ内に拡散している繊維芽球様細胞（スピンドル形細胞）はＩＣＡＭ−１結合性抗体により染色していた。外皮中のランゲルハンス／不定細胞中では染色は認められなかった。平滑筋組織中でも染色は観察されなかった。
外皮細胞の染色はへん桃線の粘膜中で一貫して見られた。肝細胞、肝管外皮、腸外皮細胞、およびじん臓中の管状外皮細胞は殆んどの情況において染色されなかったが、じん細胞がん腫を有するじん摘出片からの正常じん臓組織の切片はICAM−１の多くの隣接管状細胞の染色を示した。これらの管状外皮細胞はまた抗ＨＬＡ−ＤＲ結合性抗体によっても染色していた。
要するに、ＩＣＡＭ−１は管状外皮細胞のような非造血細胞上、および組織マクロファージおよびマイトジエン刺激Ｔリンパ芽球のような造血細胞上に発現する。ＩＣＡＭ−１は末梢血リンパ球に少量発現することが判った。
実施例１４
モノクローナル抗体アフィニティクロマトグラフィーによるＩＣＡＭ−１の精製
一般的精製手順
ＩＣＡＭ−１をヒト細胞または組織からモノクローナル抗体アフィニティークロマトグラフィーを用いて精製した。ＩＣＡＭ−１と反応性のモノクローナル抗体ＲＲ１／１を最初に精製して不活性カラムマトリックスに結合させた。この抗体は“Rothlein, R.等、J. Immunol. １３７：1270−１２７４（１９８６）”および“Dustin, M. L.等、J. Immunol. １３７：２４５（１９８６）”に記載されている。ＩＣＡＭ−１を細胞膜から細胞を非イオン性清浄剤トリトンＸ−１００中で近中性pHで溶解することによって可溶化した。可溶化ＩＣＡＭ−１を含有する細胞溶解物をカラムマトリックス材料に非特異的に結合する物質を除去するように設計されたプレカラムに通し、次いでモノクローナル抗体カラムマトリックスに通してＩＣＡＭ−１を抗体に結合させた。抗体カラムをpHをpH１１．０まで上昇させた一連の洗浄剤洗浄バッファーで洗浄した。これらの洗浄中、ＩＣＡＭ−１は抗体マトリックスに結合したままであり、結合してないまた弱く結合している不純物は除去された。次いで、結合ＩＣＡＭ−１をpH１２．５の洗浄剤バッファーを適用することによってカラムから特異的に溶出させた。
モノクローナル抗体ＲＲ１／１の精製およびセファローズＣＬ−４Ｂへの共有結合
抗ＩＣＡＭ−１モノクローナル抗体ＲＲ１／１をハイブリドーマ含有マウスの腹水またはハイブリドーマ培養上清から標準の硫酸アンモニウム沈降法およびプロティンＡアフィニティークロマトグラフィーにより精製した〔Ey等、Immunochem. １５：４２９（１９７８）〕。精製IgGまたはラットIgG（シグマケミカル社、セントルイス、ＭＯ）をセファローズＣＬ−４Ｂ（ファルマシア社、スウェーデン）にMarch等の方法〔Anal. Biochem.６０：１４９（１９７４）〕の修正法を用いて共有結合させた。要するに、セファローズＣＬ−４Ｂを蒸留水中で洗浄し、５ＭのK₂HPO₄（pH約１２）中の４０mg／mｌのＣＮＢｒで５分間活性化し、次いで０．１ｍＭ HCｌで４℃にて長く洗浄した。濾過し活性化したセファローズを等容量の精製抗体（０．１Ｍ NaHCO₃、０．１Ｍ NaCｌ中２〜１０mg／mｌ）で再懸濁させた。懸濁液を４℃で１８時間ゆるやかな端から端までの回転によりインキュベートした。次いで、上清を未結合抗体について２８０nmの吸収によりモニターし、活性化セファローズの残りの反応部位をグリシンを０．０５Ｍに加えることによって飽和させた。結合効率は通常９０％以上であった。
ヒトひ臓から調製した膜の洗浄剤可溶化
すべての手順を４℃で行った。ヘアリー細胞発血病の患者からの凍結ヒトひ臓（２００ｇフラグメント）を氷上で１ｍＭのフェニルメチルスルホニルフルオライド（ＰＭＳＦ）、０．２Ｕ／mｌのアプロチニンおよび５ｍＭのイオドアセタミドを含有する２００mｌトリス−塩水（５０ｍＭのTris、０．１４ＭのNaCｌ、４℃でpH７．４）中に溶解させた。組織を小片に切断し４℃でテクマー強力ホモジナイザーで均質化した。容量をトリス−塩水で３００mｌとし、トリス−塩水中１０％トウィーン４０（ポリオキシエチレンソルビタンモノパルミテート）１００mｌを加え最終濃度２．５％トウィーン４０を得た。
膜を調製するため、均質化物を３ストロークのドンス（Dounce）またはより好ましくはテフロンポッターエルブジャムホモジナイザーを用いて抽出し、次いで１０００ｘｇで１５分間遠心した。上清を残し、ペレットをトリス−塩水中の２．５％トウィーン４０の２５０mｌで再抽出した。1000ｘｇで１５分間の遠心後、両抽出物からの上清を一緒にし１５０，０００ｘｇで１時間遠心して膜をペレット化した。膜を２００mｌのトリス−塩水中に再懸濁させることによって洗浄し、150,000ｘｇで１時間遠心した。膜ペレットを２００mｌのトリス−塩水中に再懸濁させ、モーター駆動ホモジナイザーおよびテフロンペストルで懸濁液が濃密になるまで均質化した。容量を次いでトリス−塩水で９００mｌまでにし、Ｎ−ラウロイルサルコシンを最終濃度１％になるよう加えた。４℃で３０分の攪拌後、洗浄剤溶解物中の不溶性物質を１５０，０００ｘｇ、１時間での遠心により除去した。トリトンＸ−１００を上清に最終濃度２％に加え、溶解物を４℃で１時間攪拌した。
ＪＹＢ−リンパ芽球細胞の洗浄剤可溶化
ＥＢＶ形質転換Ｂ−リンパ芽球細胞系ＪＹを１０％ウシ胎児血清（ＦＣＳ）および１０ｍＭ ＨＥＰＥＳを含有するＲＰＭＩ１６４０中で０．８〜１．０×１０⁶細胞／mｌの密度に増殖させた。ＩＣＡＭ−１の細胞表面発現を増大させるため、ホルボール１２−ミリステート１３−アセテート（ＰＭＡ）を細胞を収穫する前に２５ng／mｌで８〜１２時間で加えた。バナジン酸ナトリウム（５０μＭ）もこの時間中に培養物に加えた。細胞を５００ｘｇで１０分間遠心することによってペレット化しハンス平衡塩溶液（ＨＢＳＳ）中で再懸濁および遠心することによって２回洗浄した。細胞（５ｌの培養物当り約５ｇ）を５０mｌの溶解バッファー（０．１４Ｍ NaCｌ、５０ｍＭ Tris、pH８．０、１％トリトンＸ−１００、０．２μ／mｌアプロチニン、１ｍＭＰＭＳＦ、５０μＭのバナジン酸ナトリウム）中に４℃で３０分間攪拌することによって溶解させた。未溶解核および不溶性片を１０，０００ｘｇで１５分間の遠心、次いで１５０，０００ｘｇでの１時間の上清の遠心および上清のワットマン３mmフィルター紙による濾過により除去した。
構造検討のためのＩＣＡＭ−１のアフィニティークロマトグラフィー
構造検討に使用するＩＣＡＭ−１の大規模精製として、１０mｌのＲＲ１／１−セファローズＣＬ−４Ｂのカラム（２．５mgの抗体／ゲルのmｌで結合）、およびＣＮＢｒ−活性化、グリシン安定化セファローズＣＬ−４ＢおよびラットIgG結合セファローズＣＬ−４Ｂ（２mg／mｌ）の２種のプレカラムを用いた。各カラムを直列につなぎ、１０カラム容量の溶解バッファー、１０カラム容量のpH１２．５バッファー（５０ｍＭトリエチルアミン、０．１％トリトンＸ−１００、４℃でpH１２．５）で予備洗浄し次いで１０カラム容量の溶解バッファーで平衡させた。１ｌのヒトひ臓の洗浄剤溶解物を０．５〜１．０mｌ／分の流速で負荷させた。２つのプレカラムはＲＲ１／１−セファローズカラムに通す前の溶解物から非特異結合物質を除去するのに用いた。
負荷後、ＲＲ１／１−セファローズのカラムおよび結合ＩＣＡＭ−１を(1)溶解バッファー、(2)２０ｍＭ Tris pH８．０／０．１４Ｍ Nacｌ／０．１％トリトンＸ−１００、(3)２０ｍＭグリシンpH10.0/０．１％トリトンＸ−１００、および(4)５０ｍＭトリエチルアミンpH１１．０／０．１％トリトンＸ−１００の各々最低５カラム容量で１mｌ／分の流速で順次洗浄した。すべての洗浄バッファーは１ｍＭのＰＭＳＦと０．２μ／mｌのアプロチニンを含んでいた。洗浄後、残留結合ＩＣＡＭ−１を５カラム容量の溶出バッファー（５０ｍＭトリエチルアミン／０．１％トリトンＸ−１００／４℃でpH１２．５）により１mｌ／３分の流速で溶出させた。溶出ＩＣＡＭ−１を１mｌフラクションに集め直ちに０．１mｌの１Ｍトリス、pH６．７を加えて中和させた。ＩＣＡＭ−１を含有するフラクションを１０μｌのアリコートのＳＤＳ−ポリアクリルアミド電気泳動〔Springer等、J. Exp. Med.１６０：１９０１（１９８４）〕により次いで銀染色〔Morrissey, J. H.,Anal. Biochem.１１７：３０７（１９８１）〕によって同定した。これらの条件下で、ＩＣＡＭ−１のバルクが約１カラム容量に溶出し、銀染色電気泳動から判断して９０％以上の純度であった（アフィニティーマトリックスから漏出した少量のIgGが主要不純物であった）。ＩＣＡＭ−１を含有するフラクションをプールしセントリコン−３０マイクロコンセントレーター（アミコン社、デンバース、ＭＡ）を用いて約２０倍に濃縮した。精製ＩＣＡＭ−１をプールのエタノール沈降アリコートのローリー（Lowry）たん白質アッセイにより定量した。約５００μｇの純ＩＣＡＭ−１を２００ｇのヒトひ臓から調製できた。
約２００μｇの精製ＩＣＡＭ−１を分離用SDS−ポリアクリルアミドゲル電気泳動により第２段階の精製に供した。ＩＣＡＭ−１を示すバンドはゲルを１Ｍ KCｌ中にソーキングすることによって可視化させた。ＩＣＡＭ−１を含むゲル領域を検査して“Hunkapiller等、Meth. Enzymol.９１：２２７−２３６（１９８３）”の方法によって電気溶出させた。精製たん白質はＳＤＳ−ＰＡＧＥおよび銀染色で判断したとき９８％以上の純度であった。
官能試験のためのＩＣＡＭ−１のアフィニティ精製
官能性試験用のＩＣＡＭ−１を前述したようなＪＹ細胞からの洗浄剤溶解物から精製したが、小スケール（１mｌのＲＲ１／１−セファローズカラム）で次の修正を加えて行った。すべての溶液は５０μＭバナジン酸ナトリウムを含んでいた。カラムを０．１％トリトンＸ−１００含有pH１１．０バッファーで洗浄後、カラムを０．１％トリトンＸ−１００に代えて１％のｎ−オクチル−ベーター−Ｄ−グルコピラノサイド（オクチルグルコシド）含有の同じバッファー５カラム容量で再洗浄した。オクチルグルコシド洗浄剤はＩＣＡＭ−１に結合したトリトンＸ−１００を除去し、トリトンＸ−１００と異なりその後透析により除去できる。次いで、ＩＣＡＭ−１を０．１％トリトンＸ−１００の代りに１％のオクチルグルコシドを含むpH１２．５バッファーで溶出させ、分析し、上述のようにして濃縮した。
実施例１５
精製ＩＣＡＭ−１の特性
ヒトひ臓から精製したＩＣＡＭ−１はＳＤＳ−ポリアクリルアミドゲル中でＭｒ７２，０００〜９１，０００の広いバンドとして浸透する。ＪＹ細胞から精製したＩＣＡＭ−１もＭｒ７６，５００〜９７，０００の広いバンドとして浸透する。これらＭｒは種々の細胞源から免疫沈降させたＩＣＡＭ−１の報告された範囲にある：ＪＹ細胞からのＭＲ＝９０，０００、骨ずい単球細胞系Ｕ９３７上の１１４，０００、および繊維芽球上の９７，０００〔Dustin等、J. Immunol. １３７：２４５（１９８６）〕。Ｍｒのこの広い範囲は広汎であるが変化し得る度合のグリコシル化に貢献する。非グリコシル化プレカーサーはＭｒ５５，０００を有する（Dustin等）。ＪＹ細胞またはヒトひ臓から精製したたん白質は、その元のアフィニティカラムへの再結合能力によりまたＲＲ１／１セファローズによる免疫沈降およびＳＤＳ−ポリアクリルアミド電気泳動により明らかなように、その抗原活性を保持している。
ＩＣＡＭ−１のペプチドフラグメントを調製するためには、約２００μｇを２ｍＭジチオスレイトール／２％ＳＤＳで還元し、次いで５ｍＭの沃化酢酸でアルキル化した。たん白質をエタノールで沈降させ、０．１Ｍ NH_4CO3／０．１mM CaCｌ₂／０．１％双性イオン剤（Zwittergent）３−１４（カルビオケミ社）中に再溶解させ、１％ w/wトリプシンで３７℃、４時間消化し、次いで１％トリプシンで３７℃、１２時間の追加の消化を行った。トリプシンペプチドを逆相ＨＰＬＣにより０．４×１５cmＣ４カラム（Vydac社）を用いて精製した。ペプチドは０．１％トリフルオロ酢酸中で０％〜６０％アセトニトリルの直線勾配によって溶出した。選択したペプチドをガス相マイクロシーケネーター（アプライドバイオシステムズ社）での配列分析に供した。この試験から得られた配列情報を第５表に示す。

実施例１６
ＩＣＡＭ−１遺伝子のクローニング
ＩＣＡＭ−１の遺伝子は任意の種々の手順を用いてクローニングできる。例えば、ＩＣＡＭ−１のトリプシンフラグメントのシーケンシング（第５表）から得られたアミノ酸配列情報を用いてＩＣＡＭ−１遺伝子に相当するオリゴヌクレオチド配列を同定し得る。また、ＩＣＡＭ−１遺伝子は抗ＩＣＡＭ−１抗体を用いてＩＣＡＭ−１を産生するクローンを検出することによってもクローニングできる。
オリゴヌクレオチドプローブを用いることによるＩＣＡＭ−１遺伝子のクローニング
遺伝子コード〔Waston, J. D., Molecular Biology of the Gene,第３版、W. A.ベンジャミン社、メロノパーク、ＣＡ（１９７７）〕を用いて、１種以上の異なるオリゴヌクレオチドを同定でき、その各々はＩＣＡＭ−１トリプシンペプチドをコードし得るものである。特定のオリゴヌクレオチドが実際に現実のＩＣＡＭ−１コード配列を構成する可能性は異常な塩基対関係および特定のコドンを現実に真核細胞に用いて（特定のアミノ酸をコードする）頻度を考慮することによって評価できる。そのような“コドン利用法則”は“Lathe, R.等、J. Melec. Biol. １８３：１〜１２（１９８５）”により開示されている。Latheの“コドン利用法則”を用いて、理論的に“最も可能性のある”ＩＣＡＭ−１トリプシンペプチド配列をコードし得るヌクレオチド配列（即ち、最低の不要物を有するヌクレオチド配列）を含有する単一オリゴヌクレオチドまたはオリゴヌクレオチドのセットを同定する。
ＩＣＡＭ−１フラグメントをコードし得る理論的に“最も可能性のある”配列を含むオリゴヌクレオチドまたはオリゴヌクレオチドのセットを用いて“最も可能性ある”配列または配列のセットにハイブリッド化し得る相補オリゴヌクレオチドまたはオリゴヌクレオチドのセットの配列を同定する。そのような相補配列を含むオリゴヌクレオチドはＩＣＡＭ−１遺伝子を同定し単離するプローブとして使用できる〔Maniatis, T.等、Molecular Cloning A Laboratory Manual,コールド スプリング ハーバー プレス社、コールド スプリング、ＮＹ（１９８２）〕。
上記文献のセクションＣに記載されているように、ＩＣＡＭ−１遺伝子を含有する可能性ある真核ＤＮＡ調製物からＩＣＡＭ−１遺伝子をクローニングすることは可能である。ＩＣＡＭ−１たん白質をコードする遺伝子を同定しクローニングするためには、ＤＮＡライブラリーをその上記のオリゴヌクレオチドプローブとハイブリッド化する能力についてスクリーニングする。正常な二倍体細胞中にはＩＣＡＭ−１の遺伝子のほんの２つのコピーしか存在しないようであるので、またICAM−１遺伝子はクローニングが望まれない大きな非転写介在配列（イントロン）を有し得る可能性があるので、好ましいのはゲノムＤＮＡよりはむしろＩＣＡＭ−１産生性細胞のｍＲＮＡから調製したｃＤＮＡライブラリーからＩＣＡＭ−１コード配列を単離することである。適当なＤＮＡまたはｃＤＮＡ調製物は酵素的に開裂させ、ランダムにせん断し、連結反応させて組換えベクターとする。次いで、これら組換えベクターの上述のオリゴヌクレオチドプローブとハイブリッド化する能力を測定する。ハイブリッド化の手順は、例えば、“Maniatis, T., Molecular Cloning A Laboratory Manual,コールド スプリング ハーバー プレス社、コールド スプリング ハーバー、ＮＹ（１９８２）”または“Haymes, B. T.等、Nucleic Acid Hybridization a Practical Approach,ＩＲＬプレス社、オックスフォード、英国（１９８５）”において開示されている。そのようなハイブリッド化ができることが判っているベクターを分析してベクターが含有するICAM−１配列の程度および性質を分析する。純粋に仮説的な考察によれば、ＩＣＡＭ−１分子をコードするような遺伝子はほんの１８ケのヌクレオチドを有するオリゴヌクレオチドを用いて明確に同定できるであろう（ハイブリッド化スクリーニングにより）。
即ち、要約すれば、ＩＣＡＭ−１ペプチド配列の現実の同定により、そのようなペプチドをコードし得る理論的に“最も可能性ある”ＤＮＡ配列またはそのような配列のセットの同定ができる。この理論的配列に相補性のオリゴヌクレオチドを構築することにより（あるいは“最も可能性ある”オリゴヌクレオチドのセットに相補的なオリゴヌクレオチドのセットを構築することにより）、ＩＣＡＭ−１遺伝子を同定し単離するプローブとして機能し得るＤＮＡ分子（またはＤＮＡ分子のセット）が得られる。
第５表のＩＣＡＭ−１ペプチド配列を用いて、ＡＡおよびＪペプチドをコードし得るオリゴヌクレオチドの“最も可能性ある”配列の配列を決定した（それぞれ、第６表および第７表）。これら配列に相補性のオリゴヌクレオチドを合成し精製してＩＣＡＭ−１遺伝子配列を単離するプローブとした。適当なサイズ選定ｃＤＮＡライブラリーをＰＭＡ誘起ＨＬ−６０細胞からおよびｐｓ刺激へそ帯静脈内皮細胞からのポリ（Ａ）⁺ＲＮＡから調製した。サイズ選定ｃＤＮＡライブラリーは“Gubler, U.等〔Gene ２５：２６３−２６９（１９８３）〕およびCorbi, A.等〔ＥＭＢＯ J.６：４０２３−４０２８（１９８７）〕の方法に従ってＰＭＡ誘起ＨＬ−６０細胞からのポリ（Ａ）⁺ＲＮＡを用いて調製した。これらの文献は参考として本明細書に引用する。
サイズ選定ｃＤＮＡライブラリーはＰＳ５μｇ/mｌで４時間刺激したへそ帯静脈内皮細胞からのポリ（Ａ）⁺を用いて調製した。ＲＮＡは上記細胞を４Ｍグアニジニウムイソシアネート中で均質化し上清をCsCｌ勾配により超遠心に供することによって抽出した〔Chirgwin, J. M.等、Biochem.１８：５２９４−５２９９（１９７９）〕。ポリ（Ａ）⁺ＲＮＡは全ＲＮＡスペイシスの混合物からオリゴ（ｄＴ）−セルロースクロマトグラフィー（タイプ３、コラボラティブ リサーチ社）を用いて単離した〔Aviv, H.等、Proc. Natl. Acad. Sci.（ＵＳＡ）６９：１４０８−１４１２（１９７２）〕。

第１のストランドｃＤＮＡを８μｇのポリ（Ａ）⁺ＲＮＡ、トリ骨ずい芽球症ウイルス逆転写酵素（ライフサイエンス社）およびオリゴ（ｄＴ）プライマーと用いて合成した。ＤＮＡ−ＲＮＡハイブリッドはラナーゼＨ（ＢＲＬ社）で消化、第２ストランドはＤＮＡポリメラーゼＩ（ニューイングランド バイオラブス社）を用いて合成した。生成物をＥｃｏＲＩリンカー（ニューイングランド バイオラブス社）でメチル化し、ＥｃｏＲＩリンカー（ニューイングランド バイオラブス社）にブラントエンド連結反応させ、ＥｃｏＲＩで消化し低融点アガロースゲル上でサイズ選定した。５００ｂｐより大きいｃＤＮＡを前以てＥｃｏＲＩ消化させ脱リン酸化させているλｇｔ１０に連結反応させた（ストラタジーン）。連結反応生成物を次いでパッケージンクした（ストラタジーンゴールド）。
次に、へそ帯静脈内皮細胞およびＨＬ−６０ｃＤＮＡライブラリーを２０，０００ＰＦμ／150mmプレートに塗布した。組換えＤＮＡを複製中でニトロセルロースフィルターに移し、０．５Ｍ NaOH／１．５Ｍ NaCｌ中で変性し１Ｍトリス、pH７．５／１．５Ｍ NaCｌ中で中和し、８０℃で２時間ベーキングした〔Benton, W. D.等、Science １９６：１８０−１８２（１９７７）〕。フィルターをプレハイブリッド化し、５×Denhardt溶液、５０ｍＭ NaPO₄および１μｇ／mｌのサケ精子ＤＮＡを含む５×ＳＳＣ中でハイブリッド化した。プレハイブリッド化は４５℃で１時間行なった。
ハイブリッド化は３２ｂｐ（′５−ＴＴＧＧＧＣＴＧＧＴＣＡＣＡＧＧＡＧＧＴＧＧＡＧＣＡＧＧＴＧＡＣ）または４７ｂｐ（５′−ＧＡＧＧＴＧＴＴＣＴＣＡＡＡＣＡＧＣＴＣＣＡＧＧＣＣＣＴＧＧＧＧＣＣＧＣＡＧＧＴＣＣＡＧＣＴＣ）抗感覚オリゴヌクレオチドを上述の方法で、それぞれ、ＩＣＡＭ−１トリプシンペプチドＪおよびＡＡ（第６表および第７表）上で用いて行なった〔Lathe, R. J. Melec. Biol. １８３：１−１２（１９８５）〕。オリゴヌクレオチドはｒ−（³²Ｐ）ＡＴＰでＴ⁴ポリヌクレオチドキナーゼおよび製造者（ニューイングランド バイオラブス社）に推奨される条件を用いて末端標識した。オーバーナイトハイブリッド化に続いて、フィルター２×ＳＳＣ／０．１％ＳＤＳで３０分間、４５℃で２回洗浄した。ファージをハイブリッド化を示すプラーグから単離し、連続再塗布および再スクリーニングにより精製した。
抗ＩＣＡＭ−１抗体の使用によるＩＣＡＭ−１の遺伝子のクローニング
ＩＣＡＭ−１の遺伝子を別法として抗ＩＣＡＭ−１抗体の使用によりクローニングできる。DNAまたはより好ましくはｃＤＮＡをＩＣＡＭ−１を発現し得る細胞から抽出し精製する。精製cDNAをフラグメント化し（せん断化、エンドヌクレアーゼ消化等により）ＤＮＡまたはｃＤＮＡフラグメントのプールを調製する。このプールからのＤＮＡまたはｃＤＮＡフラグメントを発現ベクターにクローニングして各々のメンバーが特異的クローン化ＤＮＡまたはｃＤＮＡフラグメントを含有する発現ベクターの遺伝子ライブラリーを調製する。
実施例１７
ｃＤＮＡクローンの分析
陽性クローンからのファージＤＮＡをEcoRIで消化し、１つのクローンからのｃＤＮＡをプローブとして用いてサウサーン分析により試験した。交差ハイブリッド化した最大サイズｃＤＮＡ挿入物をプラスミドベクターｐＧＥＭ４Ｚ（プロメガ社）のＥｃｏＲＩサイトにサブクローニングした。両配向でｃＤＮＡを含むＨＬ−６０サブクローンをエンドヌクレアーゼIII消化〔Henikoff, s., Gene ２８：３５１−３５９（１９８４）〕により製造者の推奨（エラスーアーベース、ブロメガ社）に従って欠落させた。漸次的に欠落させたｃＤＮＡを次いでクローニングしてジデオキシヌクレオチド鎖終端シーケンシングに製造者の推奨（シーケナーゼ、Ｕ．Ｓ．バイオケミカル社）に従って供した〔Sanger, F.等、Proc. Natl. Acad. Sci（ＵＳＡ）７４：５４６３−５４６７（１９７７）〕。ＨＬ−６０ｃＤＮＡ５′およびコード領域を両ストランド上で完全にシーケンシングし、３′領域を両ストランド上で約７０％シーケンシングした。代表的な内皮ｃＤＮＡをその長さの殆どに亘って４ｂｐ認識制限酵素フラグメントのショトガンクローニングによりシーケンシングした。
１種のＨＬ−６０および１種の内皮細胞cDNAのｃＤＮＡ配列を確立した（第８図）、３０２３ｂｐ配列は短い５′未ほん訳領域と位置２９６６に共通ポリアデヒル化シグナルを有する１．３ｋｂ３′未ほん訳領域を含む。最長開放読み枠は位置５８の第１ＡＴＧで始まり位置１６５３のＴＧＡ終端トリプレットで終る。ほん訳アミノ酸配列と合計９１個のアミノ酸の８種の異なるトリプシンペプチドから決定した配列（第８図中でアンダーラインを施している）との間の同一性は信頼できるＩＣＡＭ−１ｃＤＮＡクローンが単離されたことを確認した。ＩＣＡＭ−１トリプシンペプチドのアミノ酸配列を第８表に示す。

疎水性分析〔Kyte, J.等、J. Melec. Biol. １５７：１０５−１３２（１９８２）〕は２７残基シグナル配列の存在を示唆している。＋１グルタミンの役目は３種のＩＣＡＭ−１たん白質調製物上にＮ−末端配列を本発明で得ることができないことと一致しており；グルタミンはフイログルタミン酸に環状化しブロックしたＮ−末端をもたらす。１〜４５３のほん訳配列は主として親水性であり２４残基の疎水性の推定トランスメンブランドメインが続く。トランスメンブランドメインはその後すぐに２７残基の推定細胞質ドメイン内に含まれた数個の荷電残基が続く。
成熟ポリペプチド鎖の予示サイズは５５，２１９ダルトンであり、脱グリコシル化ＩＣＡＭ−１の観察されたサイズ５５，０００と良好に一致している〔Dustin, M. L.等、J. Immunol. １３７：２４５−２５４（１９８６）〕。８個のＮ−結合グリコシル化部位が予示される。これらの部位の２つのトリプシンペプチド配列中のアスパラギンの不存在はそれらのグリコシル化とそれらの細胞外配向を確認している。高マンノースＮ−結合カルボハイドレート当り２，５００ダルトンを想定すると、７５，０００ダルトンのサイズがＩＣＡＭ−１プレカーサーについて予示され、観察されたサイズ７３，０００ダルトン〔Dustin, M. L.等、J. Immunol. １３７：２４５−２５４（１９８６）〕に匹敵する。高マンノースのコンプレックスカルボハイドレートへの転換後、成熟ＩＣＡＭ−１糖たん白質は細胞の種類により７６〜１１４Kdである〔Dustin, M. L.等、J. Immunol. １３７：２４５−２５４（１９８６）〕。即ち、ＩＣＡＭ−１は高度にグリコシル化されているが典型的な完全膜たん白質である。
実施例１８
ＩＣＡＭ−１は免疫グロブリン超遺伝子群のインテグリン結合性１員である：
ＩＣＡＭ−１内部繰返し配列をミクロジーニ（Microgenie）たん白質配列プログラム〔Queen, C.等、Nucl. Acid Res. １２：５８１−５９９（１９８４）〕を用い次いで検査することによって行なった。ＩＣＡＭ−１のＩｇＭ、Ｎ−ＣＡＭおよびＭＡＧへの配列をミクロジーニおよびＡＬＩＧＮプログラム〔Daypoff, M. O.等、Meth. Enzmol. ９１：５２４−５４５（１９８３）〕を用いて行なった。ナショナル バイオメデカル リサーチ ファンデーション（National Biomedical Research Foundation）に保管されている４種のたん白質配列データベースをWilliamsとPearsonのＦＡＳＴＰプログラムを用いてたん白質配列の類似性について調査した〔Lipman, D. J.等、Science ２２７：１４３５−１４３９（１９８５）〕。
ＩＣＡＭ−１はインテグリンのリガンドであるので、免疫グロブリン超遺伝子群の１員であることは予期されなかった。しかしながら、ＩＣＡＭ−１配列の調査はその配列が免疫グロブリン超遺伝子群中の身内関係に提唱されたすべての規準を満たすことを示している。これらの基準を以下に説明する。
ＩＣＡＭ−１の全細胞外ドメインを第９Ａ図に配列して示した５つの相同性免疫グロブリン様ドメインから構築する。ドメイン１−４はそれぞれ８８、９７、９９および９９の残基長であり、かくして典型的なＩｇドメインサイズを有しており；ドメイン５は６８個の残基中で切り取られている。ＦＡＳＴＰプログラムを用いてのＮＢＲＦデータベースの調査はＩｇＭとＩｇＧ Ｃドメインを包含する免疫グロブリン超遺伝子群の１員、Ｔ細胞レセプターαサブユニット可変ドメイン、およびアルファ１ベータ糖たん白質と有意の相同性を示していた（第９Ｂ−Ｄ図）。
上記の情報を用いて、ＩＣＡＭ−１のアミノ酸配列を免疫グロブリン超遺伝子群の他の１員のアミノ酸配列と比較した。
Ｉｇ超遺伝子群ドメインの３つのタイプであるＶ、Ｃ１およびＣ２は分化されている。ＶとＣの両ドメインは内部ドメインジスルフィド結合により一緒に結合している２β−シートから構築されており；Ｖドメインは９つの抗平行β−ストランドを有しＣドメインは７つ有している。定常ドメインを第９Ａ図に示した特徴的残基に基づいてＣ１およびＣ２−セットに分割した。Ｃ１−セットは抗原認識中に含まれるたん白質を含んでいる。Ｃ２−セットは数個のＦ_cレセプターとＣＤ２、ＬＦＡ−３、ＭＡＧおよびＮＣＡＭを包含する細胞粘着中に含まれるたん白質とを含んでいる。ＩＣＡＭ−１ドメインはこのセット中でＩＣＡＭ−１と置き変っているＣ２−セットのドメインと最も強い相同性を有することが判った；このことは第９図のβ−ストランドＢ−Ｆについて示されているようにＣ１ドメインよりもＣ２中の保持残基により強く類似している点にも反映されている。また、ＩＣＡＭ−１ドメインはＶおよびＣ１ドメイン中の上記ストランドとよりもＣ２ドメインのβストランドＡおよびＧとより一層良好に列んでおり、全Ｃ２ドメイン強度を横切って良好な配列を可能としている。ＮＣＡＭ、ＭＡＧおよびアルファ１−β糖たん白質からのＣ２ドメインとの配列は第９Ｂ図および第９Ｃ図に示されており；同一性は２８〜３３％の範囲であった。Ｔ細胞レセプターＶα２７％同一性およびＩｇＭ Ｃドメイン３ ３４％同一性との配列も示されている（第９Ｂ、９Ｄ図）。
免疫グロブリンドメインの最も重要な特徴の１つはβシートサンドイッチを安定化するＢおよびＦβストランドを架橋するジスルフィド結合システィンであり；ＩＣＡＭ−１においてはシスティンはすべての場合に保持されているが、ドメイン４のストランドｆにおいては、上記サンドイッチに面しかついくつかの他のＶ−およびＣ２−セットドメインにおいて提案されたような接触を安定化しているロイシンが見い出される。システィン（４３、５０、５２および３７残基）間の距離はＣ２セットについて述べたとおりである。
ＩＣＡＭ−１中の鎖間ジスルフィド結合の存在について試験するために、内皮細胞ＩＣＡＭ−１を還元および非還元条件下でＳＤＳ−ＰＡＧＥに供した。内皮細胞ＩＣＡＭ−１はこれがＪＹまたは毛状ひ臓細胞ＩＣＡＭ−１よりも小さいグリコシル化異種原性を示しＭｒ中のシフトにより大きな感応性を示すことから使用した。従って、ICAM−１を１６時間ＬＰＳ（５μｇ／mｌ）刺激ヘソ帯静脈内皮細胞培養物から前述したようなイノムアフィニティークロマトグラフィーにより精製した。アセトン沈降ＩＣＡＭ−１を０．２５％２−メルカプトエタノールまたは２５ｍＭイオドアセタミド含有のサンプルバッファー〔Laemmli, U. K., Nature ２２７：６８０−６８５（１９７０）〕中に再懸濁させ１００℃に５分間もたらした。サンプルをＳＤＳ−ＰＡＧＥ４６７０および銀染色４６１３に供した。内皮細胞ＩＣＡＭ−１は還元条件下で１００ｋｄの見掛けのＭｒをまた非還元条件下で９６ｋｄを示し天然ＩＣＡＭ−１中の鎖間ジスルフィドの存在を強く示唆していた。
二次構造を予想するための一次配列の使用
〔Chon, P. Y.等、Biochem. 13：２１１−２４５（１９７４）〕は、第９Ａ図上方にａ−ｇと表示され、免疫グロブリンドメインについての予想を正確に満たしまた免疫グロブリン中のストランドＡ−Ｈの各位置（第９Ａ図下方）に相応する各ＩＣＡＭ−１ドメイン中の７つの予期されたβ−ストランドを示していた。ドメイン５はＡおよびＣストランドを欠損しているが、これらはシートの端部を形成しているので、各シートは依然として恐らくはストランドＤによって形成しておりいくつかの他のＣ２ドメインで提案されたようにストランドＣの代理をしており；またＢおよびＦストランド間の特徴的ジスルフィド結合は影響を受けないであろう。即ち、ドメインサイズ、配列相同性、推定ドメイン間ジスルフィド結合を形成する保持システィン、ジスルフィド結合の存在、および予想βシート構造の基準はすべて免疫グロブリン超遺伝子群中でのＩＣＡＭ−１の内在を満たしている。
ＩＣＡＭ−１はＣ２セットのＮＣＡＭおよびＭＡＧ糖たん白質と最も強く相同性であることが判明した。このことはＮＣＡＭとＭＡＧが共に細胞−細胞粘着を媒介するので特に興味深い。NCAMはニューロン−ニューロンおよび神経−筋相互作用において重要であり〔Cunningham, B. A.等、Science ２３６：７９９−８０６（１９８７）〕、またＭＡＧはミエリン化（myelination）中のニューロン−オリゴデントロサイトおよびオリゴデントロサイト−オリゴデントロサイト相互作用において重要である〔Poltorak, M.等、J. Cell. Biol. １０５；１８９３−１８９９（１９８７）〕。ＮＣＡＭとＭＡＧの細胞表面発現は神経系形成およびミリエン化中に、それぞれ、炎症におけるＩＣＡＭ−１の調整された誘起と類似して発展的に調整される〔Springer, T.A.等、Ann. Rev. Immunol. 5：２２３−２５２（１９８７）〕。ＩＣＡＭ−１、ＮＣＡＭ〔Cunningham, B. A.等、Science ２３６：７９９−８０６（１９８７）〕、およびＭＡＧ〔Salzer, J. L.等、J. Cell. Biol. １０４：９５７−９６５（１９８７）〕は全体構造において類似しまた相同性である、何故ならば、各々はＮ末端細胞外領域を形成する５つのＣ２ドメインから構成された完全膜糖たん白質であるからである。ただし、ＮＣＡＭにおいては、ある追加の非Ｉｇ様配列が最後のＣ２ドメインとトランスメンブランドメイン間に存在する。ICAM−１はトランスメンブランと細胞質ドメインを含むその全体長に亘ってＭＡＧと２１％の同一性を有して列んでおり；同一％の同一性がＩＣＡＭ−１とＮＣＡＭ−１の５つのドメインを比較したとき見出される。ＩＣＡＭ−１とＭＡＧの二次構造の図式的比較は第１０図に示している。ドメイン対ドメインの比較はＩＣＡＭ−１とＮＣＡＭ分子内のドメイン間の相同性のレベル（それぞれ、×±s.d.２１±２．８％および１８．６±３．８％）はＩＣＡＭ−１ドメインをＮＣＡＭおよびＭＡＧドメインと比較したときの相同性のレベル（それぞれ、２０．４±３．７および２１．９±２．７）と同じであることを示している。ＮＣＡＭ〔Cunningham, B. A.等、Science ２３６：７９９−８０６（１９８７）〕；Barthels, D.等、ＥＭＢＯ Ｊ．６：９０７−９１４（１９８７）〕およびＭＡＧ〔Lai, C.等、Proc. Natl. Acad. Sci.（USA）８４：４３７７−４３４１（１９８７）〕のＣ−末端領域における別の接合の証拠は存在するけれども、これが内皮またはＨＬ−６０ＩＣＡＭ−１クローンのシーケンシングのおいてあるいは種々のタイプのＩＣＡＭ−１たん白質バックボーンおよびプレカーサーの研究〔Dustin, M. L.等、J. Immunol. １３７：２４５−２５４（１９８６）〕において見出されたという証拠はない。
ＩＣＡＭ−１は多くの種々の細胞種とのリンパ球相互作用におけるＬＦＡ−１用のリガンドとして機能する。リンパ球は人工膜２重層に含まれたＩＣＡＭ−１と結合し、これはリンパ球上にLFA−１を必要としＩＣＡＭ−１とＬＦＡ−１の相互作用を直接示唆している（Marlin, S. D.等、Cell ５１：８１３−８１９（１９８７）〕。
ＬＦＡ−１は白血球インテグリンであり免疫グロブリン様特徴を有しない。白血球インテグリンは１種のインテグリン亜族を含む。他の２つの亜族は細胞マトリックス相互作用を媒介し、フィブロネクチン、ビトロネクチン、コラーゲンおよびフィフリノーゲンを包含するそのリガンド内の配列ＲＧＤを認識する〔Hynes, R.O., Cell ４８：５４９−５５４（１９８７）〕；Ruoslahti, E.等、Science ２３８：４９１−４９７（１９８７）〕。白血球インテグリンは白血球上のみに発現し、細胞−細胞相互作用に含まれ、わずかに公知のリガンドはＩＣＡＭ−１とｉＣ３ｂ（即ち免疫グロブリン様特徴を示さない補体成分Ｃ３のフラグメント）であり、Ｍａｃ−１によって認識される〔Kishimoto, T. K.等、Leukocyte Typing III, ＭｃＭｉ Chael, M.編、スプリンガー ベルラーグ、ニューヨーク（１９８７）；Springer, T.A.等, Ann. Rev. Immunol. 5：２２３−２５２（１９８７）；Anderson, D. C.等、Ann. Rev. Med. ３８：１７５−１９４（１９８７）〕。配列分析により、ＬＦＡ−１により認識されるICAM−１配列内の潜在的ペプチドは第９表に示す。

ＩＣＡＭ−１はインテグリンに結合する免疫グロブリン超遺伝子群の１員の最初の例である。これらの群の両方は細胞粘着において重要な役割を発揮するけれども、両者間の相互作用は以前には予期されてなかった。これに対し、免疫グロブリン超遺伝子群内の相互作用は全く一般的である。インテグリンと免疫グロブリン群との間の相互作用のさらなる例が発見されるであろうことは全く可能である。ＬＦＡ−１はＩＣＡＭ−１とは異なるリガンドを認識し〔Springer, T.A.等、Ann. Rev. Immunol. ５：２２３−２５２（1987）〕、白血球インテグリンMac−１は好中球−好中球粘着のＣ３biと異なるリガンドを認識する〔Anderson, D.C.等、Ann. Rev. Med. ３８：１７５−１９４（１９８７）〕。さらにまた、精製したＭＡＧ含有ベシクル（小胞）はＭＡＧであるニューリット（neurites）に結合し、かくしてＭＡＧは異種レセプターと異好性相互作用可能でなければならない〔Poltorak, M.等、J. Cell. Biol. １０５：１８９３−１８９９（１９８７）〕。
神経−神経および神経−筋肉細胞相互作用におけるＮＣＡＭの役割は同種親和性ＮＣＡＭ−ＮＣＡＭ相互作用に基づくことは示唆されている〔Cunningham, B.A.等、Science ２３６：７９９−８０６（１９８７）〕。ミエリン鞘形成中に軸索を取り込むシュヴアン細胞の隣近回転ループ間の相互作用におけるＭＡＧの重要な役割は異種レセプターとの相互作用または同種親和性ＭＡＧ−ＭＡＧ相互作用に基づき得る。ＮＣＡＭとの相同性および免疫グロブリン超遺伝子群内でのドメイン−ドメイン相互作用の頻繁な出現はＩＣＡＭ−１が同種親和性相互作用並びにＩＣＡＭ−１−ＬＦＡ−１異好性相互作用に係わり得る可能性を引き起こす。しかしながら、同様な密度のＬＦＡ−１とＩＣＡＭ−１を共発現するＢリンパ芽球細胞の人工または細胞単一層中のＩＣＡＭ−１への結合はＢ−リンパ芽球のＬＦＡ−１ ＭＡｂによる前処理によって完全に抑制され得るが、粘着はＩＣＡＭ−１ ＭＡｂによるＢ−リンパ芽球前処理によって影響されない。ＩＣＡＭ−１ Ｍａｂによる単一層の前処理は結合を完全に壊滅させている〔Dustin, M.L.等、J. Immunol. １３７：２４５−２５４（１９８６）；Marlin, S.D.等、Cell, ５１：８１３−８１９（１９８７）〕。これらの知見は、ＩＣＡＭ−１同種親和性相互作用が全く起った場合、その作用がＬＦＡ−１との異好性相互作用よりもかなり弱くなければならないことを示している。
白血球インテグリンが基本的に異なる方法でリガンドを認識する可能性はリガンド結合において重要でありＲＧＤ認識性インテグリン中には存在しないそれらのαサブユニット中の１８０残基配列の存在と一致する〔Corbi, A.等、ＥＭＢＯ Ｊ．６：４０２３−４０２８（１９８７）〕。Ｍａｃ−１はｉＣ３ｂ５０８６中に存在するＲＧＤ配列を認識することが提示されているけれども、ＩＣＡＭ−１中にはＲＧＤ配列はない（第８図）。これはフィブロネクチンペプチドＧＲＧＤＳＰとコントロールペプチドＧＲＧＥＳＰがＩＣＡＭ−１−ＬＦＡ−１粘着を抑制できないことと一致している〔Marlin, S.D.等、Cell. ５１：８１３−８１９（１９８７）〕。しかしながら、PRGGSおよびＲＧＥＫＥのような関連配列はＩＣＡＭ−１中に、それぞれ、ドメイン２のβ−ストランドａとｂおよびドメイン２のｃとｄ間のループに予示される領域において存在しており（第９図）、従って、認識について受け入れられ得る。興味あることは相同性ＭＡＧ分子がドメイン１と２の間にＲＧＤ配列を含むことである〔Poltorak, M.等、J. Cell. Biol. １０５：１８９３−１８９９（１９８７）；Salzer, J.L.等、J. Cell. Biol. １０４：９５７−９６５（１９８７）〕。
実施例１９
サウサーンおよびノウサーンブロット
サウサーンブロットを３種の細胞系から抽出した５μｇの遺伝子ＤＮＡを用いて行なった：ＢＬ２、バーキットリンパ腫細胞系（Dr. Gilbert Lenoirから供与）；ＪＹおよびＥｒ−ＬＣＬのＥＢＶ形質転換Ｂ−リンパ腫様細胞系。
各ＤＮＡを５×製造者が推奨する量のＢamＨ１とＥcoＲＩエンドヌクレアーゼ（ニューイングランド バイオラブス社）で消化した。０．８％アガロースゲルによる電気泳動に続いて、ＤＮＡをナイロン膜（ゼータプローブ、バイオラド社）に移した。フィルターをプレハイブリッド化およびα−（³²P）d ＸＴＰ’ｓで標識したＨＬ−６０からのＩＣＡＭ ｃＤＮＡを用いての標準手法に従ってランダムプライミングによりハイブリッド化した。ノウサーンブロット２０μｇの全ＤＮＡまたは６μｇのポリ（Ａ）⁺ＲＮＡを用いて行なった。ＲＮＡは変性し１％アガローズ−ホルムアルデヒドゲルにより電気泳動し、ゼータプローブに電気移動させた。各フィルターをプレハイブリッド化し³²Ｐ標識オリゴヌクレオチドプローブ（前述の）ＨＬ−６０ｃＤＮＡプローブを用いて前述したようにしてハイブリッド化した（Stauton, D.E.等、Embo J. ６：３６９５−３７０１（１９８７）〕。
３ｋｂ ｃＤＮＡプローブおよびＢamＨ１とＥcoＲ１で消化した遺伝子ＤＮＡを用いたサウサーンブロットはそれぞれが単一遺伝子を示しまたコード化情報の殆どが８ｋｂ内に存在することを示す２０および８ｋｂの単一主要ハイブリッド化性フラグメントを示した。３種の細胞系のブロットにおいては、制限フラグメント多形性の証拠はなかった。
実施例２０
ＩＣＡＭ−１遺伝子の発現
“発現ベクター”は、（適当な転写性および／またはほん訳性コントロール配列の存在に基づき）ベクターにクローニングされるＤＮＡ（またはｃＤＮＡ）を発現し得、それによってポリペプチドまたはたん白質を産生し得るベクターである。クローニングした配列の発現は発現ベクターを適当なホスト細胞に組み込んだときに起る。原核細胞発現ベクターを用いる場合は、適切なホスト細胞はクローニングした配列を発現し得る任意の原核細胞であろう。同様に、真核発現ベクターを用いる場合、適切なホスト細胞はクローニングした配列を発現し得る任意の真核細胞である。重要なことは、真核ＤＮＡは介在配列を含み得るので、またそのような配列は原核細胞中では正確に加工できないので、原核ゲノム発現ベクターライブラリーを産生するためには、ＩＣＡＭ−１を発現し得る細胞からのｃＤＮＡを用いることが好ましい。ｃＤＮＡを調製する方法およびゲノムライブラリーを産生する方法はManiatis, J.等により開示されている〔Molecular Cloning：A Laboratory Manual,コールド スプリング ハーバー プレス社、コールド スプリング ハーバー、ＮＹ（１９８２）〕。
上述の発現ベクター遺伝子ライブラリーを用いてホスト細胞のバンクを作る（その各々はライブラリーの１員を含む）。発現ベクターはホスト細胞に任意の種々の手段（即ち、形質転換、トランスフェクション、原形質体融合、エレクトロポーレーション等）によって組み込み得る。発現ベクター含有細胞のバンクはクローン的に増殖させ、その各員は個々にアッセイして（イムノアッセイを用いて）これらが抗ＩＣＡＭ−１抗体に結合し得るたん白質を産生するかどうかを測定する。
抗ＩＣＡＭ−１抗体に結合し得るたん白質を産生する細胞の発現ベクターはさらに分析してこれらのベクターが全ＩＣＡＭ−１遺伝子を発現（または含有）するかどうか、ＩＣＡＭ−１遺伝子のフラグメントのみを発現（または含有）するかどうかあるいは生成物が免疫学的にＩＣＡＭ−１に関係したとしてもＩＣＡＭ−１ではない遺伝子を発現（または含有）するかどうかを決定する。そのような分析は任意の都合の良い方法で行なってよいけれども、好ましいのは発現ベクターにクローニングされるＤＮＡまたはｃＤＮＡのヌクレオチド配列を決定することである。そのようなヌクレオチド配列を検査してＩＣＡＭ−１のトリプシン消化フラグメント（第５表）と同じアミノ酸配列を有するポリペプチドをコードし得るかどうかを決定する。
ＩＣＡＭ−１遺伝子をコードするＤＮＡまたはｃＤＮＡ分子を含む発現ベクターは、かくして、（ｉ）抗ＩＣＡＭ−１抗体に結合し得るたん白質の発現を行なう能力；および（ii）ＩＣＡＭ−１のトリプシンフラグメントの各々をコードし得るヌクレオチド配列の存在によって認識し得る。そのような発現ベクターのクローニングＤＮＡ分子は発現ベクターから取り出して純粋な形で単離できる。
実施例２１
精製ＩＣＡＭ−１の機能活性
細胞中では、ＩＣＡＭ−１は細胞膜と会合した表面たん白質として通常は機能する。従って、精製ＩＣＡＭ−１の機能は分子を人工脂質膜（リポソームまたはベシクル）中にたん白質を洗浄剤可溶化脂質中に溶解し次いで洗浄剤を透析により除去することによって再構成させたのち試験した。ＪＹ細胞から精製し洗浄剤オクチルグリコシド中に上述のようにして溶出したＩＣＡＭ−１をベシクル中に再構成し、ＩＣＡＭ−１含有ベシクルをガラスカバースリップまたはプラスチック培養ウェルに融合させてたん白質に結合する細胞の検出をできるようにした。
平坦膜およびプラスチック結合ベシクルの調製
ベシクルはＧay等の方法により調製した〔J. Immunol, １３６：２０２６（１９８６）〕。即ち、たまごホスファチジルクロリンとコレステロールをクロロホルム中に溶解し７：２のモル比で混合した。脂質混合物をチッ素ガス流下に回転させながら薄膜に乾燥させ、次いで１時間で凍結乾燥させてすべての痕跡量のクロロホルムを除去した。脂質膜を１％オクチルグリコシド／０．１４Ｍ NaCｌ／２０ｍＭトリス（ｐＨ７．２）中にホスファチジルクロリン最終濃度０．１ｍＭに溶解した。約１０μｇの精製ＩＣＡＭ−１またはコントロール膜糖たん白質としてのヒトグリコホリン（シグマケミカル社、セントルイス、ＭＯ）を溶解脂質の各mｌに加えた。たん白質−脂質−洗浄剤溶液を２００容量の２０ｍＭトリス／０．１４Ｍ NaCｌ、ｐＨ７．２の３回交換およびＨＢＳＳの１回交換に対して４℃で透析した。
平坦膜はBrian等の方法により調製した〔Proc. Natl. Acad. Sci. ８１：６１５９（１９８４）〕。ガラスカバースリップ（直径１１mm）を１７×洗浄剤（Linbro）の１：６希釈液中で１５分間煮沸し、一夜蒸留水中で洗浄し、７０％エタノール中に浸し、風乾させた。ＩＣＡＭ−１またはクリコホリンのいずれかを含有するベシクル懸濁液の８０μｌ小滴を２４ウェルクルスタープレートのウェル底部に入れ、上記で調製したガラスカバースリップを静かに頂部に浮かした。室温での２０〜３０分のインキュベーション後、各ウェルをＨＢＳＳで満し、カバースリップをひっくり返して平坦面を上向きにした。次いでウェルをHBSSで十分洗浄して未結合ベシクルを除去した。平坦膜表面は全く空気にさらさなかった。
ガラス表面に融合させた平坦膜による実験途中で、ＩＣＡＭ−１を含むベシクルはマルチウェル組織培養プレートのプラスチック表面に直接結合し、特異的細胞結合により明らかなような機能活性を保持していることが判明した。そのようなベシクルは以後“プラスチック結合ベシクル（PBV）と称する。というのは、プラスチックに結合した脂質ベシクルの性質を測定するのではないからである。プラスチック結合ベシクルは３０μｌのベシクル懸濁液を直接９６ウェル組織培養トレイ（ファルコン社）中のウェル底部に加え次いで平坦膜について述べたようにしてインキュベーションおよび洗浄を行うことによって調製した。
細胞粘着アッセイ
平坦膜またはプラスチック結合ベシクルを用いた細胞粘着アッセイの両方を本質的に同じ方法で行ったが、ＰＢＶアッセイの細胞数および容量は平坦膜アッセイで用いたものの１／５に減じた。
正常コントロールおよびＬＦＡ−１を発現できない白血球粘着欠損（ＬＡＤ）患者〔Anderson, D.C.等、J. Infect. Dis. １５２：668（1985）〕からのＴリンパ球を、１μｇ／mｌのコンカナバリン−Ａ（Con-A）を含む末梢血単核細胞RPMI−１６４０＋２０％ＦＣＳ中で５×１０⁵細胞／mｌで３日間培養することによって調製した。次いで、細胞をＲＰＭＩで２回；５ｍＭメチル−アルファ−ｂ−マノフィラノシドで１回洗浄して残留レクチンを細胞表面から除去した。細胞を１ng／mｌの組換えＩＬ−２を含むＲＰＭＩ／２０％ＦＣＳ中で増殖させ、培養開始後１０および２２日の間で使用した。
平坦膜またはＲＢＶに結合する細胞を検出するため、Con A芽球、Ｔ−リンパ腫ＳＫＷ−３、ＥＢＶ形質転換Ｂ−リンパ腫様細胞系ＪＹ（LFA−１陽性）およびＬＦＡ−１欠損リンパ腫様細胞系（ＢＢＮ）〔患者１由来、Springer, T.A.等、J. Exper. Med. １６０：１９０１−１９１８（１９８６）〕を１mｌのＲＰＭＩ−１６４０／１０％ＦＣＳ中の１×１０⁷細胞を１００μＣｉのNa⁵¹CrO₄で３７℃、１時間インキュベートし、次いでＲＰＭＩ−１６４０で４回洗浄し未結合標識を除去することによって放射性標識させた。モノクローナル抗体ブロッキング試験においては、細胞またはプラスチック結合ベシクルはＲＰＭＩ−１６４０／１０％ＦＣＳ中の２０μｇ／mｌの精製抗体で４℃にて３０分間前処理し、次いで４回洗浄して未結合抗体を除去した。細胞結合に関しての２価イオンの効果の実験においては、細胞をＣa²⁺、Ｍg²⁺を含まないＨＢＳＳ＋１０％透析ＦＣＳで１回洗浄し、CaCｌとMgCｌを決められた濃度に加えた。すべての実験において、細胞および平坦膜またはＰＢＶは適当な温度（４℃、２２℃または３７℃）で適当なアッセイバッファー中で予備平衡させた。
精製ＩＣＡＭ−１に結合する細胞を測定するために、⁵¹Ｃr標識細胞（平坦膜アッセイでの５×１０⁵ＥＢＶ形質転換体；ＰＢＶアッセイでの１×１０⁵ＥＢＶ形質転換体またはＳＫＷ−３細胞、２×１０⁵Ｃon−Ａ芽球）を平坦膜またはＰＢＶ上で２５Ｘgで２分間遠心し、次いで４℃、２２℃または３７℃で１時間インキュベーションした。インキュベーション後、未結合細胞を適当な温度での予備平衡させたバッファーによる充填、吸引の８回のサイクルによって除去した。結合細胞はウェル内容物の0.1N NaOH／１％トリトンＸ−１００による可溶化およびガンマーカウンターでの計数によって定量した。％細胞結合は結合細胞からのcpmを入力細胞のcpmで割ることによって決定した。平坦膜アッセイにおいては、入力cpmはカバースリップの表面積を培養ウェルの表面積と比較した比に対して集めた。
これらのアッセイにおいて、ＥＢＶ形質転換Ｂ−リンパ腫細胞、ＳＫＷ−３Ｔ−リンパ腫細胞、およびＣon−A Ｔリンパ芽球は人工膜中のICAM−１に特異的に結合した（第１１図および第１２図）。結合は、細胞が等価の量の他のヒト細胞表面糖たん白質グリコホリンを含んだコントロールの平坦膜またはベシクルに極めて貧弱にしか結合しなかったことから特異的であった。さらにまた、ＬＦＡ−１陽性ＥＢＶ形質転換体およびＣon Ａ芽球も結合したが、それらのＬＦＡ−１陰性等価物は何ら有意の程度に結合せず、結合が細胞上のＬＦＡ−１の存在に依存していることを示した。
細胞結合の特異性および細胞ＬＦＡ−１への依存性の両方はモノクローナル抗体のブロッキング試験において確認した（第１３図）。ＪＹ細胞の結合はＩＣＡＭ−１含有ＰＢＶを抗ＩＣＡＭ−１モノクローナル抗体ＲＲ１／１前処理したとき９７％まで抑制できた。同じ抗体による細胞の前処理は殆んど効果がなかった。逆に、抗ＬＦＡ−モノクローナル抗体ＲＳ１／１８は９６％まで結合を抑制したがＰＢＶでなく細胞を前処理したときはわずかであった。ＬＦＡ−３と反応性のコントロール抗体ＴＳ２／９（異なるリンパ球表面抗原）は細胞またはＰＢＶのいずれを前処理したときも有意の抑制効果はなかった。この実験は人工膜の若干量の不純物のないＩＣＡＭ−１自体が観察された細胞粘着を媒介していることおよび粘着は結合性細胞上のＬＦＡ−１に依存していることを示している。
人工膜中のＩＣＡＭ−１への細胞の結合はまたＬＦＡ−１依存性粘着系の２つの他の特性：温度依存性と２価カチオンの必要性を示した。第１４図に示すように、Ｃon−Ａ芽球はＰＢＶ中のICAM−１に３７℃で最も効果的に、２２℃で部分的に、４℃で極めて貧弱に結合した。第１５図に示すように、結合は完全に２価カチオンの存在に依存している。生理学上の濃度においては、Ｍg²⁺は単独で最高の細胞結合を示したが、Ｃa²⁺の単独は極めて低レベルの結合を示した。しかしながら、Ｃ²⁺と組合せた通常濃度の１／１０のＭg²⁺は相乗効果を有し最高の結合を示した。
要約すれば、人工膜中に含有させた精製ICAM−１に対する細胞結合の特異性、モノクローナル抗体による特異的抑制、および温度および２価カチオンの必要性はＩＣＡＭ−１がＬＦＡ−１依存性の粘着系の特異的リガンドであることを示している。
実施例２２
アレルギーおよび毒性パッチ試験反応におけるＩＣＡＭ−１とＨＬＡ−ＤＲの発現
５人の正常人の皮ふ生検をそのＩＣＡＭ−１およびＨＬＡ−ＤＲ発現について行った。ある血管中の内皮細胞は通常ＩＣＡＭ−１を発現するけれども、正常皮ふからのケラチン細胞にはＩＣＡＭ−１は発現しないことが判った。正常皮ふ生検からの任意ケラチン細胞上のＨＬＡ−ＤＲの染色は観察されなかった。ＩＣＡＭ−１とクラスII抗原の発現動力学をアレルギー性および毒性皮ふ傷害の生検の細胞において検討した。検討した６人の対象者の半分がハプテンの適用後４時間でICAM−１を発現したケラチン細胞を有していることが判った（第１０表）。ハプテンへの露出時間によりケラチン細胞上にＩＣＡＭ−１を発現する人の割合が増大し、また４８時間までにケラチン細胞当りより多くのＩＣＡＭ−１発現を示す染色強度も増大した。事実、この時点で、すべての生検のケラチン細胞の部分がＩＣＡＭ−１に対して陽性に染色した。７２時間（ハプテン除去後２４時間）で、８人の対象者の７人がケラチン細胞にICAM−１を発現しており、１人の対象者のＩＣＡＭ−１発現は４８〜７２時間の間に弱くなった。

組織学上、ハプテンの適用後４時間で採取した生検からのケラチン細胞上のＩＣＡＭ−１の染色像は通常小群生状であった。４８時間後、ICAM−１はケラチン細胞の大部分の表面上に発現し、傷害の中心および周辺間に差異はなかった。染色強度はケラチン細胞が爪角質層に近づいたとき減少した。これは傷害の中心および周辺から採られた生検において見い出された。また、この時間でも、パッチ試験は陽性であった（湿潤、紅斑、小胞）。異なるハプテンを感受性個々人に適用してもICAM−１発現における差異は見られなかった。ケラチン細胞以外にも、ＩＣＡＭ−１は傷害部位でいくつかの単核細胞および内皮細胞上にも発現した。
アレルギー皮ふ傷害のケラチン細胞上でのHLA−ＤＲの発現はＩＣＡＭ−１の発現よりも頻度は小さかった。検討した対象者のうち、ハプテン適用後２４時間までにＨＬＡ−ＤＲに陽性に染色したケラチン細胞による傷害を有するものはなかった。事実、わずかに４人の生検サンプルがＨＬＡ−ＤＲを発現したケラチン細胞を有するに過ぎず、ＨＬＡ−ＤＲに陽性でＩＣＡＭ−１に陽性でないケラチン細胞を有する生検はなかった（第１０表）。
アレルギーパッチ試験傷害と対照的に、はず油またはラウリル硫酸ナトリウムで誘発させた毒性パッチ試験傷害は試験のすべての時点でその表面にＩＣＡＭ−１を殆んど示さないケラチン細胞を有していた（第１１表）。実際に、パッチ適用後４８時間で、これはアレルギー性パッチ試験対象者における最適の時点であるが、１４人の毒性パッチ試験対象者のうちの１人が傷害中にＩＣＡＭ−１を発現するケラチン細胞を有していた。また、アレルギー性パッチ試験生検と対照的に、毒性パッチ試験傷害のケラチン細胞上にＨＬＡ−ＤＲは発現しなかった。
これらのデータはＩＣＡＭ−１が免疫系炎症中で発現し毒性系炎症では発現しないことを示しており、かくして、ＩＣＡＭ−１の発現は、疾患が免疫抑制治療剤の拒絶または尿毒性によるのかどうかの判断が難しい賢移植患者における急性腎疾患のような免疫系および毒性系炎症を区別するのに使用できる。腎生検およびＩＣＡＭ−１発現の向上の評価が免疫系拒絶と非免疫系毒性反応の区別を可能にするであろう。

実施例２３
良性皮ふ病中のＩＣＡＭ−１とＨＬＡ−ＤＲの発現
種々のタイプの炎症性皮ふ病を有する患者からの傷害の皮ふ生検からの細胞をそのＩＣＡＭ−１とＨＬＡ−ＤＲの発現について検討した。アレルバー性接触湿疹、天疱瘡、および扁平苔癬の生検中のケラチン細胞の１部はＩＣＡＭ−１を発現した。扁平苔癬は４８時間アレルギー性パッチ試験生検で見られた結果と同等か幾分強い像により最も強く染色を示した（第１２表）。アレルギー性パッチ試験の結果と同様に、最も強いＩＣＡＭ−１染色は高単核細胞浸潤部位で見られた。さらにまた、試験した１１の扁平苔癬生検のうちの８つはケラチン細胞上でＨＬＡ−ＤＲ発現について陽性であった。
発疹とじんま疹を有する患者の皮ふ生検からのケラチン細胞上のＩＣＡＭ−１の発現は少なかった。これらの疾患を有する試験した７人の患者のうち４人のみが傷害部位でＩＣＡＭ−１を発現したケラチン細胞を有していた。ＨＬＡ−ＤＲ発現は１人の患者においてのみであり、これはICAM−１と関連していた。
試験した良性炎症皮ふ病のすべてからの内皮細胞および単核細胞浸潤の部分は変化度合でICAM−１を発現していた。

実施例２４
乾癬皮ふ病のケラチン細胞上でのＩＣＡＭ−１発現
乾癬を有する５例の患者からの皮ふ生検中のＩＣＡＭ−１発現をＰＵＶＡ治療の開始前および治療途中で周期的に検討した。生検は組織学によって確認した古典的乾癬を有する５例の患者から得た。生検はＰＵＶＡ治療の前および指示された時間中に連続的に採取した。ＰＵＶＡは週に３〜４回投与した。生検は５例の患者の乾癬斑の周辺から採取し、生検以外に、これら患者の４人の臨床的に正常な皮ふからも採取した。
各新鮮皮ふ生検試料を凍結し、液体チッ素中に保存した。６ミクロンの保存切片を一夜室温で風乾し、アセトン中で１０分間固定し、直ちに染色しまたはアルミニウムホイルで包み染色するまで−８０℃で保存した。
染色は次の方法で作った。切片をモノクローナル抗体でインキュベートし、ジアミノベンジジンH₂O₂、基質を用いて３段階イムノパーオキシダーゼ法により染色した〔Stein, H.等、Adv. Cancer Res. ４２：６７−１４７、（１９８４）〕。へん桃腺およびリンパ節を抗ＩＣＡＭ−１およびＨＬＡ−ＤＲ染色用の陽性コントロールとして用いた。一次抗体の不存在下で染色した組織は陰性コントロールであった。
ＨＬＡ−ＤＲに対するモノクローナル抗体をBecton Dickinson社（カリホルニア州マウンテンビュー）から購入した。抗ＩＣＡＭ−１モノクローナル抗体はＲ６−５−Ｄ６であった。パーオキシダーゼ接合ウサギ抗マウスＩｇおよびパーオキシダーゼ接合ブタ抗ウサギＩｇはスウェーデン、コペンハーゲンのDAKAPATTSより購入した。ジアミノベンジジン−テトラヒドロクロリドはシグマ社（セントルイス）より得た。
試験の結果は数種の血管の内皮細胞が疾患および正常皮ふの両方でＩＣＡＭ−１を発現していることを示したが、染色強度およびＩＣＡＭ−１を発現する血管の数は乾癬皮ふ病変内で増大した。さらに、５例の患者からの未治療乾癬皮ふ病変のケラチン細胞中のＩＣＡＭ−１の発現像はわずかに小群の細胞染色から多数のケラチン細胞が染色されているまでに変化した。ＰＵＶＡ治療の途中では、２例の患者（患者２と３）のＩＣＡＭ−１発現は臨床的軽快に先行してあるいは同時に著しい低減を示した。患者１、４および５は、それぞれ、臨床的軽快あるいは悪化に相関してＰＵＶＡ治療中にＩＣＡＭ−１発現を減少あるいは増大させた。ＰＵＶＡ治療前後の正常皮ふからのケラチン細胞上ではＩＣＡＭ−１発現はなかった。このことはＰＵＶＡは正常皮ふからのケラチン細胞上にＩＣＡＭ−１を誘起しないことを示している。
注目すべきことは単核細胞浸潤密度はケラチン細胞上のＩＣＡＭ−１発現量と相関していることであった。このことはＩＣＡＭ−１発現も弱まったときＰＵＶＡ治療中の傷害中の単核細胞の数も減少することおよびケラセン細胞上のＩＣＡＭ−１発現がより顕著になったときＰＵＶＡ治療中の単核細胞の数が増大することの両方に関係している。内皮細胞および皮ふ単核細胞もまたＩＣＡＭ−１陽性である。臨床的に正常な皮ふにおいては、ＩＣＡＭ−１発現はケラチン細胞の標識化なしで内皮細胞に確認された。
ケラチン細胞上のＨＬＡ−ＤＲの発現は可変的であった。ＩＣＡＭ−１陽性でないＨＬＡ−ＤＲ陽性生検は存在しなかった。
要約すれば、これらの結果は、治療前では、ＩＣＡＭ−１発現はケラチン細胞上で高く、単核細胞浸潤密度と相関していることを示している。ＰＵＶＡ治療中は、ＩＣＡＭ−１染色の著しい減少が臨床的改善と平行して見られる。組織学的には、皮ふ浸潤は消失していた。臨床的悪化が治療中に見られる場合には、ケラチン細胞上のICAM−１の発現並びに皮ふ浸潤密度も増大した。臨床的軽快が治療中に見られたときは、ケラチン細胞上のＩＣＡＭ−１染色の同時の減少並びに皮ふ浸潤の減少があった。即ち、ケラチン細胞上のICAM−１の発現は皮ふの単核細胞浸潤密度に相応していた。これらのデータはＰＵＶＡ治療に対する臨床的応答は単核細胞のよりおだやかな下降と平行してケラチン細胞上のＩＣＡＭ−１発現の促進された減少をもたらすことを示している。このことはケラチン細胞上のＩＣＡＭ−１発現が皮ふ浸潤の開始および持続に応答性であること、およびＰＵＶＡ治療がＩＣＡＭ−１を下方調整し皮ふ浸潤および炎症応答を緩和していることを示している。データはまたＰＵＶＡ治療中のケラチン細胞上のＨＬＡ−ＤＲが発現が変化性であったことも示している。
乾癬傷害のケラセン細胞上のＩＣＡＭ−１発現は傷害臨床上の厳正さおよび皮ふ浸潤の大きさと相関している。即ち、ＩＣＡＭ−１は乾癬において中心的役割を発揮し、その発現の抑制および／またはその単核細胞上でのＣＤ１８コンプレックスとの相互作用の抑制は本病変の有効な治療となるであろう。さらにまた、ケラチン細胞上のICAM−１発現をモニターすることは乾癬の診断、予防、および治療経過を評価するための有効な手段となるであろう。

実施例２５
悪性皮ふ病中のＩＣＡＭ−１とＨＬＡ−ＤＲの発現
良性皮ふ状態の病変とは異なり、悪性皮ふ傷害からのケラチン細胞上のＩＣＡＭ−１の発現は変化に富んでいた（第１４表）。試験した皮ふＴ−細胞リンパ腫２３例のうち、ＩＣＡＭ−１陽性ケラチン細胞は１４例のみにおいて同定された。菌状息肉腫病変の生検からのケラチン細胞では、病気の進行がより前の段階に進むにつれてそのICAM−１発現を消化する傾向にあった。しかしながら、ＩＣＡＭ−１発現は皮ふＴ細胞リンパ腫病変の殆んどからの変化割合の単核細胞浸潤上に見られた。試験した残りのリンパ腫のうちでは、８つのうち４つがＩＣＡＭ−１を発現したケラチン細胞を有していた。試験した悪性皮ふ病を有する２９人の患者のうち、５例はＩＣＡＭ−１を発現することなしにＨＬＡ−ＤＲを発現したケラチン細胞を有していた（第１４表）。

実施例２６
ヒト末梢血単核細胞の増殖上の抗ＩＣＡＭ−１抗体の効果
ヒト末梢血単核細胞を抗原またはマイトジェンの存在および認識より誘起させ増殖させる。マイトジェン、コンカナバリンＡまたはＴ細胞結合性抗体ＯＫＴ₃のようなある種の分子は末梢血単核細胞の非特異的増殖を引き起こす。
ヒト末梢血単核細胞はこれらが特異的抗原を認識し得る細胞の細個体群（subpopulation）からなる点で不均質である。特定の特異抗原を認識し得る末梢血単核細胞がその抗原に出会った場合、単核細胞の上記細個体群の増殖は誘起される。破傷風トキソイドおよびキーホールリンペットヘモシアニンは末梢単核細胞の細個体群により認識される抗原の例であるが感応化した個体中のすべての末梢単核細胞によって認識されるものではない。
細胞−細胞粘着を必要とすることが知られている系中でのヒト末梢血単核細胞の増殖的応答を抑制する抗ＩＣＡＭ−１モノクローナル抗体Ｒ６−５−Ｄ６の能力を試験した。
末梢血単核細胞をフィコール−ペーグ（Ficoll-Paqua、ファルマシア社）勾配で製造者が推奨するようにして精製した。界面を集めたのち、細胞をＲＰＭＩ１６４０培地で３回洗浄し平底９６ウェルマイクロタイタープレート中で１０％ウシ胎児血清、２ｍＭグルタミンおよびジェンタマイシン（５０μg／mｌ）を加えたＲＰＭＩ１６４０培地中で１０⁶細胞／mｌの濃度で培養した。
抗原、Ｔ−細胞マイトジェン、コンカナバリンＡのいずれか（０．２５μｇ／mｌ）；Ｔ−細胞結合性抗体ＯＫＴ₃（０．００１μｇ／mｌ）；キーホールリンペットヘモシアニン（１０ｇ／mｌ）または破傷風トキソイド（供給源からの１：１00希釈物）を上記のようにして培養した細胞中に抗ＩＣＡＭ−１抗体（Ｒ６−５−Ｄ６；最終濃度５ｇ／mｌ）の存在または不存在下に加えた。細胞はアッセイが終了する前の３．５日（コンカナバリンＡ試験）、２．５日（ＯＫＴ₃試験）、または５．５日（キーホールリンペットヘモシアニンおよび破傷風トキソイド試験）で培養した。アッセイ終了前１８時間で、２．５μＣｉの³Ｈ−チミジンを培養物に加えた。細胞増殖を末梢血単核細胞によるＤＮＡへのチミジンの取り込みを測定することによってアッセイした。取り込まれたチミジンを集め液体シンチレーションカウンター中で計数した〔Merluzzi等、J. Immunol. １３９：１６６−１６８（１９８７）〕。これらの実験の結果は第１６図（コンカナバリンＡ試験）、第１７図（ＯＫＴ₃試験）、第１８図（キーホールリンペットヘモシアニン試験）、および第１９図（破傷風トキソイド試験）に示す。
抗ＩＣＡＭ−１抗体は単核細胞中の非特異Ｔ−細胞マイトジェン、ＣonＡ；非特異的Ｔ−細胞会合抗原、ＯＫＴ−３；および特異抗原、キーホールリンペットヘモシアニンおよび破傷風トキソイドに対する増殖的応答を抑制することが判明した。抗ＩＣＡＭ−１抗体による抑制は抗ＬＦＡ−１抗体の抑制効果と匹敵し、ＩＣＡＭ−１はＬＦＡ−１の官能性リガンドであることおよびＩＣＡＭ−１のアンタゴニストは特異的防御系応答を抑制するであろうことを示唆している。
実施例２７
混合リンパ球反応についての抗ＩＣＡＭ−１の効果
前述したように、ＩＣＡＭ−１はＬＦＡ−１依存性細胞粘着より媒介された免疫応答中の有効な細胞相互作用のために必要である。免疫応答または炎症疾患中のＩＣＡＭ−１の誘起は白血球の相互または内皮細胞との相互作用を可能にする。
２例の無関係の個人からのリンパ球を各互いの存在下で培養したときは、芽球形質転換およびリンパ球の細胞増殖が観察される。１つの集団の白血球を第２集団の白血球の存在へのこの応答は混合リンパ球反応（ＭＬＲ）として公知であり、リンパ球のマイトジェンの添加に対する応答と同類である〔Immunology The Science of Self-Nonself Discrimination, Klein, J. John Wiley & Sons社、ＮＹ（１９８２）、ｐｐ４５３−４５８〕。
抗ＩＣＡＭモノクローナル抗体のヒトＭＬＲに対する効果について試験した。これらの実験は次のようにして行った。末梢血を正常な健康ドナーから静脈穿刺により採取した。血液をヘパリン化チューブに集め、室温でPunk's G（GIBCO社）平衡塩溶液（ＢＳＳ）で１：１に希釈した。血液混合物（２０mｌ）を１５mｌのFicoll/Hypaque密度勾配（ファルマシア社、密度１．０７８、室温）上に層化し、１０００Ｘgで２０分間遠心した。次いで界面を集め、Punk's G中で３回洗浄した。細胞をヘマシトメーター上で計数し、０．５％のジェンタマイシ、１mM Ｌ−グルタミン（GIBCO社）および５％加熱不活化（５６℃、３０分）ヒトＡＢ血清（フローラボラトリーズ社）とを含むRPMI−１６４０培地（GIBCO社）（以下、ＲＰＭＩ培地と呼ぶ）中に再懸濁させた。
マウス抗−ＩＣＡＭ−１（Ｒ６−５−Ｄ６）をこの実験で用いた。すべてのモノクローナル抗体（ジャックソンイムノリサーチラボラトリーズ社、ボストン、ＭＡにより腹水から調製された）を精製ＩｇＧ調製物として用いた。末梢血単核細胞（ＰＢＭＣ）はリンブロ（Linbro）丸底マイクロタイタープレート（＃７６−０１３−０６）中で６．２５×１０⁵細胞／mｌで培地中で培養した。別々のドナーからのスチミュレーター細胞を1000Ｒで照射し、レスポンダー細胞と同じ濃度で培養した。培養当りの総容量は０．２mｌであった。コントロールはレスポンダー細胞単独およびスチミュレーター細胞単独を含んでいた。培養プレートを３７℃で５％ＣＯ₂−湿潤空気雰囲気中で５日間インキュベーとした。各ウェルを０．５μＣｉのトリチウム化チミジン（³ＨＴ）（ニューイングランドニュクレア社）で培養の最後の１８時間脈動させた。ある場合には、２経路（two-way）MLRを行った。そのプロトコールは第２ドナー細胞を照射により不活化しなかった以外は同じであった。
細胞をガラス繊維フィルター上に自動化マルチプルサンプルハーベスタ（Skatron社、ノルウェー）を用いて採取し、水およびメタノールですすいだ。フィルターをオーブン乾燥させ、アクアゾル中でベッグマン（ＬＳ−３８０１）液体シンチレーションカウンターで計数した。結果は６例の個々の培養物について平均ＣＰＭ±標準誤差として示している。
第１５表は精製抗ＩＣＡＭ−１モノクローナル抗体が２０ng／mｌで明らかな有意の抑制でもって投与量依存の形でＭＬＲを抑制していた。精製マウスＩｇＧは殆んどあるいは全く抑制効果を示さなかった。抗ＩＣＡＭ−１モノクローナル抗体によるＭＬＲの抑制は抗体を培養の最初の２４時間以内で加えたとき生じる（第１６表）

要約すれば、ＩＣＡＭ−１に対する抗体のMLRを抑制する能力はＩＣＡＭ−１モノクローナル抗体が急性移植拒絶に治療的利用性を有していることを示している。ＩＣＡＭ−１モノクローナル抗体はまたＬＦＡ−１／ＩＣＡＭ−１調整細胞−細胞相互作用に依存する関連免疫媒介不整における治療的利用性を有している。
上記の実験はＩＣＡＭ−１に対するモノクローナル抗体の添加が反応の最初２４時間中に加えたときに混合リンパ球反応（ＭＬＲ）を抑制することを示している。さらにまた、ＩＣＡＭ−１はインビトロ培養中のヒト末梢血単核細胞について状態向上なる。
さらにまた、ＩＣＡＭ−１は静止ヒト末梢血リンパ球または単核細胞上には発現しないことが見い出された。ＩＣＡＭ−１は単独培養細胞または混合リンパ球反応での無関係ドナー細胞との共培養細胞の単核細胞上で、通常のフローサイトメトリック分析を用いることにより状態向上される。単核細胞上でのこのＩＣＡＭ−１の状態向上は炎症の指示剤として、特に、ＩＣＡＭ−１が急性または慢性炎症を有するヒトの新鮮単核細胞上で発現する場合に使用できる。
活性化単核細胞に対するＩＣＡＭ−１の特異性およびＩＣＡＭ−１に対する抗体のＭＬＲを抑制する能力はＩＣＡＭ−１モノクローナル抗体が急性移植拒絶および細胞−細胞相互作用を必要とする関連免疫媒介不整における診断上および治療上の潜在力を有し得ることを示している。
実施例２８
抗ＩＣＡＭ−１および抗ＬＦＡ−１抗体の混合投与の相乗効果
実施例２７で示したように、ＭＬＲは抗ICAM−１抗体によって抑制される。ＭＬＲはまた抗ＬＦＡ−１抗体によっても抑制できる。抗ICAM−１および抗ＬＦＡ−１抗体の組合せ投与が促進されたあるいは相乗的効果を有するかどうかを決定するために、ＭＬＲアッセイ（実施例２７に記載したようにして行った）を２つの抗体の種々の濃度の存在下で行った。
このＭＬＲアッセイは抗ＩＣＡＭ−１＋抗LFA−１の組合せが、抗体単独では劇的にＭＬＲを抑制しない濃度において、ＭＬＲ応答を抑制するのに著しい効力があることを示した（第１７表）。この結果は抗ＩＣＡＭ−１抗体（またはそのフラグメント）と抗ＬＦＡ−１抗体（またはそのフラグメント）を共投与することを含む治療が改善された抗炎症治療を与える能力を有することを示している。そのような改善された治療は治療上有効である他の方法よりもより低い抗体投与量の投与を可能にし、また高濃度の個々の抗体が抗イデオタイプ応答を誘起するような場合に重要性を示す。

実施例２９
ＭＬＲにおける抗ＩＣＡＭ−１と他の免疫抑制剤との次善投与量での混合投与の付加的効果
実施例２８で示すように、ＭＬＲは抗ＩＣＡＭ−１抗体と抗ＬＦＡ−１抗体の組合せによって抑制される。抗ＩＣＡＭ−１と他の免疫抑制剤〔デキサメタソン、アゼチオピリン、シクロスポリンＡまたはステロイド（例えば、プレドニソン等の）のような〕との混合投与も改善された効果を有するかどうかを見るために、ＭＬＲアッセイを、実施例２７のプロトコールによるようにして、他の免疫抑制剤と組合せたＲ６−５−Ｄ６の次善濃度（即ち、薬剤を単独で対象物に対して投与する最適濃度よりも低いであろう濃度）を用いて行った。
データはＲ６−５−Ｄ６の抑制効果が次善投与量のデキサメタソン（第１８表）、アゼチオピリン（第１９表）およびシクロスポリンＡ（第２０表）の抑制効果を少なくとも付加されていることを示している。このことは抗ＩＣＡＭ−１が公知の免疫抑制剤の必要投与量の低減、即ち、その有毒副作用の低減において有効であり得ることを意味する。抗ＩＣＡＭ−１抗体（またはそのフラグメント）を用いてそのような免疫抑制を得るのに、この抗体（またはそのフラグメント）と単一の追加の免疫抑制剤または２種以上の追加の免疫抑制剤の組合せとの投与を行うことが可能である。

実施例３０
移植した同種異系臓器の拒絶を抑制するのにおける抗ＩＣＡＭ−１抗体の効果
同種異系移植臓器の拒絶を抑制するのにおける抗ＩＣＡＭ−１抗体の効果を示すために、カニクイザルにCosimi等の方法〔Transplant. Proc. １３：４９９−５０３（１９８１）〕に従って同種異系の腎臓を移植した、ただし、麻酔薬としてバリウム（valium）とケタミンを用いる修正を加えた。
即ち、腎臓移植を本質的に次のようにして行った。異型腎アログラフトを３〜５kgのカニクイザルに、バリウムとケタミンによる麻酔の誘起後、本質的にMarquetにより開示されたようにして行った〔Marquet等、Medical Primatology, Part II、Basel, Karger, p. １２５（１９７２）〕。大動脈または大動脈のパッチ上のドナー腎臓の末端−側面アナストモーシス（anastpmoses）を７−０プロレン（Prolene）縫合線を用いて構築した。ドナー尿管をスパチュラ処理し外のう的方法によってブラッダー中に埋め込んだ〔Taguchi, Y.等、Dausset等編、Advances in Transplantation、バルチモア、ウィリアムス アンド ウィルキンス、ｐ３９３（１９６８）〕。腎機能を１週間毎にまたは２週間毎の血清クリアチニン測定によって評価した。さらに、頻ぱんなアログラフト生検を組織病理学検査用に採取し、完全解剖をすべての死んだ受容体で行った。殆んどの受容体において、両側性腎摘出を移植時に行い、その後の尿毒症死をアログラフト生存の終点とみなした。いくつかの受容体においては、片側の生腎摘出と対側尿管ライゲーションを移植時点で行った。アログラフト拒絶が生じたとき、同原尿管上のライゲーチュアを除去し、正常腎機能の再生と受容動物の免疫学的モニターを続ける機会を得た。
モノクローナル抗体Ｒ６−５−Ｄ６を１２日間毎日投与したが、移出前２日から１〜２mg／kg／日の投与量で開始した。クレアチニンの血清量を周期的に試験して拒絶をモニターした。同種異系腎の免疫系拒絶に対する抗ＩＣＡＭ−１抗体の効果は第２１表に示す。

上記の結果はＲ６−５−Ｄ６が同種異系移植を受け入れたサルの寿命延長に有効であることを示している。
実施例３１
移植臓器の急性拒絶を抑制するのにおける抗ＩＣＡＭ−１抗体の効果
抗ＩＣＡＭ−１抗体が移植拒絶の急性モデルにおいて有効であることを示すために、Ｒ６−５−Ｄ６を治療中のまたは急性腎拒絶モデルにおいても試験した。このモデルにおいて、サル腎臓を移植し（実施例３０のプロトコールを用いて）、15mg／kgのシクロスポリンＡ（ＣｙＡ）を筋注で組織周辺的に安定な腎機能が得られるまで投与した。次に、ＣｙＡ投与量を血液クレアチニン値の上昇で示されるような拒絶が生じるまで２．５mg／kg増分量で２週間毎に低減した。この時点で、Ｒ６−５−Ｄ６は１０日投与し、生存時間をモニターした。重要なことは、このプロトコールにおいては、ＣｙＡの投与量が急性拒絶状態が生ずると同時に変化しないので次善のまゝであるということに留意することである。このモデルにおいては、抗体援助を有しない組織学的対照（Ｎ＝５）は拒絶状態の開始から５〜１４日間生存する。これまで、６匹の動物をこのプロトコールにおいてＲ６−５−Ｄ６を用いて試験した（第２２表）。これら動物の２匹はまだ生存している（Ｒ６−５−Ｄ６の投与後にＭ１２：３１日間、およびＭ５：４７日間）。２匹の動物はＲ６−５−Ｄ６治療開始後３８日および５５日間生き、２匹の動物は急性拒絶以外の原因で死亡した（一匹はＣｙＡ毒性で死亡し、一匹は麻酔下でＲ６−５−Ｄ６を投与している間に死亡した）。このモデルはＲ６−５−Ｄ６を最初から用いた臨床状態とより密接に近似している。

実施例３２
ＩＣＡＭ−１の末端切取り誘導体の遺伝子構築および発現
天然状態において、ＩＣＡＭ−１は５つの免疫グロブリン様ドメインの細胞外領域トランスメンブランドメインおよび細胞質ドメインを含有する細胞膜結合たん白質である。望ましいのはICAM−１から分泌した可溶物を発現できる点でトランスメンブランドメインおよび／または細胞質ドメインを欠落するＩＣＡＭ−１の官能性誘導体を構築することであった。これらの官能性誘導体はＩＣＡＭ−１遺伝子のオリゴヌクレオチド関連変異誘発およびその後の変異遺伝子による投入後のサル細胞中での発現により構築した。
アミノ酸置換および／または末端切取り（Truncated）誘導体を与えるＩＣＡＭ−１遺伝子の突然変異はKunkel, T.の方法により発生させた〔Proc. Natl. Acad. Sci.（U.S.A.）８２：４８８−４９２（１９８５）〕。上記のようにして調製したＩＣＡＭ−１ｃＤＮＡを制限エンドヌクリアーゼＳal１およびKpm１で消化し、得られた１．８kb ＤＮＡフラグメントをプラスミドベクターＣＤＭ８にサブクローン化した〔Seed, B.等、Proc. Natl. Acad. Sci.（U.S.A.）８４：3365−３３６９（１９８７）〕。次に、E. Coli（ＢＷ３１３／Ｐ３）のdut ^-、ung ^-株をｐＣＤ１．８Ｃと表示する上記構築物で形質転換した。単一ストランドウラシル含有鋳型を形質転換体からヘルパーファージＲ４０８（ストレータジーンＲ）で感染させることによって救援（rescue）した。次いで、変異体ＩＣＡＭ−１ cＤＮＡを不整合ベースを有するオリゴヌクレオチドとの第２ストランド合成および引き続くung ⁺宿主（ＭＣ1061／Ｐ３）の得られたヘテロ二本鎖体による形質転換を開始することによって産生させた。変異体を上記変異オリゴヌクレオチドを導入した新たに創生したエンドヌクレアーゼ制限サイトについてスクリーニングすることによって単離した。変異体ＩＣＡＭ−１たん白質は標準ＤＥＡＥ−デキストラン法〔Selden, R.F.等、Current Protocols in Molecular Biology（Ausebel, F.M.等編集）、９．２．１−９．２．６（１９８７）〕を用いて真核発現ベクターＣＤＭ８中で上記変異体ＤＮＡによるＣos−７細胞の移入によって発現させた。
トランスメンブランドメインおよび細胞質ドメインを欠落するが５つの免疫グロブリン様ドメインのすべてを含有するＩＣＡＭ−１の末端切取り官能性誘導体を調製した。３０bp変異体オリゴヌクレオチド（CTC TCC CCC CGG TTC TAG ATT GTC ATC ATC）を用いて位置４５２および４５３のアミノ酸チロシン（Ｙ）およびグルタミン酸（Ｅ）のコドンを、それぞれ、フェニルアラニン（Ｆ）およびほん訳停止コドン（ＴＡＧ）に形質転換した。変異体はその特異的Ｘba１制限サイトにより単離し、Ｙ⁴⁵²Ｅ／Ｆ、ＴＡＧと表示した。
変異体たん白質を発現させるために、ＣＯＳ細胞に３種の変異体サブクローン（♯２、♯７および♯８）を移入した。これら３種の変異体サブクローンによる移入の３日後、培養上清および細胞溶解物を抗ＩＣＡＭ−１モノクローナル抗体ＲＲ１／１による免疫沈降およびＳＤＳ−ＰＡＧＥによって分析した。ＩＣＡＭ−１は変異体サブクローン♯２および♯８を移入した細胞の培養上清からは沈降したがこれら細胞の清浄剤溶解物からは沈降しなかった。培養上清中に見い出されるICAM−１の分子量はＩＣＡＭ−１の膜体に比し約６kd低下していたが、これは変異体ＤＮＡから予想した大きさに一致する。即ち、このＩＣＡＭ−１の官能性誘導体は可溶性たん白質として分泌している。これに対し、ＩＣＡＭ−１は対照の天然ICAM−１を移入した細胞の培養上清からは免疫沈降せず、ＩＣＡＭ−１の膜体はＣＯＳ細胞からは発現しなかったことを示していた。さらにまた、ICAM−１は陰性対照の偽移入細胞からの培養上清または細胞溶解物からは免疫沈降してこなかった。
移入細胞から分泌した末端切取りＩＣＡＭ−１をＩＣＡＭ−１特異性抗体（Ｒ６−５−Ｄ６）によるイムノアフィニティクロマトグラフによって精製し、細胞結合アッセイでの官能活性について試験した。清浄剤オクチルグルコサイドの存在下での精製後、天然ＩＣＡＭ−１または末端切取り型分泌体を含有する調製物を０．２５％オクチルグルコサイドの最終濃度に希釈した（清浄剤の臨界ミセル濃度以下の濃度）。ＩＣＡＭ−１のこれら調製物をプラスチック９６−ウェルプレート（Nunc社）の表面に結合させて固相に結合したＩＣＡＭ−１を調製した。未結合物を洗い出したのち、表面上にＬＦＡ−１を含有するＳＫＷ−３細胞の約７５〜８０％および約８３〜８５％が、それぞれ、ＩＣＡＭ−１の天然体および末端切取り体に特異的に結合した。これらのデータは分泌型の末端切取り可溶性ＩＣＡＭ−１官能性誘導体が免疫学的反応性および天然ＩＣＡＭ−１の特徴であるICAM−１依存性粘着を仲介する能力の両方を保持していることを示している。
細胞質ドメインのみを欠落するＩＣＡＭ−１の官能性誘導体を同様な方法で調製した。２５bpオリゴヌクレオチド（TC AGC ACG TAC CTC TAG AAC CGC CA）を用いてアミノ酸４７６（Ｙ）のコドンをＴＡＧほん訳停止コドンに変えた。この変異体はＹ⁴⁷⁶／ＴＡＧと表示した。この変異体を移入したＣos細胞の免疫沈降分析およびＳＤＳ−ＰＡＧＥは陰性ＩＣＡＭ−１よりも約３kd小さい分子量を有するＩＣＡＭ−１の膜結合体を検出した。上記変異体移入Ｃos細胞の間接免疫螢光分析はＬＰＳ刺激ヒト内皮細胞上に発現した天然ＩＣＡＭ−１と同様な点状染色像を示した。また、上記変異体ＤＮＡを移入した細胞は天然ＩＣＡＭ−１ＤＮＡを移入したＣos細胞と同じような形でプラスチック表面上の精製ＬＦＡ−１に特異的に結果した（第２３表）。

実施例３３
ＩＣＡＭ−１官能性ドメインのマッピング
ＩＣＡＭ−１の研究はその分子が７とのドメインを有することを見い出した。これらドメインのうちの５つは細胞外であり（ドメイン５は細胞表面に最も近く、ドメイン１は細胞表面から最も遠い）、１つのドメインはトランスメンブランドメインであり、１つのドメインは細胞質である（即ち、細胞内に存在する）。どのドメインがICAM−１のＬＦＡ−１に結合する能力に貢献するかをみるために、エピトープマッピング（地図作製）試験を用いた。そのような試験を行うためには異なる欠落変異体を作製し、そのＬＦＡ−１を結合する能力について特徴決定した。別に、ＩＣＡＭ−１のＬＦＡ−１に結合する能力を干渉することが知られている抗ＩＣＡＭ抗体を用いた試験を行った。かかる抗体の適当な例にはＲＲ１／１〔Rothlein, R.等、J. Immunol. １３７：1270−１２７４（１９８６）〕、Ｒ６．５（Springer, T.A.等、米国特許出願第０７／２５０，４４６号）、ＬＢ−２〔Clark, E.A.等、Leukocyte Typing I（A. Bernard等編集）、Springer-Verlag、３３９−３４６（１９８４）〕、またはＣＬ203〔Staunton, D.E.等、Cell. ５６：８４９−８５３（１９８９）〕がある。
ＩＣＡＭ−１の欠落変異体は種々の任意の方法によって調製できる。しかしながら、そのような変異体は部位特異的変異誘発によりまたは他の組換え手段（特定のたん白質領域をコードする配列を欠落させたＩＣＡＭ−１発現性遺伝子配列を構築することによるような）により産生させるのが好ましい。そのような変異体を産生するのに適する手順は当該技術において周知である。そのような手順を用いて、３つのＩＣＡＭ−１欠落変異体を調製した。第１の変異体はアミノ酸残基F185〜Ｐ２８４を欠落する（即ち、ドメイン３の欠落）。第２の変異体はアミノ酸残基Ｐ２８４〜Ｒ４５１を欠落する（即ち、ドメイン４および５の欠落）。第３の変異体はＹ４７６より後のアミノ酸残基を欠落する（即ち、細胞質ドメインの欠落）。そのような試験の結果はドメイン１、２および３が抗ＩＣＡＭ−１抗体およびＬＦＡ−１とのＩＣＡＭ−１相互作用中に主として含まれていることを示す。
実施例３４
ＬＦＡ−１結合に対してのＩＣＡＭ−１変異の効果
ＩＣＡＭ−１がＬＦＡ−１に反応し結合する能力はＩＣＡＭ−１分子のドメイン１中に存在するＩＣＡＭ−１アミノ酸残基によって仲介される（第８、９および１０図）。しかしながら、そのような反応はＩＣＡＭ−１のドメイン２および３中に存在するアミノ酸の貢献によって促進される。即ち、本発明の好ましい官能性誘導体の内には、ＩＣＡＭ−１のドメイン１、２および３を含有するＩＣＡＭ−１分子の可溶性はフラグメントが存在する。より好ましいのはＩＣＡＭ−１のドメイン１および２を含有するＩＣＡＭ−１分子の可溶性フラグメントである。最も好ましいのはICAM−１のドメイン１を含有するＩＣＡＭ−１の可溶性フラグメントである。第１ＩＣＡＭ−１ドメイン内の幾つかのアミノ酸残基はＩＣＡＭ−１とＬＦＡ−１の反応中に含まれている。これらアミノ酸の他のアミノ酸による置換はＩＣＡＭ−１のＬＦＡ−１に結合する能力を変化させる。これらのアミノ酸残基およびその置換体を第２５図に示す。第２５図は得られた変異体ＩＣＡＭ−１分子のＬＦＡ−１に結合する能力についてのそのような変異の効果を示す。第２３〜２５図においては、各残基はアミノ酸の１文字コードについて記載し、次いでＩＣＡＭ−１分子中のその残基の位置を示している。即ち、例えば“Ｅ９０”はＩＣＡＭ−１の位置９０でのグルタミン酸残基を称す。同様に、“Ｅ９０Ｖ”は位置９０のグルタミン酸残基と位置９１のバリン残基とからなるジペプチドを示す。置換配列はスラッシュ（“／”）マークの右に示されている。ＩＣＡＭ−１のＶ４、Ｒ１３、Ｑ２７、Ｑ５８およびＤ６０Ｓ６１残基はＬＦＡ−１結合中に含まれる。
これらアミノ酸の置換はＩＣＡＭ−１のＬＦＡ−１への結合能力を変化させる。例えば、Ｖ４のＧによる置換はＬＦＡ−１により少なくしか結合しない変異体ＩＣＡＭ−１分子の形成をもたらす（第２５図）。ＩＣＡＭ−１のＲ１３残基のＥによる置換は実質的に小さいＬＦＡ−１への結合能力を有する変異体分子の形成を与える（第２５図）。ＩＣＡＭ−１のＱ５８残基のＨによる置換は実質的に通常のＬＦＡ−１への結合能力を有する変異体分子を与える（第２５図）。ＩＣＡＭ−１のＤ６０Ｓ残基のＫＬによる置換は実質的に小さいＬＦＡ−１への結合能力を有する変異体分子を与える（第２５図）。
第２ドメイン中のグリコシル化部位もまたLFA−１結合中に含まれる（第２３図）。Ｎ１０３のＫによるまたはＡ１５５ＮのＳＶによる置換はＬＦＡ−１を実質的に結合し得ない変異体ICAM−１分子の形成をもたらす。対照的に、グリコシル化部位Ｎ１７５のＡによる置換はその変異体ＩＣＡＭ−１のＬＦＡ−１結合能に実質的に影響しないようであった。
第３ＩＣＡＭ−１ドメインの変異はＩＣＡＭ−１−ＬＦＡ−１結合性を認知し得る程変化させないようであった（第２４図）。
実施例３５
増大した生物学的半減アフィニティとクリアランス能力を有するＩＣＡＭ−１の多量体形
ＩＣＡＭ−１のドメイン１と２をその免疫グロブリン長鎖のヒンジ領域に結合させたキノラ分子を構築する。好ましい構築物はＩＣＡＭ−１ドメイン２のＣ末端をヒンジ領域対しての丁度Ｎ−末端の免疫グロブリン長鎖のセグメントに結合させて、ヒンジ領域により付与されたセグメントフレキシビリティを可能にする。ＩＣＡＭ−１ドメイン１と２はかくして抗体のＦabフラグメントを置換するであろう。ＩｇＧ類の長鎖への結合および動物細胞の生成はキメラ分子の産生をもたらすであろう。ＩｇＡまたはＩｇＭに由来する長鎖を含有する分子の生成は２〜１２個のＩＣＡＭ−１分子を含有するより高多量体の分子の産生をもたらすであろう。ＩＣＡＭ−１長鎖キメラ分子を産生する動物細胞中での丁鎖遺伝子の共発現は４〜６ケのＩＣＡＭ−１分子を含有するＩｇＡ分子と約１０ケのＩＣＡＭ−１分子を含有するＩｇＭのケースとを主として与えるＩｇＡとＩｇＭ多量体の適当な集合体を与えるであろう。これらのキメラ分子は幾つかの利点を有する。第１は、Ｉｇ分子を循環系中で長く滞在するよう設計し、これにより生物学的半減期を改善できる。
さらにまた、これら加工分子の多量体性は、治療情状のいかんによって、これら分子がより高い結合活性によってライノウイルス並びに細胞表面ＬＦＡ−１と反応できるようにし、かくして有効投与量を与えるべき投与に必要な組換えたん白質の量を大いに低減させる。ＩｇＡおよびＩｇＭは鼻におけるような粘膜部位の分泌物中に通常存在する高グリコシル化分子である。その高親水性は粘膜中で結合する微生物およびウイルスを保持するのを助長して細胞への結合を防止しまた上皮細胞膜バリヤーの交差を防止する。即ち、これらの分子は増大した治療効力を有する。ＩｇＭと特にＩｇＡとは粘膜環境で安定でありＩＣＡＭ−１構築物の安定性を増大させる。そのようなＩＣＡＭ−１官能性誘導体を血液流中に投与する場合、この誘導体も生物学的半減期を増大する。ＩｇＡは補体を固定せず、従って、それが有害である用途において理想的であろう。ＩｇＧのＨ鎖キメラを望む場合には、補体への結合並びにＦｃレセプターとの反応中に含まれる領域を変異させることが可能であろう。
実施例３６
ＩＣＡＭ−１変異体の発現
オリゴヌクレオチド特異性変異誘発
１．８kb Ｓal１−Ｋpm１フラグメント中のICAM−１ cＤＮＡのコード領域を発現ベクターCDMB中にサブクローニングした〔Seed, B.等、Proc. Natl. Acad. Sci.（U.S.A.）８４：３３６５〜３３６９（１９８７）〕。Kunkel, T.の方法〔Proc. Natl. Acad. Sci.（U.S.A.）８２：488−４９２（１９８５）〕およびStaunton D.等の変法（Staunton D.E.等、Cell ５２：９２５−９３３（１９８８）〕に従って、この構築物（pＣＤ１．８）を用いてオリゴヌクレオチド特異性変異誘発で使用する単一ストランドウラシル含有鋳型を調製した。
要するに、E. Coli株ＸＳ１２７をpＣＤ１．８で形質転換した。単一コロニーを１３μｇ／mｌのアンピシリンと８μｇ／mｌのテトラサイクリンを含有する１mｌのルリア ブロス（ＬＢ）培地（Difco社）中で近飽和まで増殖させた。100μｌの培養物をＲ４０８ヘルパーファージ（Strategene社）で１０の感染多重度（ＭＯＩ）で感染させ、アンピシリンとテトラサイクリンを含む１０mｌのＬＢ培地を３７℃で１６時間培養で加えた。１０，０００rpmで１分間の遠心および上清の０．２２μm濾過の後、ファージ懸濁液をE. Coli ＢＷ３１３／Ｐ３を感染し、次いで、これをアンピシリンおよびテトラサイクリンを加えたＬＢ寒天（Difco社）プレート上に塗布した。コロニーを採取し、アンピシリンとテトラサイクリンを含む１mｌのＬＢ培地中で近飽和に増殖させ、ヘルパーファージでＭＯＩ１０で感染させた。次いで、培養容量を２５０mｌに増大させ、細胞を一夜培養した。単一ストランドＤＮＡを標準のファージ抽出により単離した。
変異体オリゴヌクレオチドをリン酸化し、第２ストランド合成反応にpＣＤ１．８鋳型と共に用いた〔Staunton D.E.等、Cell ５２：９２５−９３３（１９８８）〕。
移 入
ＣＯＳ細胞を１６〜２４時間で５０％の集密性となるように１０cmの組織培養プレート中にシード付けした。次いで、ＣＯＳ細胞をＴＢＳで１度洗浄し、１０％のＮu血清（Collaborative社）、５μｇ／mｌのクロロキーネ（chloroquine）、３μｇの変異プラスミドおよび２００μｇ／mｌのＤＥＡＥ−デキストラン サルフェートを含有する４mｌのＲＰＭＩで４時間培養した。細胞をその後１０％のＤＭＳＯ／ＰＢＳ次いでＰＢＳで洗浄し、培養培地中で１６時間培養した。培養培地を新鮮培地で入れ替え、４８時間で、後移入ＣＯＳ細胞をトリプシン／ＥＤＴＡ（Gibco社）処理により懸濁し、ＨＲＶ結合用の２、１０cmプレート並びに２４ウェル組織培養プレート中に細分した。７２時間で、細胞を１０cmプレートから５mM ＥＤＴＡ／ＨＢＳＳで採集し、ＬＦＡ−１コーティングプラスチックへの粘着および免疫螢光分析用に加工した。
ＬＦＡ−１およびＨＲＶ結合性
ＬＦＡ−１をＳＫＷ−３からＴＳ２／４ＬＦＡ−１ mＡbセファローズ上でイムノアフィニティクロマトグラフにより精製し、２mMのMgCｌ₂および１％のオクチルグルコシドの存在下にpＨ１１．５で溶出させた。ＬＦＡ−１（１０μｇ／２００μｌ／６cmプレート）を微生物学的ペトリ皿に２mMのMgCｌ₂を含むＰＢＳ（リン酸塩緩衝塩水）中でオクチルグルコシドを０．１％に希釈し４℃で一夜インキュベートすることによって結合させた。各プレートを１％ＢＳＡ（ウシ血清アルブミン）でブロックし、２mMのMgCｌ₂、０．２％ＢＳＡ、０．０２５％のアザイド、および５０μｇ／mｌのジェンタマイシンを含有するＰＢＳ中で保存した。
５％ＦＣＳ（ウシ胎児血清）、２mMのMgCｌ₂、０．０２５％のアザイドを含有するＰＢＳ（バッファー）中の⁵¹Ｃr標識ＣＯＳ細胞をＬＦＡ−１コーティングマイクロタイタープレート中で５μｇ／mｌのＲＲ１／１およびＲ６．５の存在または不存在下に２５℃、１時間インキュベートした。粘着してない細胞をバッファーで３回洗浄することにより除去した。粘着細胞はＥＤＴＡの添加により１０mMに溶出し、γ−計算した。
結 果
ＲＲ１／１、Ｒ６．５、ＬＢ−２、またはＣＬ２０３のような抗ＩＣＡＭ−１抗体は同定されている。これら抗体がＩＣＡＭ−１機能を抑制し得るならば、これらの抗体はＩＣＡＭ−１分子の特定の部位（これがまたＩＣＡＭ−１機能に重要である）に結合し得なければならない。即ち、前述のICAM−１の欠落変異体を調製し、上記の抗ICAM−１抗体が上記欠落体に結合し得る程度を測定することによって、欠落ドメインが機能上重要であるかどうかを見ることができる。
ＩＣＡＭ−１は複合膜たん白質であり、その細胞外ドメインは５つのＩｇ様Ｃ−ドメインからなるものと予測される。ＬＦＡ−１を結合するのに含まれるドメインを同定するには、ドメイン３、およびドメイン４と５（カルボキシル末端）をオリゴヌクレオチド特異性変異誘発により欠落させ、ＣＯＳ細胞中で発現後官能的に試験した。さらに、全細胞質ドメインを欠落させてＩＣＡＭ−１反応へのその潜在的影響を確認した。期待したように、細胞質ドメイン欠落体、Ｙ４７６／^*はＲＲ1/1、Ｒ６．５、ＬＢ−２およびＣＬ２０３の反応性の喪失を示さないのに対し、ドメイン３の欠落体Ｆ１８４−Ｒ４５１はＣＬ２０３反応性の減少および喪失をもたらした（第２０図）。即ち、ＣＬ２０３エピトープはドメイン４中に存在するようであるが、ＲＲ１／１、Ｒ６．５およびＬＢ−２は２ケのアミノ末端ドメイン中に存在するようである。
３つの欠落変異体はすべてＬＦＡ−１に対し野生型レベルの粘着性を示した（第２１図）。ドメイン１、２および３中の予測β−ターンでのアミノ酸置換体も調製しＣＯＳ細胞中での発現後機能性試験した。Ｒ６．５エピトープはドメイン２の配列Ｅ１１１ＧＧＡに局在化しまたドメイン１中にＥ３９を含み得るが、ＲＲ１／１とＬＢ−２は共にドメイン１のＲ１３に依存している（第２２図）。さらに、ＲＲ１／１結合性は配列Ｄ７１ＧＯＳ中での変異により減少した。Ｎ１０３およびＮ163のＮ−結合グリコシル化部位を削減する変異はＲＲ１／１、Ｒ６．５とＬＢ−２、ＬＦＡ−１ HRV結合性を減少させた。これらの変異はＩＣＡＭ−１ダイマーを発生させるような加工を行うようである。
ドメイン２または３の他の変異は変化型ＬＦＡ−１粘着をもたらさなかった（第２３および２４図）。ドメイン１のアミノ酸、Ｒ１３とＤ６０の両方はＬＦＡ−１と結合することによって含まれる（第２５図）。
即ち、ＬＦＡ−１およびＨＲＶ結合性はICAM−１のアミノ末端Ｉｇ様ドメインの機能であるようである。第２６図はＩＣＡＭ−１末端ドメインの配列を示す。
本発明をその特定の実施態様について説明して来たが、更なる修正が可能であることは理解し得ることであり、本出願は、一般に、本発明の原理に従いかつ本発明が関係する技術内での公知または慣用的実施に含まれるようなまた特許請求するような本質的な特徴に適用し得るような本明細書の記載からの回避を包含するすべての変形、用途または適用を含むものとする。
【図面の簡単な説明】
第１図は正常細胞とＬＦＡ−１欠損細胞間の粘着を図式的に示す。
第２図は正常細胞／正常細胞粘着過程を図式的に示す。
第３図は５０ng／mｌのＰＭＡの不存在（×）または存在（○）下の細胞凝集の動力学を示す。
第４図はＬＦＡ−１^-細胞とＬＦＡ−１⁺細胞間の凝集を示す。図中に示すようにカルボキシフルオレスセインジアセテート標識ＥＢＶ−形質転換細胞（１０⁴）を１０⁵未標識同元細胞（黒枠）またはＪＹ細胞（白枠）とＰＭＡの存在下に混合した。１．５時間後、凝集物中または遊離の標識細胞を実施例２の定量アッセイを用いて計数した。凝集物中の標識細胞の％を示す。２つの内の１つの代表的な試験を示している。
第５図はＪＹ細胞からのＩＣＡＭ−１とＬＦＡ−１の免疫沈降を示す。ＪＹ細胞のトリトンＸ−１００溶解物（レーン１および２）または対照溶解バッファー（レーン３および４）をＩＣＡＭ−１に結合し得る抗体（レーン１および３）またはＬＦＡ−１に結合し得る抗体（レーン２および４）で免疫沈降させた。パネルＡは還元条件下での結果を示し、パネルＢは非還元条件下で得られた結果を示す。分子量標準物はレーンＳに示した。
第６図はヒト皮ふ繊維芽細胞上のＩＣＡＭ−１発現に対してのＩＬ−１とガンマーインターフェロンの効果の動力学を示す。ヒト皮ふ繊維芽細胞は８×１０⁴細胞／０．３２cm²ウェルの密度に増殖させた。ＩＬ−１（１０μ／mｌ、黒丸）または組換えガンマーインターフェロン（１０μ／mｌ、白四角）を加え、図中に示した時間で、４℃に冷却し間接結合アッセイを実施した。標準偏差は１０％を越えなかった。
第７図はＩＣＡＭ−１へのＩＬ−１とガンマーインターフェロン効果の濃度依存性を示す。ヒト皮ふ繊維芽細胞は８×１０⁴細胞／０．３２cm²／ウェルの密度に増殖させた。ＩＬ−２（白丸）、組換えヒトＩＬ−１（白四角）、組換マウスＩＬ−１（黒四角）および組換えベータインターフェロン（白三角）を図中に示した稀釈率で４時間（ＩＬ−１）または１６時間（ベータおよびガンマーインターフェロン）インキュベートした。示した結果は４回測定の平均を示し、標準偏差は１０％を越えなかった。
第８図はＩＣＡＭ−１ cＤＭＡのヌクレオチドおよびアミノ酸配列を示す。第１ＡＴＧは位置５８にある。ＩＣＡＭ−１トリプシンペプチドに相当する翻訳配列はアンダーラインを施してある。疎水性推定シグナルペプチドおよびトランスメンブラン配列は肉太アンダーラインを施している。Ｎ−結合グリコシル化部位は囲っている。位置２９７６のポリアデニリル化シグナルAATAAAはオーバーラインを施している。図示した配列はＨＬ−６０ cＤＮＡクローン用である。内皮細胞cＤＮＡはその長さの大部分に亘ってシーケンシングし小さな差異のみを示した。
第９図はＩＣＡＭ−１相同ドメインおよび免疫グロブリン超遺伝子群への相関を示す。（Ａ）５つの相同ドメインの配列（Ｄ_1-5）：列んだ２以上の同じ残基は囲っている。ＮＣＡＭドメインに２回以上含まれる残基、およびセットＣ₂およびＣ₁のドメイン中に含まれる残基はＩＣＡＭ−１内部繰返しによって配列している。ＩＣＡＭ−１のドメイン中の予想βストランドの位置は棒線および配列上の小文字でマークしており、また免疫グロブリンｃドメイン中のβ−ストランドの公知の位置は棒線および配列下の大文字でマークしている。ＩＣＡＭ−１ドメイン内の推定ジスルフィド架橋の位置はＳ−Ｓによって記している。ICAM−１に相同性のたん白質ドメインの（Ｂ−Ｄ）配列：各たん白質はＦＡＳＴＰプログラムを用いてＮＢＲＦデータベースを調査することによって先ず配列する。各たん白質配列はＭＡＧ、ＮＣＡＮ、Ｔ細胞レセプターαサブユニットＶドメイン、ＩｇＭμ鎖およびα−１−Ｂ−糖たん白質である。
第１０図はＩＣＡＭ−１とＭＡＧの二次構造の比較図である。
第１１図は平坦膜中でＩＣＡＭ−１に結合しているＬＦＡ−１陽性ＥＢＶ−形質転換Ｂ−リンパ芽球腫（lymphoblastoid）細胞を示す。
第１２図はプラスチック結合ベシクル中のICAM−１に結合しているＬＦＡ−１陽性Ｔ−リンパ芽球およびＴ−リンパ球を示す。
第１３図はプラスチック結合ベシクル中のICAM−１に結合しているＪＹ Ｂ−リンパ芽球腫の細胞またはベシクルのモノクローナル抗体による前処理による結合抑制を示す。
第１４図はプラスチック結合ベシクル中のICAM−１へのＴ−リンパ芽球の結合に対しての温度の効果を示す。
第１５図はプラスチック結合ベシクル中のICAM−１へのＴ−リンパ芽球の結合における二価のカチオンの必要性を示す。
第１６図は末梢血液単核細胞がＴ−細胞会合抗原ＯＫＴ３の認識に応答して増殖する能力に及ぼす抗粘着性抗体の効果を示す。“ＯＫＴ３”は抗原の添加を示す。
第１７図は、末梢血液単核細胞が、非特異的Ｔ−細胞分裂促進物質である、コンカナバリンＡの認識に応答して増殖する能力に及ぼす抗粘着性抗体の効果を示す。“ＣＯＮＡ”はコンカナバリンＡの添加を示す。
第１８図は、末梢血液単核細胞が、鍵穴カサガイヘモシアニン（keyhole limpet hemocyanin）抗原の認識に応答して増殖する能力に及ぼす抗粘着性抗体の効果を示す。“ＫＬＨ”は、鍵穴カサガイヘモシアニンの、細胞への添加を示している。
第１９図は末梢血液単核細胞が、破傷風菌トキソイド抗原の認識に応答して増殖する能力に及ぼす抗粘着性抗体の効果を示す。“ＡＧＮ”は破傷風菌トキソイド抗原の、細胞への添加を示す。
第２０図はＩＣＡＭ−１欠落変異体へのモノクローナル抗体ＲＲ１／１、Ｒ６．５、ＬＢ２、およびＣＬ２０３の結合を示す。
第２１図はＩＣＡＭ−１欠落変異体のＬＦＡ−１への結合を示す。
第２２図は抗ＩＣＡＭ−１モノクローナル抗体ＲＲ１／１、Ｒ６．５、ＬＢ２、およびＣＬ２０３により認識したエピトープを示す。
第２３図はＬＦＡ−１へのＩＣＡＭ−１ドメイン２変異体の結合能力を示す。
第２４図はＬＦＡ−１へのＩＣＡＭ−１ドメイン３変異体の結合能力を示す。
第２５図はＬＦＡ−１へのＩＣＡＭ−１ドメイン１の結合能力を示す。
第２６図はＩＣＡＭアミノ末端ドメインの配列を示す。 Industrial application fields
The present invention relates to an intercellular adhesion molecule such as ICAM-1, which is included in a process in which a group of lymphocytes recognizes and adheres to a cellular substrate, permeates into an inflammatory site and reacts with a cell during an inflammatory reaction. The invention further relates to ligand molecules that can bind to such intercellular adhesion molecules, screening assays for these ligands, and uses of the intercellular adhesion molecules, the ligand molecules and the screening assays.
Conventional technology
Leukocytes must be able to attach to the cell matrix to properly protect the host against external enemies such as bacteria or viruses. Eisen, H.W.Micro-biology3rd edition, Philadelphia, PA, published by Harpe & Rows, (1980), 290-295 and 381-418 ". Leukocytes can bind to endothelial cells and undergo ongoing inflammation from the circulatory system In addition, leukocytes must adhere to antigen-providing cells to produce a normal specific immune response, and lymphocytes can attach to appropriate target cells to cause viral infections or tumors. Cell degradation must occur.
Recently, leukocyte surface molecules involved in mediating such adhesion have been identified using hybridoma technology. In short, human T-cells [Davignon, D. et al.Proc. Natl. Acad. Sci. USA 78: 4535-4539 (1981)] and mouse spleen cells [Springer, T. et al.,Eur. J. Immunol. 9: 301-306 (1979)] were identified as binding to the leukocyte surface and inhibiting the aforementioned adhesion-related functions [Springer, T. et al.,Fed. Proc. 44: 2660-2663 (1985)]. The molecules identified by these antibodies are called Mac-1 and lymphocyte function associated antigen 1 (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and granular lymphocytes. LFA-1 is a heterodimer found in most lymphocytes [Springer, T.A. et al.,Immunol. Rev.68: 111-135 (1982)]. These two molecules, and the third molecule p150, 95, which has a tissue distribution similar to Mac-1, play a role in cell adhesion [Keizer, G. et al.Eur. J. Immunol.15: 1142-1147 (1985)].
The above leukocyte molecules were found to be a member of a related group of glycoproteins [Sanchez-Madrid, F. et al.J. Exper. Med. 1581785-1803 (1983); Keizer, G.D.Eur. J. Immunol. 15: 1142-1147 (1985)]. This glycoprotein group consists of heterodimers having one alpha chain and one beta chain. Although the alpha chains of each antigen are different from each other, the beta chains have been found to be highly protected [Sanchez-Madrid, F. et al.J. Exper. Med. 158: 1785-1803 (1983)]. The beta chain of the glycoprotein group (sometimes referred to as “CD18”) was found to have a molecular weight of 95 KD, while the alpha chain was found to vary between 150 KD and 180 KD [Springer, T. et al. ,Fed. Proc.44: 2660-2663 (1985)]. Although membrane protein alpha subunits do not have the broad homology of beta subunits, approximate analysis of glycoprotein alpha subunits shows that there is substantial similarity between the two . A study of similarity between alpha and beta subunits of LFA-1-related glycoproteins has been made by Sanchez-Madrid, F. et al. [J. Exper. Med. 158: 586-602 (1983);J. Exper. Med. 185: 1785-1803 (1983)].
A group of people has been identified that cannot express normal amounts of any one of the above adherent protein groups on the leukocyte surface [Anderson, D.C. et al.Fed. Proc.44: 2671-2677 (1985); Anderson, D.C.J. Infect. Dis, 152: 668 689 (1985)]. Lymphocytes from these patients showed an in vivo defect similar to that of a healthy person in which the LFA-1 group of molecules was neutralized by antibodies. Furthermore, the above patients are unable to measure a normal immune response because their cells cannot adhere to the cell matrix [Anderson, D.C., et al.Fed. Proc.44: 2671-2677 (1985); Anderson, D.C.J. Infect. Dis, 152: 668-689 (1985)]. These data show that the immune response is alleviated when lymphocytes cannot adhere in the normal form due to the lack of functional adhesion molecules of the LFA-1 group.
In summary, the ability of lymphocytes to maintain animal health and vitality requires that lymphocytes can adhere to other cells (such as endothelial cells). This adhesion has been found to require cell-cell contact involving specific receptor molecules present on the cell surface of lymphocytes. These receptors allow lymphocytes to adhere to other lymphocytes or endothelium and other non-vascular cells. Cell surface receptor molecules have been found to be highly related to each other. Humans whose lymphocytes are deficient in the cell surface receptor molecules show chronic and recurrent infections and other clinical symptoms including defective antibody responses.
Since lymphocyte adhesion occurs in the process of foreign tissue being recognized and rejected, understanding this process is of significant value in the fields of organ transplantation, tissue transplantation, allergy and oncology.
The content of the invention
The present invention relates to an intercellular adhesion molecule-1 (ICAM-1) and a functional derivative thereof (functional derivative). The invention further relates to antibodies and antibody fragments that can suppress ICAM-1 function, and other inhibitors to ICAM-1 function, and assays that can identify such inhibitors. The invention further relates to diagnostic and therapeutic uses for all of the above molecules.
More particularly, the present invention encompasses the intercellular adhesion molecule ICAM-1 or a functional derivative thereof substantially free of natural impurities. The invention further relates to such molecules that can bind to molecules present on the surface of lymphocytes.
The invention further relates to detectably labeled intercellular adhesion molecule ICAM-1 and derivatives thereof.
The invention further encompasses recombinant DNA molecules that are capable of expressing ICAM-1 or functional derivatives thereof.
The present invention also encompasses a method for recovering ICAM-1 in substantially pure form comprising the following steps:
(a) solubilizing ICAM-1 from a cell membrane expressing ICAM-1 to prepare a solubilized ICAM-1 preparation;
(b) introducing the solubilized ICAM-1 preparation into an affinity matrix containing an antibody capable of binding to ICAM-1.
(c) binding ICAM-1 to the antibody of the affinity matrix;
(d) removing from the matrix all compounds that cannot bind to antibodies, and
(e) recovering ICAM-1 in substantially pure form by eluting it from the matrix.
The invention further encompasses antibodies that can bind to a molecule selected from the group consisting of ICAM-1 and functional derivatives of ICAM-1. The invention also includes hybridoma cells capable of producing such antibodies.
The present invention further provides a monoclonal antibody R₆-5-D₆Hybridoma cells capable of producing
The present invention further includes a method for producing a desired hybridoma cell that produces an antibody capable of binding to ICAM-1, comprising the following steps.
(a) immunizing an animal with cells expressing ICAM-1;
(b) fusing the spleen cells of the animal with a myeloma cell line;
(c) preparing antibody-secreting hybridoma cells from the fused spleen and myeloma cells; and
(d) screening hybridoma cells to hybridoma cells capable of producing a desired ICAM-1-binding antibody.
The present invention also includes a hybridoma cell obtained by the above method and an antibody produced from the hybridoma cell.
The present invention also relates to a method for identifying a non-immunoglobulin antagonist of intercellular adhesion, which method comprises the following steps:
(a) incubating a non-immunoglobulin agent, which may be an antagonist of intercellular adhesion, with a lymphocyte preparation containing a plurality of cells capable of aggregation; and
(b) examining the lymphocyte preparation to determine whether the presence of the non-immunoglobulin agent inhibits cell aggregation of the lymphocyte preparation; Identify as an antagonist.
The present invention also relates to a method of treating inflammation resulting from the response of a mammal's specific defense system, the method comprising a subject in need of such treatment in an amount sufficient to suppress said inflammation. Providing an inflammatory agent, the anti-inflammatory agent comprising: an antibody capable of binding to ICAM-1, a fragment of an antibody capable of binding to ICAM-1, ICAM-1, a functional derivative of ICAM-1, and ICAM-1 Selected from the group consisting of non-immunoglobulin antagonists.
The present invention further includes the above-described method for treating inflammation, wherein the non-immunoglobulin antagonist of ICAM-1 is a non-immunoglobulin antagonist of ICAM-1 other than LFA-1.
The present invention also relates to a method of inhibiting the metastasis of hematopoietic tumor cells that require a functional member of the LFA-1 group for penetration, which method inhibits said metastasis in patients in need of such treatment. Providing an anti-inflammatory agent in an amount sufficient to produce an antibody capable of binding to ICAM-1, an antibody fragment capable of binding to ICAM-1, ICAM-1, ICAM-1 functionality Selected from the group consisting of sex derivatives and non-immunoglobulin antagonists of ICAM-1.
The present invention further includes a method for suppressing metastasis of the above hematopoietic tumor cells, wherein the non-immunoglobulin antagonist of ICAM-1 is a non-immunoglobulin antagonist of ICAM-1 other than LFA-1.
The invention also encompasses a method of inhibiting the growth of ICAM-1-expressing tumor cells, the method comprising providing a patient in need of such treatment with an amount of a toxin sufficient to inhibit the growth. This toxin is derived from a toxin-derived antibody that can bind to ICAM-1, a toxin-derived antibody fragment that can bind to ICAM-1, a toxin-derived member of the LFA-1 group molecule, and a member of the LFA-1 group molecule Selected from the group consisting of functional derivatives.
The present invention also relates to a method of inhibiting the growth of LFA-1-expressing tumor cells, which method provides a sufficient amount of toxin to inhibit such growth in a patient in need of such treatment. The toxin is selected from the group consisting of toxin-derived ICAM-1 and toxin-derived functional derivatives of ICAM-1.
The present invention further relates to a method of diagnosing the presence and location of inflammation resulting from the response of a specific defense system of a mammalian subject concerned with inflammation, the method comprising:
(a) administering to said subject a composition containing a detectably labeled binding ligand capable of identifying cells expressing ICAM-1; and
(b) detecting the binding ligand,
including.
The present invention further relates to a method of diagnosing the presence and location of inflammation resulting from the response of a specific defense system of a mammalian subject concerned with inflammation, the method comprising:
(a) incubating a sample of the subject's tissue with a composition containing a detectably labeled binding ligand capable of identifying cells expressing ICAM-1, and
(b) detecting the binding ligand,
including.
The invention also relates to a method of diagnosing the presence and location of such cells in a mammalian subject having a concern with ICAM-1-expressing tumor cells, the method comprising:
(a) administering to the subject a composition comprising a detectably labeled binding ligand capable of binding to ICAM-1, the ligand comprising an antibody and an antibody fragment capable of binding to ICAM-1. Chosen, and
(b) detecting the binding ligand,
including.
The invention also relates to a method of diagnosing the presence and location of such cells in a mammalian subject having a concern with ICAM-1-expressing tumor cells, the method comprising:
(a) incubating a sample of the subject tissue with a composition containing a detectably labeled binding ligand capable of binding to ICAM-1, an antibody fragment capable of binding the ligand to ICAM-1 Selected from the group consisting of, and
(b) detecting the binding ligand,
including.
The invention also relates to a method of diagnosing the presence and location of such cells in a subject of concern having tumor cells that express a member of the LFA-1 group molecule, the method comprising:
(a) administering to the subject a composition comprising a detectably labeled binding ligand capable of binding to one member of an LFA-1 group molecule, the ligand comprising ICAM-1 and ICAM-1 functionality Being selected from the group consisting of derivatives, and
(b) detecting the binding ligand,
including.
The invention also relates to a method of diagnosing the presence and location of such cells in a subject of concern having tumor cells that express a member of the LFA-1 group molecule, the method comprising:
(a) incubating a sample of the subject's tissue in the presence of a detectably labeled binding ligand capable of binding to one member of the LFA-1 group of molecules, the ligands being ICAM-1 and ICAM-1 Being selected from the group consisting of functional derivatives of
(b) detecting the binding ligand bound to a member of the LFA-1 group of molecules present in the tissue sample;
including.
The present invention further provides:
(a) selected from the group consisting of an antibody capable of binding to ICAM-1, a fragment of an antibody capable of binding to ICAM-1, an ICAM-1, a functional fragment of ICAM-1, and a non-immunoglobulin antagonist of ICAM-1. Anti-inflammatory agents, and
(b) at least one immunosuppressive agent selected from dexametheson, azethiopyrine and cyclosporin A
Are also included.
Preferred embodiment
One aspect of the invention relates to the discovery of naturally binding ligands for LFA-1. Molecules such as LFA-1 group molecules are involved in the process of intercellular adhesion and are referred to as “adhesive molecules”.
The natural binding ligand of the present invention is indicated as “Intercellular Adhesion Molecule-1” or “ICAM-1.” ICAM-1 is a 76-97 kd glycoprotein. ICAM-1 is not a heterodimer. The present invention relates to ICAM-1 and its “functional derivatives”. A “functional derivative” of ICAM-1 is a compound having a biological activity (functionally or structurally) substantially similar to that of ICAM-1. The term “functional derivative” is intended to encompass a “fragment”, “variant”, “homolog” or “chemical derivative” of a molecule. A “fragment” of a molecule such as ICAM-1 refers to any polypeptide subset of the molecule. Particularly preferred are fragments of ICAM-1 that have ICAM-1 activity and are soluble (ie, not membrane bound). A “variant” of a molecule such as ICAM-1 refers to a molecule that is substantially similar in structure or function to the entire molecule or fragment thereof. A molecule is said to be “substantially similar” if both molecules have a substantially similar structure to another molecule or if both molecules have biological activity as well. That is, if two molecules have similar activity, these molecules are considered to be mutants, and the term is used herein if one structure of the molecule is not found in the other molecule or the amino acid residue. Used even when base sequences are not the same. An “analog” or “homolog” of a molecule such as ICAM-1 refers to a molecule that is functionally substantially similar to the whole molecule or a fragment thereof. As used herein, a molecule is said to be a “chemical derivative” of another molecule if it contains additional chemical components that are not normally part of the molecule. Such components can improve the solubility, absorbability, biological half-life, etc. of the molecule. These components also reduce the toxicity of the molecule and eliminate or attenuate unwanted side effects of the molecule. Ingredients that can mitigate such effects are “Remington's Pharmaceutical Sciences(1980) "." Toxin-derived "molecules constitute a special class of" chemical derivatives "." Toxin-derived "molecules are molecules having a toxin component (such as ICAM-1 or antibodies). Binding of such molecules to the cell carries the toxin component in close proximity to the cell and thereby promotes cell death, although any suitable toxin component can be used, for example, ricin toxin, diphtheria toxin It is preferred to use toxins such as radioisotope toxins, membrane-channel forming toxins, etc. Methods for attaching such components to molecules are well known in the art.
Antigenic molecules such as members of the ICAM-1 or LFA-1 group molecules are naturally expressed on the surface of lymphocytes. That is, introduction of such cells, such as by intraperitoneal injection into a suitable animal, will result in the production of antibodies that can bind to one member of the ICAM-1 or LFA-1 group molecules. In some cases, the serum of such animals can be removed and used as a source of polyclonal antibodies that can bind to these molecules. Preferably, however, splenocytes are removed from such animals, such spleen cells are fused with a myeloma cell line, and the resulting fusion cells bind to one member of an ICAM-1 or LFA-1 group molecule. To establish a hybridoma cell that produces a monoclonal antibody capable of.
The hybridoma cells obtained by the above method can be screened by various methods to identify desired hybridoma cells that produce antibodies that can bind to either members of the ICAM-1 or LFA-1 group molecules. In one preferred screening assay, such molecules are identified by their ability to suppress aggregation of Epstein-Barr virus transformed cells. Antibodies that can suppress such aggregation are further screened to determine if these antibodies suppressed such aggregation by binding to a member of an ICAM-1 or LFA-1 group molecule. Any means that can distinguish ICAM-1 from LFA-1 group molecules can be used for such screening. For example, antigens bound to antibodies can be analyzed as by immunoprecipitation and polyacrylamide gel electrophoresis. If the bound antigen is a member of the LFA-1 group of molecules, the immunoprecipitated antigen will be found as a dimer, whereas if the bound antigen is ICAM-1, a single molecular weight species will be immunized. It will come down. Also, antibodies that bind to one member of the LFA-1 group molecules are screened for the ability of antibodies that bind to cells such as granulocytes that express LFA-1 but not ICAM-1 from antibodies that bind to ICAM-1. Can also be distinguished. The ability of an antibody to bind to granulocytes (known to inhibit cell aggregation) indicates that the antibody can bind to LFA-1. Absence of such binding may suggest an antibody that can recognize ICAM-1. The ability of an antibody to bind to cells such as granulocytes can also be detected by methods commonly used by ordinary experts. Such methods include immunoassays, cell aggregation, filter binding tests, antibody precipitation and the like.
The anti-aggregation antibody of the present invention also measures its ability to specifically bind to cells expressing ICAM-1 (such as activated endothelial cells) and its ability to bind to cells that do not express ICAM-1. Can also be identified. As will be readily appreciated by those skilled in the art, each of the above assays can be modified, i.e., performed in a different order to provide a variety of potential screening assays, each of these assays being capable of binding to ICAM-1. And one member of the LFA-1 group molecule can be identified and differentiated.
The anti-inflammatory agent of the present invention is a natural antibody [for example, a polyclonal antibody capable of binding animals to ICAM-1 by inducing animals, plants, fungi, bacteria, etc. to produce non-immunoglobulin antagonists of ICAM-1. Synthetic methods [eg, using the Merrifield method for polypeptide synthesis, ICAM-1, a functional derivative of ICAM-1, or a protein antagonist of ICAM-1 (immune Such as by synthesizing either globulin or non-immunoglobulin), by hybridoma technology (eg, producing monoclonal antibodies that can bind to ICAM-1), or by recombinant technology (eg, the present invention). Anti-inflammatory agents of various hosts (eg yeast, bacteria, fungi In a cultured animal cell etc.) or from a recombinant vector or a viral vector] The selection of the method to be used depends on factors such as convenience and the desired yield. It is not necessary to produce a specific anti-inflammatory agent using only one of the techniques, and the methods, techniques and techniques described above may be combined to obtain a specific anti-inflammatory agent.
A.Identification of LFA-1 binding partner (ICAM-1)
1. Assay for LFA-1-dependent aggregation
Many Epstein-Barr virus transformed cells show aggregation. This aggregation can be promoted in the presence of phorbol esters. It has been found that such homotypic aggregation (ie, aggregation involving only one cell type) is blocked by anti-LFA-1 antibody [“Rothlein, R. et al.,J. Exper. Mes. 163: 1132-1149 (1986), which is incorporated herein by reference.] That is, the degree of LFA-1-dependent binding is determined by assessing the degree of spontaneous or phorbol ester-dependent aggregation. Can be determined.
Agents that interfere with LFA-1-dependent aggregation can be identified by using an assay that can measure whether the agent inhibits spontaneous or phorbol ester-dependent aggregation of Epstein-Barr virus transformed cells. Most Epstein-Barr virus transformed cells can be used in such assays as long as they can express the LFA-1 receptor molecule. Such cells include “Springer, T.A., etc.J. Exper. Med. 160: 1901-1918 (1984) ", which is incorporated herein by reference. Although any such cells can be used in the LFA-1-dependent binding assays of the present invention, preferred Is to use cells of the JY cell line [Terhost, CT, etc.Proc. Natl. Acad. Sci. USA, 73: 910 (1976)]. These cells can be cultured in any suitable culture medium, most preferably by culturing the cells in RMPI 1640 medium supplemented with 10% fetal bovine serum and 50 μg / ml gentamicin (Gibco Laboratories, New York). is there. These cells are in conditions suitable for animal cell growth (ie, generally at a temperature of 37 ° C., 5% CO 2.₂In an atmosphere of 95% relative humidity, etc.).
2. Binding of LFA-1 to ICAM-1
Human individuals whose lymphocytes lack the group of LFA-1 receptor molecules have been identified [Anderson, D.C. et al.Fed. Proc.44: 2671-2677 (1985); Anderson, D.C.J. Infect. Dis.152: 668-689 (1985)]. Such people are said to suffer from leukocyte adhesion deficiency (LAD). Such human EBV transformed cells do not aggregate either spontaneously or in the presence of phorbol esters in the agglutination assay described above. Aggregation is observed when such cells are mixed with LFA-1 expressing cells [Rothlein, R. et al.J. Exper. Med. 163: 1132-1149 (1986)] (FIG. 1). Importantly, these aggregates were not formed when these cells were incubated in the presence of anti-LFA-1 antibody. That is, although aggregation required LFA-1, the ability of LFA-1-deficient cells to form aggregates with LFA-1-containing cells is not an LFA-1 binding partner but rather a previously discovered cell adhesion molecule. Suggested that there was. FIG. 1 shows the mechanism of intercellular adhesion.
B.Intercellular adhesion molecule-1 (ICAM-1)
A novel intercellular adhesion molecule ICAM-1 was prepared by the procedure of “Rothlein, R. et al.J. Immunol. 137: 1270-1274 (1986), which is hereby incorporated by reference, partially characterized. To detect ICAM-1 molecules, monoclonal antibodies were prepared from spleen cells of mice immunized with human cells genetically deficient in LFA-1 expression. The obtained antibody was screened for its ability to suppress aggregation of LFA-1 expressing cells (FIG. 2). Specifically, ICAM-1 molecule mice were immunized with EBV transformed B cells from LAD patients that do not express the LFA-1 antigen. Spleen cells from these animals were removed and fused with myeloma cells to become monoclonal antibody-producing hybridoma cells. Next, EBV-transformed B cells from a normal person expressing LFA-1 are incubated in the presence of the monoclonal antibody of the hybridoma cells, and EBV-transformed B cells are phorbol ester-mediated, LFA-1-dependent, spontaneous Any monoclonal antibody that could suppress aggregation was identified. Since the hybridoma cells were derived from cells that did not contain any LFA-1 antigen, no monoclonal antibody against LFA-1 was produced. Therefore, any antibody that has been shown to suppress aggregation must be capable of binding to an antigen that was not LFA-1 but was involved in the LFA-1 adhesion process. Although any method can be used for obtaining such a monoclonal antibody, BALB / C mice are preferably used in Rothlein, R. et al.J. Immunol. 137: I270-1274 (1986)) by immunizing with Epstein-Barr virus transformed peripheral blood mononuclear cells from LFA-1 deficient using the route and schedule disclosed by Is to get. Such cells are disclosed by Springer, T. A. et al. [J. Exper. Med. 160: 1901-1918 (1984)].
In a preferred method for the production and detection of antibodies capable of binding to ICAM-1, the mouse expresses by EBV transformed B cells that express both ICAM-1 and LFA-1, or more preferably ICAM-1. Immunize with TNF-activated endothelial cells that do not express LFA-1. In the most preferred method of preparing hybridoma cells producing anti-ICAM-1 antibodies, Balb / C mice are sequentially immunized with JY cells and specific U937 cells (ATCC CRL-1593). Spleen cells from such animals are removed and fused with myeloma cells to develop antibody-producing hybridoma cells. The resulting antibodies are screened for their ability to suppress LFA-1-dependent, phorbol ester-induced aggregation of EBV transformed cell lines such as JY cells expressing both LFA-1 and ICAM-1. Rothlein, R. etc. (J. Immunol. 137: 1270-1274 (1987)], an antibody capable of suppressing such aggregation is SKW._Three[Dustin, M., etc.J. Exper. Med. 165: 672-692 (1987), its ability to spontaneously aggregate in the presence of phorbol esters is inhibited by antibodies that can bind to LFA-1, but not by anti-ICAM-1 antibodies] Test for ability of cell line to inhibit phorbol ester-induced aggregation. SKW can suppress phorbol ester-induced aggregation of cells such as JY cells_ThreeAn antibody that cannot suppress phorbol ester-induced aggregation of cells such as is probably an anti-ICAM-1 antibody. An antibody capable of binding to ICAM-1 can suppress LFA-1-dependent aggregation of LFA-expressing cells (such as JY cells), but expresses LFA-1, but mostly ICAM-1 or Screening for antibodies that cannot bind to cells that do not express at all (such as normal granulocytes) or antibodies that can bind to cells that express ICAM-1 but do not express LFA-1 (such as TNF-activated endothelial cells) Can also be identified.
Another method is to immunoprecipitate cells expressing ICAM-1, LFA-1 or both using an antibody that suppresses LFA-1-dependent aggregation of cells such as JY cells, and SDS-PAGE or equivalent The method is to measure some molecular properties of molecules precipitated by antibodies. An antibody can be considered an anti-ICAM-1-antibody if its properties are the same as those of ICAM-1. The ICAM-1 cell surface molecule was purified and characterized using the monoclonal antibody prepared by the method described above. ICAM-1 was purified from human cells or tissues using monoclonal antibody affinity chromatography. In such a method, a monoclonal antibody reactive with ICAM-1 is bound to an inert column matrix. Any method of performing such a linkage can be used, but preferred is “Oetthen, H. C. et al.J. Biol. Chem. 259: 12034 (1984) ". When cell lysate is passed through the matrix, the ICAM-1 molecules present are adsorbed and retained on the matrix. By changing the pH or ion concentration of the column Bound ICAM-1 molecules can elute from the column, although any suitable matrix can be used, but preferably, Sepharose (Pharmacia) is used as the matrix material. Is well known in the art.
In methods understood by those skilled in the art, the assays described above can be used to identify compounds that can attenuate or inhibit the rate or degree of cell adhesion.
ICAM-1 is on vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and non-hematopoietic cells such as fibroblasts and tissue macrophages, mitogen-stimulated T lymphoblasts, germinal centers B in the tonsils. Cell surface glycoprotein expressed on hematopoietic cells such as cells and dendritic cells, lymph nodes, and Peyer's patches. ICAM-1 is highly expressed in vascular endothelial cells of the T cell region in the tonsils that show lymph nodes and reactive proliferation. ICAM-1 is expressed in small amounts on peripheral blood lymphocytes. Phorbol ester-stimulated specification of certain osteogenic monocyte cell lines greatly increases ICAM-1 expression. That is, ICAM-1 is preferentially expressed in inflammatory sites and is not generally expressed in quiescent cells. ICAM-1 expression on dermal fibroblasts is increased 3 to 5 fold over 4 hours or 10 hours, respectively, with interleukin 1 or Gunner interferon at a level of 10 μ / ml. The induction depends on protein and mRNA synthesis and is reversible.
ICAM-1 exhibits molecular weight heteroantigenicity in different cell types with a molecular weight of 97 kd on fibroblasts, 114 Kd on osteoblastic cell lines, and 90 Kd on B lymphoblast cell JY. ICAM-1 biosynthesis has been shown to contain an intercellular precursor of approximately 73 Kd. The non-N-glycosylated form resulting from tunicamycin treatment (inhibiting glycosylation) has a molecular weight of 55 Kd.
ICAM-1 isolated from phorbol ester stimulated U937 cells or from fibroblasts gives the same major product with a molecular weight of 60 Kd after chemical deglycosylation. The ICAM-1 monoclonal antibody interferes with adhesion of phytohemagglutinin blasts to the LFA-1-deficient cell line. Pretreatment of fibroblasts (not lymphocytes) with a monoclonal antibody conjugated to ICAM-1 suppresses lymphocyte-fibroblast adhesion. Pretreatment of lymphocytes with antibodies to LFA-1 (without fibroblasts) has also been found to suppress lymphocyte-fibroblast adhesion.
Thus, ICAM-1 is a binding ligand for the CD18 complex on leukocytes. ICAM-1 is temporally consistent with infiltration of lymphocytes into inflamed areas in vivo on fibroblasts and endothelial cells in vitro by inflammatory mediators such as IL-1, gamma interferon and tumor necrosis factor Inducible (Dustin, ML, etc.J. Immunol. 137: 245-254, (1986): Prober, J. S. et al.J. Immunol. 137: 1893-1896, (1986)]. Furthermore, ICAM-1 is also present on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, other epithelial cells and fibroblasts and also on tissue macrophages, mitogen-stimulated T lymphoblasts, germinal centers B in the tonsils. It is also expressed on hematopoietic cells such as cells and dendritic cells, lymph nodes and Peyer's patches (Dustin, ML, etc.J. Immunol. 137: 245-254, (1986)]. ICAM-1 is also expressed on benign inflammatory keratinocytes such as allergic eczema, lichen planus, rash, hives and bullous diseases. Allergic skin reactions induced by application of haptens to the skin of patients who are allergic also produced large amounts of ICAM-1 expression on keratinocytes. In contrast, toxic patches on the skin did not produce ICAM-1 expression on keratinocytes. ICAM-1 is present on keratinocytes from skin injury biopsies from various skin irregularities, and ICAM-1 expression was induced in injury from allergic patch tests, whereas keratinocytes from toxicity patch test injuries Did not express ICAM-1.
ICAM-1 is thus a cellular substrate to which lymphocytes can adhere, so that lymphocytes can penetrate the site of inflammation and / or transmit various effector functions that contribute to this inflammation. Such functions include antibody production and lysis of virus-infected target cells. As used herein, the term “inflammation” is meant to include specific or non-specific defense system responses. The term “specific defense system” is intended to refer to a component of the immune system that reacts with the presence of a specific antigen. Inflammation is said to result from a specific defense system response if it is caused by, mediated by or accompanied by a specific defense system response. Examples of inflammation resulting from specific defense system responses include rubella virus, autoimmune disease, T-cell-mediated delayed hypersensitivity (eg, in patients who are “positive” in the Mantaux test), psoriasis Etc.
A “non-specific defense system response” is a response mediated by leukocytes incapable of immune memory. Such cells include granulocytes and macrophages. As used herein, inflammation is said to result from a non-specific defense system response if the inflammation is caused by, mediated by, or accompanied by a response of a non-specific defense system. . Examples of inflammation, at least in part derived from a non-specific defense system, include asthma, adult respiratory distress syndrome (ARDS) or septic or traumatic secondary multiple organ loss group, myocardial or other tissue reperfusion (Reperfusion), injury, acute glomerulonephritis, reactive arthritis, dermatitis due to acute inflammatory components, acute purulent peritonitis or other central nervous system inflammatory diseases, burns, hemodialysis, leukapheresis, ulcers There are inflammations associated with conditions such as ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion related syndrome, and cytokine-induced toxicity.
In accordance with the present invention, ICAM-1 functional derivatives, particularly derivatives such as those containing fragments or mutants of ICAM-1 having domains 1, 2 and 3, are treated for non-specific defense system reactions as described above. Or it can be used in treatment. More preferred in such treatment or therapy is an ICAM-1 fragment or mutant containing domain 2 of ICAM-1. Most preferred in such treatment or therapy is an ICAM-1 fragment or mutant containing domain 1 of ICAM-1.
C.Cloning of the ICAM-1 gene
Any number of methods can be used to clone the ICAM-1 gene. One such method is to analyze a shuttle vector library of cDNA inserts (derived from ICAM expressing cells) for the presence of inserts containing the ICAM-1 gene. Such an analysis can be performed by transferring cells with the vector and then assaying for ICAM-1 expression. A preferred method of cloning this gene involves determining the amino acid sequence of the ICAM-1 molecule. To do this, the ICAM-1 protein can be purified and analyzed by an automated sequenator. The molecule can also be degraded by cyanogen bromide or by proteases such as papain, chymotrypsin or trypsin [Oike, Y. et al.,J. Biol. Chem. 257: 9751-9758 (1982); Liu, C. et al.Int. J. Pept.Protein Res. 21: 209-215 (1983)]. Although the entire amino acid sequence of ICAM-1 can be determined, it is preferred to determine the sequence of the peptide fragment of the molecule. If the peptide is longer than 10 amino acids, its sequence information is generally sufficient for cloning genes such as the gene for ICAM-1.
The sequence of amino acid residues in a peptide is also indicated in this specification using a commonly used three-character display or single-character display. The list of these three-character and single-character displays is “Biochemistry, Lehninger, A., published by Worth Publishers, New York, (1970) ". When writing such sequences vertically, the amino end group should be at the top of the column and the carboxy end group should be Similarly, when writing in the horizontal direction, the amino terminus should be on the left and the carboxy terminus should be on the right. The amino acid residues in the peptide can be separated by a hyphen. Such a hyphone is only intended to facilitate the existence of the array.
-Gly-Ala-Ser-Phe-
The amino acid sequence indicated as follows indicates that the Ala residue is bonded to the carboxy group of Gly and that the Ser residue is bonded to the carboxy group of the Ala residue and the amino group of the Phe residue. This representation also shows that the amino acid sequence contains the tetrapeptide Gly-Ala-Ser-Phe. This representation does not limit the amino acid sequence to this one tetrapeptide; (1) a tetrapeptide having one or more amino acid residues linked to amino or carboxy; (2) bound to both amino and carboxy termini And tetrapeptides having one or more amino acid residues, and (3) tetrapeptides having no additional amino acid residues.
Once one or more suitable peptide fragments have been sequenced, the DNA sequence that can encode them is examined. Since the genetic code is degenerate, one or more codons can be used to code for specific amino acids [Watson, J. D.,Molecular Biology of the Gene3rd edition, W. A. Benjamin, California Menlo Park, (1977), pp 356-357]. The peptide fragments are analyzed to identify the amino acid sequence that can be encoded by the oligonucleotide with the least degree of degeneracy. This is preferably done by identifying sequences containing amino acids encoded by only a single codon. In some cases, such an amino acid sequence can be encoded by only a single oligonucleotide, but in many cases the amino acid sequence can be encoded by any set of similar oligonucleotides. Importantly, all members of the set contain an oligonucleotide that can encode the peptide fragment, thus potentially containing the same nucleotide sequence as the gene encoding the peptide fragment, whereas one member of the set Only is to contain a nucleotide sequence identical to the nucleotide sequence of this gene. This member is present in the set and can hybridize to DNA even in the presence of other members of the set, so in the same way that a single oligonucleotide is used to clone the gene encoding the peptide. Undegraded oligonucleotide sets can be used.
In exactly the same manner as described above, an oligonucleotide (or set of oligonucleotides) having a nucleotide sequence that is complementary to an oligonucleotide sequence or sequence set that can encode a peptide fragment may be used.
Identify an appropriate oligonucleotide or set of oligonucleotides (or those complementary to such oligonucleotides or oligonucleotides) that can encode a fragment of the ICAM-1 gene (using the procedure described above) and for DNA It is synthesized and hybridized using means well known in the art, preferably with a human cell-derived cDNA preparation capable of expressing the ICAM-1 gene sequence. Nucleic acid hybridization technology is described in “Molecular Cloning, a Laboratory Manual, Coldspring Harbor, NY (1982)” by Maniatis, T. et al., And “Nucleic acid Hybrization, a Practical approach, IRL Press, Washington,” by Haymes, BD, etc. DC (1985) ", which are incorporated herein by reference. The DNA or cDNA source used is preferably rich in ICAM-1 sequences. Such enrichment is most easily from cDNA obtained by extracting RNA from cells cultured under conditions that induce ICAM-1 synthesis (such as U937 etc. grown in the presence of phorbol esters). It can be done.
Like or similar to the method described above, human aldehyde dehydrogenase gene [Hsu, L. C., etc.Proc, Natl, Acad, Sci, USA82: 3771-3775 (1985)], fibronectin [Suzuki, S. et al.,Eur, Mol, Biol,Organ, J. 4: 2519-2524 (1985)] human estrogen receptor gene [Walter, P. et al.,Proc,Natl, Acad, Sci, USA82: 7889-7893 (1985)], tissue type plasminogen activator [Pennica, D. et al.,Nature 301:214-221 (1983)] and human placental alkaline phosphatase complementary DNA [Kam, W. et al.,Proc, Natl, Acad, Sci, USA82: 8715-8719 (1985)].
In another preferred method of cloning the ICAM-1 gene, a library of expression vectors is prepared by cloning DNA or preferably cDNA from cells capable of expressing ICAM-1 in the expression vector. This library is then screened for members that can bind to anti-ICAM-1 antibodies and express a protein having a nucleotide sequence that can encode a polypeptide having the same amino acid sequence as ICAM-1 or an ICAM-1 fragment.
The cloned ICAM-1 gene obtained by the above method is operably linked to an expression vector and incorporated into bacteria or eukaryotic cells to produce ICAM-1 protein. Such manipulation techniques are disclosed by Maniatis, T. et al. (Supra) and are well known in the art.
D.Use of an assay for LFA-1-dependent aggregation
The aforementioned assays that can measure LFA-1-dependent aggregation can be used to identify agents that act as antagonists that suppress the degree of LFA-1-dependent aggregation. Such antagonists can act by conferring LFA-1 or ICAM-1 capacity related to aggregation. That is, such agents include immunoglobulins such as antibodies that can bind to LFA-1 or ICAM-1. In addition, non-immunoglobulin (ie, chemical) agents can also determine whether they are antagonists of LFA-1 aggregation using the above assay.
E.Use of an antibody capable of binding to an ICAM-1 receptor protein
1.Anti-inflammatory agent
CD₁₈Monoclonal antibodies against each member of the complex bind to the endothelium [Haskard, D. et al.J. Immunol. 137: 2901-2906 (1986)], homotype adhesive [Rothlein, R. et al.,J. Exp. Med. 163: 1132-1149 (1986)] Antigen and mitogen-induced proliferation of lymphocytes [Davigon, D. et al.Proc, Natl, Acad, Sci, USA78: 4535-4539 (1981)], antibody formation [Fisher, A. et al.,J. Immunol. 1363198-3203 (1986), and cytotoxic T cells [Krensky, A. M. et al.,J. Immunol. 132: 2180-2182 (1984)], macrophages [Strassman, G. et al., J. Biol.Immunol. 136: 4328-4333 (1986)], and all cells involved in antibody-dependent cytotoxic reactions [Kohi, S. et al., J. Chem.Immunol. 133: 2972-2978 (1984)], which inhibits many adhesion-dependent functions of leukocytes, including all leukocyte effector functions such as lytic activity. In all of these functions, the antibody suppresses the ability of leukocytes to adhere to the appropriate cell substrate (this ability suppresses the end result).
As described above, binding of an ICAM-1 molecule to one member of an LFA-1 group molecule is extremely important in cell adhesion. In the process of adhesion, lymphocytes can continuously monitor animals for the presence of foreign antigens. Although these processes are usually desirable, they are also responsible for organ transplant rejection, tissue transplant rejection, and many autoimmune patients. Therefore, some means capable of attenuating or suppressing cell adhesion is highly desirable for organ transplant, tissue transplant recipients or autoimmune patients.
Monoclonal antibodies capable of binding to ICAM-1 are extremely suitable as mammalian anti-inflammatory agents. Clearly, such drugs differ from common anti-inflammatory agents in that they can selectively suppress adhesion and do not exhibit other side effects such as nephrotoxicity that can be found with conventional drugs. Thus, monoclonal antibodies that can bind to ICAM-1 can be used to prevent organ or tissue rejection or to correct autoimmune responses in mammals without the risk of such side effects.
Importantly, the use of monoclonal antibodies capable of recognizing ICAM-1 allows organ transplantation to be performed even in individuals with HLA imbalances.
2.Suppressor of delayed hypersensitivity reaction
Since ICAM-1 molecules are mostly expressed at sites of inflammation such as those involved in delayed hypersensitivity reactions, antibodies that can bind to ICAM-1 molecules (especially monoclonal antibodies) are responsible for the attenuation or elimination of such reactions. Has therapeutic potential. This potential therapeutic application can be exploited in one of two ways. First, a composition containing a monoclonal antibody against ICAM-1 can be administered to a patient who exhibits a delayed type hypersensitivity reaction. For example, such a composition could be administered to a person who has come into contact with an antigen such as a poisonous woodpecker or a poisonous oak. In a second embodiment, a monoclonal antibody that can bind to ICAM-1 is administered to a patient along with an antigen to prevent subsequent inflammatory reactions. That is, co-administration of an antigen and an ICAM-1-binding monoclonal antibody can be temporarily allowed in humans after administration of the antigen.
3. Treatment of chronic inflammatory diseases
Since LAD patients deficient in LFA-1 do not show an inflammatory response, it is believed that antagonism of the LFA-1 natural ligand, ICAM-1, also suppresses the inflammatory response. The ability of antibodies against ICAM-1 to suppress inflammation is discoid lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, diabetic Reynard syndrome, rheumatism-like It provides the basis for therapeutic use in the treatment of chronic inflammation such as arthritis and autoimmune diseases. In general, monoclonal antibodies that can bind to ICAM-1 can be used to treat diseases currently treatable by steroid therapy.
4. Diagnostic and judgmental applications
Since ICAM-1 is mostly expressed in the inflamed part, monoclonal antibodies that can bind to ICAM-1 can be used as a means of imaging or visualizing the site of patient infection and inflammation. In such applications, monoclonal antibodies are detectably labeled using radioisotopes, affinity labels (such as biotin, avidin, etc.), fluorescent labels, paramagnetic atoms, and the like. Procedures for performing such labeling are well known in the art. The clinical application of antibodies in diagnostic imaging is “Grossman, H. B.,Urol, Clin, North Amer. 13: 465-474 (1986) "," Unger, E. C., etc.Invest, Radiol, 20: 693-700 (1985) "and" Khaw, B. A. et al.,Science 209: 295-297 (1980) ".
The presence of inflammation can also be detected by the use of binding ligands such as mRNA, cDNA or DNA that bind to the ICAM-1 gene sequence of cells expressing ICAM-1 or to the ICAM-1 mRNA sequence. Techniques for performing such hybridization assays are disclosed by Maniatais, T. (supra).
Detection of such detectably labeled antibody lesions may indicate sites of inflammation or tumor expression. In one embodiment, the inflammation test is performed by removing a tissue or blood sample and incubating such sample in the presence of the detectably labeled antibody. In a preferred embodiment, this method is performed in a non-invasive manner using magnetic imaging, fluorography and the like. Such diagnostic tests can be used to monitor organ transplant recipients for early detection of potential tissue rejection. Such assays can also be performed to determine an individual's response to rheumatoid arthritis or other chronic inflammatory diseases.
5. Assist in introducing antigenic substances to be administered for therapeutic or diagnostic purposes
For example, an immune response to a therapeutic or diagnostic agent such as bovine insulin, interferon, tissue-type plasminogen activator or mouse monoclonal antibody substantially reduces the therapeutic or diagnostic value of such agents, and in fact serum Diseases such as illness can occur. Such a situation can be improved by using the antibody of the present invention. In this embodiment, such antibodies are administered in combination with a therapeutic or diagnostic agent. The addition of antibody prevents the recipient from recognizing the drug and thus prevents the recipient from initiating an immune response to the drug. The absence of such an immune response provides the patient with the ability to accept additional doses of therapeutic or diagnostic agents.
F.Use of intercellular adhesion molecule-1 (IMAC-1)
ICAM-1 is a binding partner of LFA-1. Thus, ICAM-1 or functional derivatives thereof can be used interchangeably with antibodies that can bind to LFA-1 in the treatment of disease. That is, in solubilized form, such molecules can be used to suppress inflammation, organ rejection, transplant rejection, and the like. ICAM-1 or functional derivatives thereof can be used in the same manner as anti-ICAM antibodies to reduce the immunogenicity of therapeutic or diagnostic agents.
ICAM-1, functional derivatives thereof, and antagonists thereof can be used to block the metastasis or growth of tumor cells that express ICAM-1 or LFA-1 on their surface. Various methods can be used to achieve such purpose. For example, hematopoietic cell penetration requires LFA-1-ICAM-1 binding. Thus, antagonists of such binding inhibit the above penetration and block tumor cell metastasis from leukocytes. A patient can also be administered a toxin-derived molecule that can bind to one member of an ICAM-1 or LFA-1 group molecule. When such a toxin-derived molecule binds to one member of an ICAM-1 or LFA-1 group molecule, the presence of the toxin kills tumor cells and thereby suppresses tumor growth.
G.Use of non-immunoglobulin antagonists of ICAM-1-dependent adhesion
ICAM-1-dependent adhesion can be suppressed by non-immunoglobulin antagonists that can bind to ICAM-1 or LFA-1. One example of a non-immunoglobulin antagonist of ICAM-1 is LFA-1. An example of a non-immunoglobulin antagonist that binds to LFA-1 is ICAM-1. By using the aforementioned assay, additional non-immunoglobulin antagonists can be identified and purified. Non-immunoglobulin antagonists of ICAM-1-dependent adhesion can be used for the same purpose as antibodies against LFA-1 or antibodies against ICAM-1.
H.Administration of the composition of the invention
The therapeutic effect of ICAM-1 is obtained by administering to the patient whole ICAM-1- or any therapeutically active peptide fragment thereof.
ICAM-1 and functional derivatives thereof can be obtained synthetically or by protein degradation using recombinant DNA. The therapeutic benefits of ICAM-1 can be enhanced by the use of functional derivatives of ICAM-1 having additional amino acid residues to improve binding to the carrier or to improve the activity of ICAM-1. The scope of the present invention further includes functional derivatives of ICAM-1 lacking certain amino acid residues or containing other amino acid residues as long as such derivatives exhibit the ability to act on cell adhesion. .
The antibodies and ICAM-1 molecules of the present invention can be said to be “substantially free of natural impurities” if the preparation containing them is substantially free of substances normally found in nature with these products.
The invention also extends to antibodies capable of binding to ICAM-1 and fragments (polyclonal or monoclonal) having biological activity thereof. Such antibodies can be prepared by animal, tissue culture or recombinant DNA means.
To administer an antibody or fragment thereof that can bind to ICAM-1 to a patient, or to receive and administer ICAM-1 (or a fragment, variant or derivative thereof) to a patient, the dosage is the patient's year It depends on factors such as age, weight, height, gender, general medical conditions, medical history, etc. In general, it is desirable to administer to a patient at an antibody dosage in the range of about 1 Pg / kg to 10 mg / kg (patient weight), whereby lower or higher dosages can be administered. When administering an ICAM-1 molecule or a functional derivative thereof to a patient, it is preferable to administer such a molecule at a dosage also in the range of about 1 Pg / kg to 10 mg / kg (based on patient weight), Lower or higher doses can be used. As will be described later, the above effective dose can be reduced when the anti-ICAM-1 antibody is co-administered with the anti-LAM-1 administration. In the present invention, it can be said that one compound can be co-administered to a patient when the second compound and the administration of the two compounds are in conditions such that both compounds can be detected simultaneously in the patient's serum.
Both the antibody capable of binding to ICAM-1 and ICAM-1 itself can be administered intravenously, intramuscularly, subcutaneously, enterally or parenterally to the patient. When administering antibody or ICAM-1 by injection, the administration may be by continuous infusion or by one or multiple boluses.
The anti-inflammatory agent of the present invention is administered to a recipient subject in an amount sufficient to suppress inflammation. The amount can be said to be sufficient to “suppress” inflammation when the dosage, route of administration, etc. of the drug are sufficient to attenuate or prevent inflammation.
The anti-inflammatory agent of the present invention can be administered either before the onset of inflammation (to suppress expected inflammation) or after the onset of inflammation.
The anti-ICAM-1 antibody or fragment thereof can be administered alone or in combination with one or more additional immunosuppressive agents (particularly for organ or tissue transplant recipients). Administration of such compounds can be for either “prophylaxis” or “treatment” purposes. When administered prophylactically, these immunosuppressive agents are administered prior to an inflammatory response or symptom (eg, prior to, during or immediately after organ or tissue transplantation and before organ rejection symptoms). Prophylactic administration of these compounds helps to prevent or reduce any subsequent inflammatory response, such as transplant organ or tissue rejection. When administered therapeutically, these immunosuppressive compounds are administered at (or shortly after) the onset of actual inflammation symptoms (eg, organ or tissue rejection). The therapeutic administration of these compounds helps to reduce any actual inflammation (such as rejection of transplanted organs or tissues). Thus, the anti-inflammatory agents of the present invention can be administered either before the onset of inflammation (so as to suppress expected inflammation) or after the onset of inflammation.
A composition is said to be “pharmaceutically acceptable” if its administration is acceptable to the recipient patient. Such an agent can be said to be administered in a “therapeutically effective amount” if the dosage is physiologically significant. A drug is physiologically significant if its presence results in a detectable change in the accepting patient's physiology.
The antibodies and ICAM-1 molecules of the invention can be formulated by known methods of preparing pharmaceutically useful compositions, whereby these substances or functional derivatives thereof can be combined as a mixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and preparations thereof (eg, including other human proteins such as human serum albumin) are described in, for example, “Remington's Pharmaceutical Sciences (16th Edition, Dsol, A. Ed., Mark Easton (PA), (1980) ". In order to prepare pharmaceutically acceptable compositions suitable for effective administration, such compositions may be prepared with an effective amount of an anti-ICAM-1 antibody or an appropriate amount of a carrier vehicle. Contains an ICAM-1 molecule, or a functional derivative thereof.
Additional pharmaceutical methods can be used to control the duration of activity. Controlled release formulations can be obtained by using polymers that complex or absorb anti-ICAM-1 antibodies or ICAM-1, or functional derivatives thereof. Controlled delivery is a suitable macromolecule (eg, polyester, polyamino acid, polyvinylpyrrolidone, ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and a method of incorporation to control the concentration and release of the macromolecule Can be achieved by selecting Another possible way to control sustained activity with controlled release formulations is to use anti-ICAM-1 antibodies or ICAM-1 molecules, or functional derivatives thereof, polyesters, polyamino acids, hydrogels, polylactone acids or ethylene-vinyl acetate copolymers. It is to mix in the particle | grains of high molecular substances like. Further, instead of mixing these drugs into the polymer particles, these drugs are prepared in microcapsules prepared by, for example, coacervation or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and (methyl methacrylate) microcapsules, Alternatively, it can be encapsulated in colloidal drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules or macroemulsions. Such methods are also described in “Remington's Pharmaceutical Sciences (1980)”.
Example
Having generally described the invention so far, the following examples will make it easier to understand the invention. These examples are for purposes of illustration and are not intended to limit the invention.
Example 1
Animal cell culture
In general, each EBV transformation and hybridoma cell of the present invention was stored in RMPI 1640 medium supplemented with 20 mM L-glutamine, 50 μg / ml gentamicin, and 10% fetal bovine serum. Cells are 5% CO at 37 ° C₂The cells were cultured in a 95% humidity atmosphere.
To establish Epstein-Barr virus (EBV) transformants, 10% in RPMI 1640 medium supplemented with 20% fetal calf serum (FCS) and 50 μg / ml gentamicin.⁶T-cell exsanguinated peripheral mononuclear cells / ml were incubated with EBV-containing supernatant of B95-8 cells for 16 hours [Thorley-Lawson, D. A. et al.,J. Exper. Med. 146: 495 (1977)]. A 0.2 ml cell aliquot was placed in 10 microtiter wells. The medium was replaced with RPMI 1640 medium (20% fetal calf serum and 50 μm / l gintamycin added) until cell growth was seen. Cells grew in most wells and spread in the same medium. 10 phytohemagglutinin (PHA) blasts in RPMI 1640 medium (with 20% fetal calf serum) containing 1: 800 diluted PHA-P (Difco Laboratories, Detroit, MI)⁶Established in cells / ml. The PHA system spread in the interleukin 2 (IL-2) conditioned medium and weakly pulsated by PHA [Cantrell, D. A. et al.J. Exper. Med. 158: 1895 (1983)]. The above procedure is “Springer, T. etc.J. Exper. Med. 160: 1901-1918 (1984) ", the description of which is hereby incorporated by reference. The cells obtained by the above procedure are then screened with anti-LFA-1 antibody, which contains the LFA-1 antigen. Such antibodies were determined as “Sanchey Madrid, F. et al.J. Exper. Med. 158: 1785 (1983) ".
Example 2
Cell aggregation and adhesion assays
To assess the degree of cell adhesion, an aggregation assay was used. The cell line used for this assay was washed twice with RPMI 1640 medium containing 5 mM Hepes buffer (Sigma Chemical Co., St. Louis) and washed 2 × 10⁶Resuspended to a concentration of cells / ml. Flat bottom 96 well microtiter plates (No. 3596; Coastar, Cambridge, Mass.) 50 μl of appropriate monoclonal antibody supernatant, complete medium with or without 50 μl of purified monoclonal antibody, 50 μl of 200 ng / ml phorbol ester 2 × 10 2 in complete medium with ball myristate acetate (PMA) and 100 μl complete medium⁶Cells / ml concentration of cells were added. This is 50 ng / ml PMA and 2 × 10^FiveThe final concentration of cells / well was given. Cells were allowed to settle spontaneously and the degree of aggregation was counted at each time point. Frequency is 0-5⁺Was in the range. Where 0 indicates essentially no cells in dense concentration; 1⁺Have less than 10% of the cells in the aggregate; 2⁺Indicates that less than 50% of the cells are aggregated; 3⁺Indicates that up to 100% of the cells were in a small loose concentration; 4⁺Indicates that up to 100% of cells have aggregated in close concentration; 5⁺Indicates that 100% of the cells were in large very dense aggregates. To obtain a more quantitative assessment of cell adhesion, reagents and cells were added to a 5 ml polystyrene tube in the same order as above. The tube was placed at 37 ° C. in a platform on a grinder shaker. After 1 hour at about 200 rpm, 10 μl of the cell suspension was placed in a hemocytometer and the number of free cells was quantified. % Aggregation was determined by the following equation:

The number of cells in the above equation is the number of cells per ml in a control tube containing only unincubated cells and complete medium. The number of free cells in the above equation is equal to the number of non-aggregated cells per ml from the test tube. The above procedure is “Rothlein, R. et al.J. Exper. Med. 163: 1132-1149 (1986) ".
Example 3
LFA-1-dependent cell aggregation
The quantitative aggregation assay described in Example 2 was performed using the Epstein-Barr virus transformed cell line JY. PMA was added to the medium in the microtiter plate and cell aggregation was observed. The time-lapse video recorder showed active membrane waves and pseudopod movements with moving JY cells at the bottom of the microtiter well. Contact between adjacent cell pseudopods often resulted in cell-cell adhesion. When sticking persisted, the cell contact area moved to the uropod. Contact could be maintained despite vigorous cell movement and cell pulling in the opposite direction. The main difference between PMA-treated and untreated cells appeared in the stability of the contact once formed. In PMA, cell crowding appeared and there was viscosity growth as additional cells sticking around.
As a second means for measuring adhesion, the quantitative assay described in Example 2 was used. The cell suspension was shaken for 2 hours at 200 rpm, transferred to a hemocytometer, and cells not included in the aggregates were counted. In the absence of PMA, 42% (SD = 20%, N = 6) JY cells were in aggregates after 2 hours, whereas JY cells incubated with 50 μg / ml PHA under the same conditions aggregated. It had 87% (SD = 8%, N = 6) cells in the product. Aggregation kinetic studies showed that PMA promoted aggregation rate and strength at all test times (FIG. 3).
Example 4
Inhibition of cell aggregation using anti-LFA-1 monoclonal antibody
To test the effect of anti-LFA-1 monoclonal antibodies on PMA-induced cell aggregation, such antibodies were added to cells incubated according to the quantitative aggregation assay of Example 2. This monoclonal antibody was found to suppress the formation of cell aggregates both in the presence and absence of PMA. F (ab ') of monoclonal antibody against alpha chain of LFA-1₂Both Fab 'fragments could suppress cell aggregation. On the other hand, essentially 100% of the cells formed aggregates in the absence of the anti-LFA-1 antibody, and when the antibody was added, 20% or less of the cells were found in the aggregate. The results of this experiment were disclosed by Rothlein, R. et al.J. Exper. Med. 163: 1132-1149 (1986)].
Example 5
Cell aggregation requires the LFA-1 receptor:
EBV transformed lymphoblast cells were prepared from patients by the method described in Example 1. The cells were screened against a monoclonal antibody capable of recognizing LFA-1 and found to be LFA-1 deficient.
The quantitative aggregation assay described in Example 2 was performed using the above LFA-1 deficient cells. The cells did not spontaneously aggregate in the presence of PMA.
Example 6
Discovery of ICAM-1
The LFA-1-deficient cells of Example 5 were labeled with carboxyfluorescein diacetate [Patarroyo, M. et al.Cell. Immunol. 63: 237-248 (1981)]. The labeled cells were mixed with the same or JY cells at a ratio of 1:10, and the ratio of fluorescein labeled cells in the aggregate was determined as “Rothlein, R. et al.J. Exper. Med. 163: 1132-1149 (1986) ". It was found that LFA-1-deficient cells can co-aggregate with LFA-1-expressing cells (Fig. 4).
In order to determine if LFA-1 is only important for the formation or maintenance of aggregates, an antibody capable of binding to LFA-1 was added to the previously formed aggregates. It has been found that the addition of antibody strongly disrupts the previously formed aggregation. A time course video recorder showed that the addition of monoclonal antibody to the pre-formed aggregates began to divide within 2 hours (Table 1). After addition of monoclonal antibody to LFA-1, the mobilization movement and shape change of individual cells during aggregation continued unchanged. Individual cells gradually dissociated from the periphery of the aggregate, and by 8 hours the cells were almost dispersed. According to the video time course, the disruption of the previously formed aggregation by the LFA-1 monoclonal antibody was equivalent to the aggregation process in the absence of the LFA-1 monoclonal antibody performed in reverse time.

Example 7
Necessity of divalent ions in LFA-1-dependent aggregation
LFA-1-dependent adhesion between cytotoxic T cells and target requires the presence of magnesium [Martz, E.,J. Cell., Boll.84: 584, 598 (1980)], PMA-induced JY cell aggregation was tested for divalent cation dependence. JY cells did not aggregate in medium without calcium or magnesium ions (using the assay of Example 2). Addition of divalent magnesium aggregated at a concentration as low as 0.3 mM. The addition of calcium ion alone had little effect. However, calcium ions have been found to enhance the ability to support PMA-induced aggregation of magnesium ions. It was found that when 1.25 mM calcium ions were added to the medium, aggregation was aided with a magnesium ion concentration as low as 0.02 mM. These data indicate that LFA-1-dependent aggregation of cells requires magnesium ions, and that calcium ions increase aggregation with magnesium ions, although themselves are insufficient.
Example 8
Isolation of hybridoma cells capable of expressing anti-ICAM-1 monoclonal antibody
Monoclonal antibodies capable of binding to ICAM-1 are referred to as “Rothlein, R. et al.J. Immunol.137: 1270-1274 (1986) ", which is incorporated herein by reference. Three BALB / C mice were isolated from EBV-transformed peripheral blood from LFA-1-deficient patients. Immunized intraperitoneally with nuclear cells (Springer, TA et al., J. Exper. Med. 160: 1901 (1984)) about 10 in 1 ml RPML 1640 medium.⁷Cells were used for each immunization. Immunization was performed 45 days, 29 days and 4 days before removal of spleen cells from the mice to produce the desired hybridoma cell line. Three days prior to removal of spleen cells, mice were added 10% in 0.15 ml medium.⁷Cells were administered (intravenous injection).
Isolated spleen cells from the above animals were mixed with P3 × 73Ag8.653 myeloma cells at a ratio of 4: 1 “Galfre, G. et al.Nature266: 550 (1977) ". Each aliquot of the resulting hybridoma cells was placed in a 96-well microtiter plate. Each hybridoma supernatant was screened for inhibition of aggregation and one inhibitory hybridoma (more than 600 All of the well tests were cloned and subcloned by limited dilution, designated as RP1 / 1.1.1 (hereinafter “RP1 / 1”).
Monoclonal antibody RP1 / 1 was found to consistently suppress PMA-stimulated aggregation of LFA-1-expressing cell line JY. RP1 / 1 monoclonal antibody was equivalent or slightly smaller than monoclonal antibodies against some LFA-1α or β subunits and inhibited aggregation. In contrast, a monoclonal antibody against control HLA (which is expressed in large amounts on JY cells) did not inhibit aggregation. The antigen bound to the monoclonal antibody RP1 / 1 is defined as intercellular adhesion molecule-1 (ICAM-1).
Example 9
Use of anti-ICAM-1 monoclonal antibodies to characterize ICAM-1
In order to limit the properties of ICAM-1, in particular to determine whether ICAM-1 differs from LFA-1, cell proteins were immunoprecipitated using the monoclonal antibody RP1 / 1. Immunoprecipitation was performed according to the method of “Rothlein, R. et al. [J. Immunol. 137: 1270-1274 (1986)]. JY cells were freshly added with 1 mM phenylmethylsulfonyl fluoride, 0.2% / ml trypsin inhibitor aprotinin 1% Triton X-100, 0.14 m NaCl, 10 mM Tris, pH 8.0 (lysis buffer). 5 × 10⁷Lysed at 4 ° C. for 20 minutes at cells / ml. The lysate was centrifuged at 10,000 × g for 10 minutes and pretreated with 50 ml of a 50% suspension of CNBr activated glycine-suppressed Sepharose Cl-4B for 1 hour at 4 ° C. 1 ml of the lysate was immunoprecipitated overnight at 4 ° C. with 20 μl of a 50% suspension of monoclonal antibody (1 mg / ml) conjugated to Sepharose Cl-4B [Springer, TA et al., J. Exper. Med. 160: 1901 (1984)]. Sepharose-binding monoclonal antibodies were prepared using the CNBr activation method of Sepharose Cl-4B in carbonate buffer according to the method of “March, S. et al.Anal.Biochem. 60149 (1974)]. The washed immunoprecipitate was subjected to SDS-PAGE and silver staining according to the procedure of “Morrissey, J. H. Anal. Biochem. 117: 307 (1981)”.
SDS sample buffer [HD, M. K., etc.J. Biol. Chem. 258: 636 (1983)], each sample was divided in half and subjected to electrophoresis (SDS-8% PAGE) under each condition of reduction (Fig. 5A) or non-reduction (Fig. 5B). ). Bands with molecular weights of 50 Kd and 25 Kd corresponded to the long and short immunoglobulin chains from the monoclonal antibody Sepharose (FIG. 5A, lane 3). A variable amount of other bands in the 25-50 Kd molecular weight range were also observed, but not in the sediment from hairy leukemia cells, which gave only a 90 Kd molecular weight band. The LFA-1 177 Kdα and 95 Kdβ subunits permeated differently from ICAM-1 under both reducing (FIG. 5A, lane 2) and non-reducing (FIG. 5B, lane 2) conditions.
To measure the effect of monoclonal antibody RP1 / 1 on PHA-lymphoblast aggregation, the quantitative aggregation assay described in Example 2 was used. That is, T cell blast cells were stimulated with PHA for 4 days, washed thoroughly, and then cultured for 6 days in the presence of 1L-2 conditioned medium. PHA was taken up in this 6-day culture and did not contribute to the aggregation assay. In three assays with different T cell blast preparations, the ICAM-1 monoclonal antibody consistently suppressed aggregation (Table 2).

The LFA-1 monoclonal antibody was consistently more inhibitory than the ICAM-1 monoclonal antibody, while the HLA-A, B and LFA-3 monoclonal antibodies had no effect. These results indicate that of the tested monoclonal antibodies, only those monoclonal antibodies that can bind to LFA-1 or ICAM-1 can suppress cell adhesion.
Example 10
Preparation of monoclonal antibodies against ICAM-1
Immunity
Balb / c mice are 2 x 10 in RPMI medium⁷Immunization intraperitoneally (i.p.) was performed 103 days and 24 days before fusion with 0.5 ml of JY cells. On days 4 and 3 prior to fusion, mice were immunized i.p. with PMA-differentiated U937 cells in 0.5 ml RPM1 medium.
Differentiation of U937 cells
U937 cells (ATCC CRL-1593) in 5 × 10 5 RPMI in 10% fetal bovine serum, 1% glutamine and 50 ml of 2 ng / ml phorbol-12-myristate acetate (PMA) containing dietamycin (complete medium)^FiveDifferentiated by incubating in sterile polypropylene containers at / ml. On the third day of this incubation,¹/₂The volume of medium was removed and replaced with fresh complete medium containing PMH. On day 4, cells were removed, washed and prepared for immunization.
fusion
Spleen cells from immunized mice were fused with P3 × 63Ag8.653 myeloma cells at a ratio of 4: 1 according to Galfre et al.Nature266: 550 (1977)]. After fusion, the cells are placed in a 96 well flat bottom microtiter plate 10^FivePlaced in spleen cells / well.
Selection of anti-ICAM-1 positive cells
One week later, 50 μl of the supernatant was screened in the quantitative aggregation assay of Example 2 using both JY and SKW-3 as aggregation cell lines. Cells from the supernatant that inhibit JY cell aggregation but not SKW-3 aggregation were selected and cloned twice using limited dilution.
This experiment allowed identification and cloning of three hybridoma lines producing anti-ICAM-1 monoclonal antibodies. The antibodies produced by these hybridoma systems are IgG, respectively._2a, IgG_2bAnd IgM. IgG_2aThe hybridoma cell line producing the anti-ICAM-1 antibody was designated R6'5'D6'E9'B2. The antibody produced by this preferred hybridoma cell line was designated R6'5'D6'E9'B2 (hereinafter referred to as "R6-5-D6").
Example 11
Expression and standardization of ICAM-1
To measure ICAM-1 expression, a radioimmunoassay was performed. In this assay, purified RP1 / 1 was iodinated using iodogen to a specific activity of 10 μCi / μg. Endothelial cells were grown in 96 well plates and processed as described in each experiment. Each plate was cooled to 4 ° C. by placing it in a cold room for 0.5-1 hour instead of directly on ice. Each monolayer is washed 3 times with cold complete medium and then at 4 ° C. for 30 minutes¹²⁵Incubated with I RP1 / 1. The monolayer was then washed 3 times with complete medium. Join¹²⁵I was released using 0.1N NaOH and counted.¹²⁵The specific activity of I RP1 / 1 was adjusted using unlabeled RP1 / 1 to obtain a linear signal over the antigen density range used in this study. Nonspecific binding was measured in the presence of 1,000-fold excess of unlabeled RP1 / 1, and subtracted specific binding was obtained from total binding.
ICAM expression measured using the radioimmunoassay described above is increased by 1L-1, TNF-, LPS and INF gamma on human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) (Table 3). ). Saphenous vein endothelial cells were used in this study to confirm results from umbilical vein endothelial cells in cultured vena cava endothelial cells derived from adult tissues. The basic expression of ICAM-1 is twice as high on saphenous vein endothelial cells than on umbilical vein endothelial cells. Exposure of umbilical vein endothelial cells to recombinant IL-1 alpha, IL-1 beta, and TNF increases ICAM expression 10-20 fold. IL-1 alpha, TNF and LPS are maximal potency inducers, and IL-1 is less potent at weight-based and saturating concentrations of response (Table 3). IL-1 beta at 100 ng / ml increased ICAM-1 expression 9-fold on HUVEC and 7.3-fold on HSVEC, with a half-maximal increase at 15 ng / ml. 50 ng / ml rTNF increased ICAM-1 expression 16-fold on HUVEC and 11-fold on HSVEC, with a half-maximal effect of 0.5 ng / ml. Interferon gamma showed a significant increase of 5.2 fold on HUVEC or 3.5 fold on HSVEC at 10,000 U / ml in ICAM-1 expression. The effect of LPS at 10 μg / ml was similar in strength and strength to rTNF. The combination of these mediator pairs produced an additional effect or slightly less than the additional effect on ICAM-1 expression (Table 3). Cross titration of rTMF with rIL-1 beta or rIFN gamma showed no synergistic effect at sub-optimal or best concentration effects.
Since LPS increased ICAM-1 expression on endothelial cells on a basis that is sometimes found in the media, the possibility that basal ICAM-1 expression could be LPS-based was tested. When several serum batches were tested, it was found that low endotoxin serum gave a low ICAM-1 basal expression of up to 25%. All results presented here were for endothelial cells grown in low endotoxin serum. However, addition of the LPS neutralizing antibiotic polymyxin B at 10 μg / ml reduced ICAM1 expression to an additional 25% (Table 3). Increased ICAM-1 expression upon treatment with IL-1 or TNF had no effect due to the presence of 10 μg / ml polymyxin B consistent with the low endotoxin values in these preparations (Table 3).

Upregulation of ICAM-1 expression on HVEC and HSVEC-HUVEC or HSVEC was seeded from a confluent monolayer at 1: 3 in 96 well plates and grown in fusion. Cells were then treated with each substance or medium for 16 hours and RIA was performed methodatically. All values were repeated 4 times.
Example 12
Interleukin 1 and gamma interferon-induced kinetics of ICAM-1
The kinetics of interleukin-1 and gamma interferon effects on ICAM-1 expression on skin fibroblasts are described by Dustin, M. L. et al.¹²⁵Measured using I goat anti-mouse IgG binding assay [J. Immunol.137: 245-254 (1986); this document is incorporated herein by reference. To perform this binding assay, human skin fibroblasts were placed in a 96-well microtiter plate at 2-8 × 10^FourCells / well (0.32cm²). Cells were washed twice with RPMI 1640 medium supplemented as described in Example 1. The cells were further divided into Hanks Balanced Salt Solution (HBSS), 10 mM HEPES, 0.05% NaN_ThreeAnd washed once with 10% heat-inactivated fetal bovine serum. Washing with this binding buffer was performed at 4 ° C. 50 μl of the above binding buffer and 50 ml of the appropriate hybridoma supernatant with X63 and W6 / 32 were added to each well as negative and positive controls, respectively. After incubation at 4 ° C for 30 minutes, the wells are washed twice with binding buffer to give a second antibody¹²⁵I-goat anti-mouse IgG was added at 50 nCi in 100 μl.¹²⁵I-goat anti-mouse antibody was prepared according to the method of Fraker, P. J., etc. using iodogen (Pierce) [Biochem. Biophys. Res. Commun. 80: 849 (1978)]. After 30 minutes at 4 ° C., the wells were washed twice with 200 μl binding buffer and the cell layer was solubilized by adding 100 μl 0.1 N NaOH. This treatment and 100 μl washes were counted in a Beckman 5500 gamma counter. The specific count of binding per minute was calculated as [cpm with monoclonal antibody]-[cpm with X63]. All steps including induction with specific reagents were repeated 4 times.
The effect of interleukin 1 with a half-life of 2 hours in ICAM-1 induction was more rapid than that of gamma-interferon with a half-life of 3.75 hours (FIG. 6). The time to return to quiescent levels of ICAM appeared to be due to cell cycle or cell proliferation. In quiescent cells, interleukin 1 and gamma interferon effects are stable for 2-3 days, whereas in log phase culture, ICAM-1 expression is close to baseline at 2 days after removal of these inducers.
FIG. 7 shows dose response curves of ICAM-1 induction by recombinant mouse and human interleukin 1 and ICAM-1 induction by recombinant gamma interferon. Gamma interferon and interleukin 1 were found to have similar concentration dependence with almost the same effect at 1 ng / ml. Human and mouse recombinant interleukin 1 have similar curves but show a much lower effect than the human interleukin 1 formulation in inducing ICAM-1 expression.
Cycloheximide (inhibitor of protein synthesis) and actinomycin D (inhibitor of mRNA synthesis) abolish the effects of interleukin 1 and gamma interferon on ICAM-1 expression on fibroblasts (Table 4). Furthermore, only tunicamycin (an inhibitor of N-linked glycosylation) suppressed the interleukin 1 effect by 43%. These results indicate that protein and mRNA synthesis are required for interleukin 1 and gamma interferon-induced increases in ICAM-1 expression, but N-linked glycosylation is not required.

Example 13
ICAM-1 tissue distribution
Histochemical studies were performed on frozen tissues of human organs to determine the distribution of ICAM-1 in the thoracic line, lymph nodes, intestine, skin, kidney and liver. To perform such analysis, frozen tissue sections (4 μm thick) of normal human tissue were fixed in acetone for 10 minutes and monoclonal antibody, avidin-biotin disclosed by Cerf-Bensussan, N. et al. With RR1 / 1. Staining by immunoperoxidase method using a complex method (Vector Laboratories, Burlingame, CA) [J. Immunol.130: 2615 (1983)]. Following incubation with the antibody, sections were sequentially incubated with biotinylated horse anti-mouse IgG and abitin-biotinylated peroxidase complex. Each section was finally immersed in a solution containing 3-amino-9-ethyl-carbazole (Aldrich Chemical Co., Milwaukee, Wis.) To perform a color reaction. Each section was then fixed in 4% formaldehyde for 5 minutes and counterstained with hematoxylin. Controls included sections incubated with an irrelevant monoclonal antibody instead of the RR1 / 1 antibody.
ICAM-1 was found to have almost the same distribution as the major tissue affinity complex (MHC) class II antigen. Most of the blood vessels (both large and small) in all tissues showed endothelial cell staining with ICAM-1 antibody. Vascular endothelial staining is more intense in the inter-vesicular (paracortical) regions in the lymph nodes, tonsils and payers compared to blood vessels in the kidney, liver and normal skin. In the liver, staining was mostly limited to sinusoidal capillary lining cells, and hepatocytes and endothelial cells lining most portal veins and arteries were not stained.
In the pleural sac, large cell diffusion staining and tree staining patterns were observed. In the cortex, the stained image was nest-like and mainly dendritic. Thymocytes were not stained. In peripheral lymphocyte tissue, germinal center cells of secondary lymphoid follicles were strongly stained. In some lymphoid follicles, the stained image was almost dendritic and there was no staining that could be recognized by lymphocytes. A faint staining of cells in the mantle region was also observed. Furthermore, dendritic cells with cytoplasm expansion (finger interreticular cells) and a small number of lymphocytes in vesicles or paracortical regions were stained with ICAM-1-binding monoclonal antibody.
Cell-like macrophages were stained within the lymph node and small intestinal thin film propria. Fibroblast-like cells (spindle-shaped cells) diffusing in most stroma of the organs examined were stained with ICAM-1-binding antibodies. No staining was observed in Langerhans / indeterminate cells in the envelope. No staining was observed even in smooth muscle tissue.
Outer cell staining was consistently seen in the tonsil mucosa. Although hepatocytes, hepatic duct sheaths, intestinal dermis cells, and tubular dermis cells in the kidney were not stained in most circumstances, a section of normal kidney tissue from a kidney excision with a renal cell carcinoma is ICAM- One staining of many adjacent tubular cells was shown. These tubular coat cells were also stained with anti-HLA-DR binding antibodies.
In summary, ICAM-1 is expressed on non-hematopoietic cells such as tubular ectodermal cells and on hematopoietic cells such as tissue macrophages and mitogen-stimulated T lymphoblasts. ICAM-1 was found to be expressed in small amounts in peripheral blood lymphocytes.
Example 14
Purification of ICAM-1 by monoclonal antibody affinity chromatography
General purification procedure
ICAM-1 was purified from human cells or tissues using monoclonal antibody affinity chromatography. Monoclonal antibody RR1 / 1 reactive with ICAM-1 was first purified and bound to an inert column matrix. This antibody is known as “Rothlein, R. et al.J. Immunol. 137: 1270-1274 (1986) "and" Dustin, M. L. et al.J. Immunol. 137: 245 (1986) ". ICAM-1 was solubilized from the cell membrane by lysing the cells in the nonionic detergent Triton X-100 at near neutral pH. The containing cell lysate was passed through a precolumn designed to remove non-specifically bound substances to the column matrix material, and then passed through a monoclonal antibody column matrix to bind ICAM-1 to the antibody. Washed with a series of detergent wash buffers with the pH raised to pH 11.0, during which the ICAM-1 remained bound to the antibody matrix, removing unbound and weakly bound impurities. The bound ICAM-1 was then specifically eluted from the column by applying pH 12.5 detergent buffer.
Purification of monoclonal antibody RR1 / 1 and covalent binding to Sepharose CL-4B
Anti-ICAM-1 monoclonal antibody RR1 / 1 was purified from ascites fluid or hybridoma culture supernatant of hybridoma-containing mice by standard ammonium sulfate precipitation and protein A affinity chromatography [Ey et al.Immunochem.15: 429 (1978)]. Purified IgG or rat IgG (Sigma Chemical Co., St. Louis, MO) is shared with Sepharose CL-4B (Pharmacia, Sweden) using a modified method of March et al. [Anal. Biochem. 60: 149 (1974)]. Combined. In short, Sepharose CL-4B was washed in distilled water and 5M K₂HPO_FourActivated with 40 mg / ml CNBr in (pH approx. 12) for 5 min, then washed long with 0.1 mM HCl at 4 ° C. Filtered activated Sepharose was added to an equal volume of purified antibody (0.1 M NaHCO 3_Three2-10 mg / ml in 0.1 M NaCl). The suspension was incubated at 4 ° C. for 18 hours by gentle end-to-end rotation. The supernatant was then monitored for unbound antibody by absorbance at 280 nm and the remaining reactive sites of activated Sepharose were saturated by adding glycine to 0.05M. The coupling efficiency was usually 90% or higher.
Detergent solubilization of membranes prepared from human spleen.
All procedures were performed at 4 ° C. Frozen human spleen (200 g fragment) from a patient with hairy cell haemorrhage is placed on ice in 200 ml Tris-saline (50 mM) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 U / ml aprotinin and 5 mM iodoacetamide. Tris, 0.14M NaCl, pH 7.4) at 4 ° C. The tissue was cut into small pieces and homogenized at 4 ° C with a Techmer strong homogenizer. The volume was adjusted to 300 ml with Tris-brine, and 100 ml of 10% Tween 40 (polyoxyethylene sorbitan monopalmitate) in Tris-brine was added to obtain Tween 40 with a final concentration of 2.5%.
To prepare the membrane, the homogenate was extracted using a 3-stroke Dounce or more preferably a Teflon Potter Elb Jam homogenizer and then centrifuged at 1000 × g for 15 minutes. The supernatant was left and the pellet was re-extracted with 250 ml of 2.5% Tween 40 in Tris-saline. After centrifugation at 1000 xg for 15 minutes, the supernatants from both extracts were combined and centrifuged at 150,000 xg for 1 hour to pellet the membrane. The membrane was washed by resuspension in 200 ml Tris-saline and centrifuged at 150,000 × g for 1 hour. The membrane pellet was resuspended in 200 ml Tris-saline and homogenized with a motor-driven homogenizer and Teflon pestol until the suspension became dense. The volume was then brought to 900 ml with Tris-saline and N-lauroyl sarcosine was added to a final concentration of 1%. After stirring for 30 minutes at 4 ° C., insoluble material in the detergent lysate was removed by centrifugation at 150,000 × g for 1 hour. Triton X-100 was added to the supernatant to a final concentration of 2% and the lysate was stirred at 4 ° C. for 1 hour.
Solubilization of JYB-lymphoblast cells
The EBV transformed B-lymphoblast cell line JY is 0.8-1.0 x 10 in RPMI 1640 containing 10% fetal calf serum (FCS) and 10 mM HEPES.⁶Grown to a density of cells / ml. To increase cell surface expression of ICAM-1, phorbol 12-myristate 13-acetate (PMA) was added at 25 ng / ml for 8-12 hours before harvesting the cells. Sodium vanadate (50 μM) was also added to the culture during this time. Cells were pelleted by centrifugation at 500 xg for 10 minutes and washed twice by resuspension and centrifugation in Hans Balanced Salt Solution (HBSS). Cells (approx. 5 g per 5 l culture) in 50 ml lysis buffer (0.14 M NaCl, 50 mM Tris, pH 8.0, 1% Triton X-100, 0.2 μ / ml aprotinin, 1 mM MPMSF, 50 μM sodium vanadate) It was dissolved by stirring for 30 minutes at 4 ° C. Undissolved nuclei and insoluble debris were removed by centrifugation at 10,000 xg for 15 minutes, followed by centrifugation of the supernatant at 150,000 xg for 1 hour and filtration of the supernatant through Whatman 3 mm filter paper.
Affinity chromatography of ICAM-1 for structural studies
For large scale purification of ICAM-1 used for structural studies, a 10 ml RR1 / 1-Sepharose CL-4B column (bound with 2.5 mg antibody / gel ml) and CNBr-activated, glycine stabilized Two precolumns, Sepharose CL-4B and rat IgG-conjugated Sepharose CL-4B (2 mg / ml) were used. Each column was connected in series and pre-washed with 10 column volumes of lysis buffer, 10 column volumes of pH 12.5 buffer (50 mM triethylamine, 0.1% Triton X-100, pH 12.5 at 4 ° C.) and 10 column volumes Of lysis buffer. 1 l of human spleen detergent lysate was loaded at a flow rate of 0.5-1.0 ml / min. Two precolumns were used to remove non-specific binding material from the lysate before passing through the RR1 / 1-Sepharose column.
After loading, RR1 / 1-Sepharose column and bound ICAM-1 were (1) lysis buffer, (2) 20 mM Tris pH 8.0 / 0.14 M Nacl / 0.1% Triton X-100, (3) 20 mM Glycine pH 10.0 / 0.1% Triton X-100 and (4) 50 mM triethylamine pH 11.0 / 0.1% Triton X-100 were each washed sequentially with a minimum of 5 column volumes at a flow rate of 1 ml / min. All wash buffers contained 1 mM PMSF and 0.2 μ / ml aprotinin. After washing, residual bound ICAM-1 was eluted with 5 column volumes of elution buffer (50 mM triethylamine / 0.1% Triton X-100 / 4 ° C., pH 12.5) at a flow rate of 1 ml / 3 minutes. The eluted ICAM-1 was collected in a 1 ml fraction and immediately neutralized by adding 0.1 ml of 1 M Tris, pH 6.7. The fraction containing ICAM-1 was subjected to 10 μl aliquot of SDS-polyacrylamide electrophoresis [Springer et al.J. Exp. Med. 160: 1901 (1984)] followed by silver staining [Morrissey, J. H.,Anal. Biochem. 117: 307 (1981)]. Under these conditions, the bulk of ICAM-1 eluted at about 1 column volume and was more than 90% pure as judged by silver staining electrophoresis (a small amount of IgG leaking from the affinity matrix was the main impurity. ) Fractions containing ICAM-1 were pooled and concentrated approximately 20 times using a Centricon-30 microconcentrator (Amicon, Denver, Mass.). Purified ICAM-1 was quantified by a lowry protein assay in an ethanol precipitation aliquot of the pool. About 500 μg of pure ICAM-1 could be prepared from 200 g of human spleen.
About 200 μg of purified ICAM-1 was subjected to the second stage purification by SDS-polyacrylamide gel electrophoresis for separation. The band representing ICAM-1 was visualized by soaking the gel in 1M KCl. The gel region containing ICAM-1 was examined and electroeluted by the method of “Hunkapiller et al., Meth. Enzymol. 91: 227-236 (1983)”. The purified protein was more than 98% pure as judged by SDS-PAGE and silver staining.
Affinity purification of ICAM-1 for sensory testing
ICAM-1 for sensory testing was purified from detergent lysates from JY cells as described above, but on a small scale (1 ml RR1 / 1-Sepharose column) with the following modifications. All solutions contained 50 μM sodium vanadate. After washing the column with 0.1% Triton X-100-containing pH 11.0 buffer, the column was replaced with 0.1% Triton X-100 and 1% n-octyl-beta-D-glucopyranoside (octylglucoside) Re-wash with 5 column volumes of the same buffer containing. Octyl glucoside detergent removes Triton X-100 bound to ICAM-1 and, unlike Triton X-100, can then be removed by dialysis. ICAM-1 was then eluted with pH 12.5 buffer containing 1% octyl glucoside instead of 0.1% Triton X-100, analyzed and concentrated as described above.
Example 15
Properties of purified ICAM-1
ICAM-1 purified from human spleen penetrates as a broad band of Mr 72,000 to 91,000 in SDS-polyacrylamide gel. ICAM-1 purified from JY cells also penetrates as a broad band of Mr 76,500 to 97,000. These Mrs are in the reported range of ICAM-1 immunoprecipitated from various cell sources: MR = 90,000 from JY cells, 114,000 on osteoblastic monocyte cell line U937, and fibroblasts 97,000 [Dustin et al.,J. Immunol. 137: 245 (1986)]. This wide range of Mr contributes to a wide but variable degree of glycosylation. The non-glycosylated precursor has Mr 55,000 (Dustin et al.). Protein purified from JY cells or human spleen retains its antigenic activity due to its ability to recombine to its original affinity column and as evidenced by immunoprecipitation with RR1 / 1 Sepharose and SDS-polyacrylamide electrophoresis. is doing.
To prepare a peptide fragment of ICAM-1, approximately 200 μg was reduced with 2 mM dithiothreitol / 2% SDS and then alkylated with 5 mM iodoacetic acid. Protein is precipitated with ethanol, 0.1M NH_4CO3/0.1mM CaCl₂/0.1% zwittergent 3-14 (Calbiochemi), digested with 1% w / w trypsin at 37 ° C. for 4 hours, then 1% trypsin at 37 ° C. for 12 hours Of additional digestion. The tryptic peptide was purified by reverse phase HPLC using a 0.4 × 15 cm C4 column (Vydac). The peptide was eluted with a linear gradient from 0% to 60% acetonitrile in 0.1% trifluoroacetic acid. The selected peptides were subjected to sequence analysis with a gas phase micro-sequenator (Applied Biosystems). The sequence information obtained from this test is shown in Table 5.

Example 16
Cloning of the ICAM-1 gene
The gene for ICAM-1 can be cloned using any of a variety of procedures. For example, the amino acid sequence information obtained from sequencing of the ICAM-1 trypsin fragment (Table 5) can be used to identify the oligonucleotide sequence corresponding to the ICAM-1 gene. The ICAM-1 gene can also be cloned by detecting a clone producing ICAM-1 using an anti-ICAM-1 antibody.
Cloning of the ICAM-1 gene by using oligonucleotide probes
Gene code [Waston, J. D.,Molecular Biology of the Gene,Third Edition, W. A. Benjamin, Merono Park, CA (1977)] can be used to identify one or more different oligonucleotides, each of which can encode an ICAM-1 tryptic peptide. The possibility that a specific oligonucleotide actually constitutes the actual ICAM-1 coding sequence takes into account the frequency of unusual base pairing and the actual use of specific codons in eukaryotic cells (encoding specific amino acids) Can be evaluated. Such “codon usage rules” are disclosed by “Lathe, R. et al., J. Melec. Biol. 183: 1-12 (1985)”. Using Lathe's "codon usage rule", a single containing a nucleotide sequence that can theoretically encode the "most likely" ICAM-1 tryptic peptide sequence (ie, the nucleotide sequence with the least unwanted) An oligonucleotide or set of oligonucleotides is identified.
Complementary oligonucleotides that can be hybridized to a “most likely” sequence or set of sequences using a theoretically “most likely” sequence or set of oligonucleotides that can encode an ICAM-1 fragment The sequence of the set of oligonucleotides is identified. Oligonucleotides containing such complementary sequences can be used as probes to identify and isolate the ICAM-1 gene [Maniatis, T. et al.Molecular Cloning A Laboratory Manual,Cold Spring Harbor Press, Cold Spring, NY (1982)].
As described in section C of the above document, it is possible to clone the ICAM-1 gene from a eukaryotic DNA preparation that may contain the ICAM-1 gene. In order to identify and clone the gene encoding the ICAM-1 protein, the DNA library is screened for its ability to hybridize with the above-described oligonucleotide probes. Since only two copies of the ICAM-1 gene appear to be present in normal diploid cells, the ICAM-1 gene may also have large non-transcribed intervening sequences (introns) that are not desired for cloning. Since it may be preferred, it is preferred to isolate the ICAM-1 coding sequence from a cDNA library prepared from mRNA of ICAM-1 producing cells rather than genomic DNA. An appropriate DNA or cDNA preparation is enzymatically cleaved, randomly sheared and ligated into a recombinant vector. The ability of these recombinant vectors to hybridize with the above-described oligonucleotide probes is then measured. Hybridization procedures are described in, for example, “Maniatis, T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)” or “Haymes, BT, etc., Nucleic Acid Hybridization a Practical Approach, IRL. Press, Oxford, UK (1985) ". A vector known to be capable of such hybridization is analyzed to analyze the extent and nature of the ICAM-1 sequence it contains. According to purely hypothetical considerations, a gene such as that encoding an ICAM-1 molecule could be clearly identified (by hybrid screening) using an oligonucleotide with only 18 nucleotides.
That is, in summary, the actual identification of an ICAM-1 peptide sequence allows the identification of a theoretically “most likely” DNA sequence or set of such sequences that can encode such a peptide. By constructing oligonucleotides complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the “most likely” set of oligonucleotides), the ICAM-1 gene is identified and simply A DNA molecule (or set of DNA molecules) is obtained that can function as a separating probe.
The ICAM-1 peptide sequences in Table 5 were used to determine the “most likely” sequence of oligonucleotides that could encode AA and J peptides (Tables 6 and 7, respectively). Oligonucleotides complementary to these sequences were synthesized and purified to provide probes for isolating the ICAM-1 gene sequence. Appropriately sized cDNA library from poly (A) from PMA-induced HL-60 cells and from ps-stimulated umbilical vein endothelial cells⁺Prepared from RNA. The size selection cDNA library is described in “Gubler, U. et al.Gene 25: 263-269 (1983)] and Corbi, A. et al.EMBO J.6: 4023-4028 (1987)], poly (A) from PMA-induced HL-60 cells.⁺Prepared using RNA. These documents are incorporated herein by reference.
Size-selected cDNA library is poly (A) from navel vein endothelial cells stimulated with PS 5 μg / ml for 4 hours⁺It was prepared using. RNA was extracted by homogenizing the cells in 4M guanidinium isocyanate and subjecting the supernatant to ultracentrifugation with a CsCl gradient [Chirgwin, J. M. et al.Biochem. 18: 5294-5299 (1979)]. Poly (A)⁺RNA was isolated from a total RNA space mixture using oligo (dT) -cellulose chromatography (type 3, Collaborative Research) [Aviv, H. et al.,Proc. Natl.Acad. Sci. (USA) 69: 1408-1412 (1972)].

8 μg of poly (A) for the first strand cDNA⁺Synthesized using RNA, avian osteoblastic virus reverse transcriptase (Life Sciences) and oligo (dT) primers. The DNA-RNA hybrid was digested with Ranase H (BRL), and the second strand was synthesized using DNA polymerase I (New England Biolabs). The product was methylated with an EcoRI linker (New England Biolabs), blunt-end ligated to EcoRI linker (New England Biolabs), digested with EcoRI and sized on a low melting point agarose gel. A cDNA larger than 500 bp was ligated to λgt10 previously digested with EcoRI and dephosphorylated (Stratagene). The ligation product was then packaged (Stratagene Gold).
Next, the umbilical vein endothelial cells and the HL-60 cDNA library were applied to a 20,000 PFμ / 150 mm plate. Recombinant DNA was transferred in replication to nitrocellulose filters, denatured in 0.5 M NaOH / 1.5 M NaCl, neutralized in 1 M Tris, pH 7.5 / 1.5 M NaCl, and baked at 80 ° C. for 2 hours. [Benton, WD et al., Science 196: 180-182 (1977)]. Filter pre-hybridized, 5x Denhardt solution, 50 mM NaPO_FourAnd 5 × SSC containing 1 μg / ml salmon sperm DNA. Prehybridization was performed at 45 ° C. for 1 hour.
Hybridization was performed using 32 bp ('5-TTGGGCTGGGTCACAGGAGGGTGGAGCAGGGTGAC) or 47 bp (5'-GAGGGTTCTCAAACAGCTCCAGGCCCCTGGGGCCCGCAGGTCCCAGCTC) antisensory oligonucleotides on Table ICAM-1 and Table 7 on ICAM-1 [Lathe,RJ Melec. Biol. 183: 1-12 (1985)]. The oligonucleotide is r- (³²P) T at ATP^FourEnd labeling was performed using polynucleotide kinase and conditions recommended by the manufacturer (New England Biolabs). Following overnight hybridization, the filter was washed twice with 2 × SSC / 0.1% SDS for 30 minutes at 45 ° C. Phages were isolated from plaques indicating hybridization and purified by sequential re-application and re-screening.
Cloning of ICAM-1 gene by using anti-ICAM-1 antibody
The ICAM-1 gene can alternatively be cloned by the use of an anti-ICAM-1 antibody. DNA or more preferably cDNA is extracted and purified from cells capable of expressing ICAM-1. Purified cDNA is fragmented (by shearing, endonuclease digestion, etc.) to prepare a pool of DNA or cDNA fragments. A DNA or cDNA fragment from this pool is cloned into an expression vector to prepare an expression vector gene library in which each member contains a specific cloned DNA or cDNA fragment.
Example 17
Analysis of cDNA clones
Phage DNA from positive clones was digested with EcoRI and tested by Southern analysis using cDNA from one clone as a probe. The cross-hybridized maximum size cDNA insert was subcloned into the EcoRI site of plasmid vector pGEM4Z (Promega). HL-60 subclone containing cDNA in both orientations was digested with endonuclease III [Henikoff, s.,Gene 28: 351-359 (1984)] according to the manufacturer's recommendations (Eraser Base, Bromega). Gradually missing cDNA was then cloned and subjected to dideoxynucleotide chain termination sequencing according to manufacturer's recommendations (Sequenase, US Biochemical) [Sanger, F. et al.,Proc.Natl.Acad.Sci (USA) 74: 5463-5467 (1977)]. The HL-60 cDNA 5 'and coding region were sequenced completely on both strands, and the 3' region was sequenced approximately 70% on both strands. A representative endothelial cDNA was sequenced by shotgun cloning of a 4 bp recognition restriction fragment over most of its length.
The cDNA sequence of one HL-60 and one endothelial cell cDNA was established (FIG. 8). The 3023 bp sequence is a short 5 'untranslated region and a 1.3 kb 3' unshared signal with a common polydehilation signal at position 2966. Includes translation area. The longest open reading frame begins at the first ATG at position 58 and ends with the TGA end triplet at position 1653. The identity between the translational amino acid sequence and the sequence determined from 8 different tryptic peptides of a total of 91 amino acids (underlined in FIG. 8) is isolated from a reliable ICAM-1 cDNA clone Confirmed that it was. The amino acid sequence of the ICAM-1 tryptic peptide is shown in Table 8.

Hydrophobic analysis (Kyte, J. et al.,J. Melec. Biol.157: 105-132 (1982)] suggests the presence of a 27 residue signal sequence. The role of +1 glutamine is consistent with the inability of the present invention to obtain an N-terminal sequence on three ICAM-1 protein preparations; glutamine has a blocked N-terminus that has been cyclized and blocked by phloglutamic acid. Bring. The translation sequence from 1 to 453 is mainly hydrophilic, followed by a hydrophobic putative transmembrane domain of 24 residues. The transmembrane domain is immediately followed by several charged residues contained within a 27 residue putative cytoplasmic domain.
The predicted size of the mature polypeptide chain is 55,219 daltons, in good agreement with the observed size of deglycosylated ICAM-1 55,000 [Dustin, M. L. et al.J. Immunol.137: 245-254 (1986)]. Eight N-linked glycosylation sites are predicted. The absence of asparagine in the two tryptic peptide sequences at these sites confirms their glycosylation and their extracellular orientation. Assuming 2,500 daltons per high mannose N-linked carbohydrate, a size of 75,000 daltons was predicted for the ICAM-1 precursor and the observed size of 73,000 daltons [Dustin, ML et al., J. Immunol 137: 245-254 (1986)]. After conversion of high mannose to complex carbohydrate, mature ICAM-1 glycoprotein is 76-114 Kd depending on the cell type [Dustin, M. L. et al., J. Immunol. 137: 245-254 (1986)]. That is, ICAM-1 is a highly complete glycosylated but typical complete membrane protein.
Example 18
ICAM-1 is an integrin-binding member of the immunoglobulin supergene family:
ICAM-1 internal repeats were performed by subsequent inspection using the Microgenie protein sequence program [Queen, C. et al., Nucl. Acid Res. 12: 581-599 (1984)]. ICAM-1 was sequenced into IgM, N-CAM and MAG using the Microgeni and ALIGN programs [Daypoff, M. O. et al., Meth. Enzmol. 91: 524-545 (1983)]. Four protein sequence databases stored at the National Biomedical Research Foundation were investigated for protein sequence similarity using Williams and Pearson's FASTP program [Lipman, D. J. et al.,Science 227: 1435-1439 (1985)].
Since ICAM-1 is a ligand for integrin, it was not expected to be a member of the immunoglobulin supergene group. However, a survey of the ICAM-1 sequence shows that the sequence meets all the criteria proposed for relatives in immunoglobulin supergenes. These criteria are described below.
The entire extracellular domain of ICAM-1 is constructed from the five homologous immunoglobulin-like domains shown in sequence in FIG. 9A. Domains 1-4 are 88, 97, 99 and 99 residues long, respectively, and thus have a typical Ig domain size; domain 5 is truncated in 68 residues. Investigation of the NBRF database using the FASTP program reveals significant homology with members of the immunoglobulin supergene family, including IgM and IgG C domains, T cell receptor α subunit variable domains, and alpha 1 beta glycoproteins. (FIG. 9B-D).
Using the above information, the amino acid sequence of ICAM-1 was compared with the amino acid sequence of the other member of the immunoglobulin supergene group.
Three types of Ig supergene group domains, V, C1 and C2, are differentiated. Both V and C domains are constructed from 2β-sheets joined together by internal domain disulfide bonds; the V domain has 9 antiparallel β-strands and 7 C domains. The constant domain was divided into C1 and C2-sets based on the characteristic residues shown in FIG. 9A. The C1-set contains proteins that are included during antigen recognition. C2-set is several F_cIt contains receptors and proteins contained in cell adhesion including CD2, LFA-3, MAG and NCAM. The ICAM-1 domain was found to have the strongest homology with the C2-set domain that replaced ICAM-1 in this set; this is shown for β-strand BF in FIG. This is reflected in the fact that the retention residue in C2 is more similar than the C1 domain. Also, the ICAM-1 domain is better aligned with β strands A and G of the C2 domain than with the above strands in the V and C1 domains, allowing for better alignment across the entire C2 domain strength. Sequences with the C2 domain from NCAM, MAG and alpha 1-β glycoprotein are shown in FIGS. 9B and 9C; identity ranged from 28-33%. The sequence with T cell receptor Vα 27% identity and IgM C domain 3 34% identity is also shown (FIGS. 9B, 9D).
One of the most important features of the immunoglobulin domain is the disulfide-bonded cysteine that crosslinks the B and Fβ strands that stabilize the β-sheet sandwich; in ICAM-1, the cysteine is retained in all cases, but the domain In 4 strands f, leucine is found that faces the sandwich and stabilizes the contact as proposed in several other V- and C2-set domains. The distance between cysteine (43, 50, 52 and 37 residues) is as described for the C2 set.
To test for the presence of interchain disulfide bonds in ICAM-1, endothelial cells ICAM-1 were subjected to SDS-PAGE under reducing and non-reducing conditions. Endothelial cell ICAM-1 was used because it shows less glycosylated heterogeneity than JY or hairy spleen cell ICAM-1 and is more sensitive to shifts in Mr. Therefore, ICAM-1 was purified from LPS (5 μg / ml) stimulated umbilical vein endothelial cell culture for 16 hours by Inom affinity chromatography as described above. Sample buffer [Laemmli, U. K., containing 0.25% 2-mercaptoethanol or 25 mM iodoacetamide with acetone-precipitated ICAM-1Nature 227: 680-685 (1970)] and brought to 100 ° C. for 5 minutes. Samples were subjected to SDS-PAGE 4670 and silver stain 4613. Endothelial cells ICAM-1 showed an apparent Mr of 100 kd under reducing conditions and 96 kd under non-reducing conditions, strongly suggesting the presence of interchain disulfides in native ICAM-1.
Use of primary sequence to predict secondary structure
[Chon, P. Y., etc.Biochem. 13:211-245 (1974)] is labeled a-g at the top of FIG. 9A and accurately fulfills the expectation for the immunoglobulin domain and at each position of strands A-H in the immunoglobulin (bottom of FIG. 9A). The seven expected β-strands in each corresponding ICAM-1 domain were shown. Domain 5 lacks the A and C strands, but since they form the end of the sheet, each sheet is still probably formed by strand D and was proposed in several other C2 domains As a surrogate for strand C; and the characteristic disulfide bond between the B and F strands will not be affected. That is, domain size, sequence homology, retention cysteines that form putative interdomain disulfide bonds, the presence of disulfide bonds, and the expected β-sheet structure criteria all meet the ICAM-1 intrinsic in immunoglobulin supergenes Yes.
ICAM-1 was found to be most strongly homologous to the C2 set of NCAM and MAG glycoproteins. This is particularly interesting because both NCAM and MAG mediate cell-cell adhesion. NCAM is important in neuron-neuron and nerve-muscle interactions [Cunningham, B. A. et al.,Science 236799-806 (1987)], and MAG is important in neuron-oligodentrosite and oligodentrosite-oligodentrosite interactions during myelination [Poltorak, M. et al.,J. Cell. Biol. 105; 1893-1899 (1987)]. Cell surface expression of NCAM and MAG is evolutionarily regulated, similar to the coordinated induction of ICAM-1 in inflammation, respectively, during neurogenesis and myelination [Springer, T.A. et al.Ann. Rev.Immunol. 5: 223-252 (1987)]. ICAM-1, NCAM [Cunningham, BA, etc.,Science 236: 799-806 (1987)], and MAG [Salzer, J. L. et al.,J. Cell. Biol. 104: 957-965 (1987)] are similar and homologous in overall structure because each is a complete membrane glycoprotein composed of five C2 domains that form the N-terminal extracellular region. It is. However, in NCAM, some additional non-Ig-like sequences exist between the last C2 domain and the transmembrane domain. ICAM-1 is lined up with 21% identity with MAG over its entire length including the transmembrane and cytoplasmic domains; the same percent identity identifies the five domains of ICAM-1 and NCAM-1. Found when compared. A schematic comparison of the secondary structures of ICAM-1 and MAG is shown in FIG. Domain-to-domain comparison shows the level of homology between ICAM-1 and domains within the NCAM molecule (x ± sd21 ± 2.8% and 18.6 ± 3.8%, respectively) And the same level of homology as compared to the MAG domain (20.4 ± 3.7 and 21.9 ± 2.7, respectively). NCAM [Cunningham, B. A., etc.Science 236: 799-806 (1987)]; Barthels, D. et al.,EMBO J.M. 6: 907-914 (1987)] and MAG [Lai, C. et al.,Proc. Natl. Acad. Sci. (USA) 84: 4377-4341 (1987)], although there is evidence of additional conjugation in the C-terminal region, this may be in the sequencing of endothelium or HL-60 ICAM-1 clones or various types of ICAM-1 protein backbones. And precursor research (Dustin, ML, etc.J. Immunol. 137: 245-254 (1986)].
ICAM-1 functions as a ligand for LFA-1 in lymphocyte interactions with many different cell types. Lymphocytes bind to ICAM-1 contained in the artificial membrane bilayer, which requires LFA-1 on lymphocytes and directly suggests an interaction between ICAM-1 and LFA-1 (Marlin, SD Et al., Cell 51: 813-819 (1987)].
LFA-1 is a leukocyte integrin and does not have immunoglobulin-like characteristics. Leukocyte integrins contain one integrin subfamily. The other two subfamilies mediate cell-matrix interactions and recognize the sequence RGD within its ligand, including fibronectin, vitronectin, collagen and fibrinogen [Hynes, R.O.,Cell 48: 549-554 (1987)]; Ruoslahti, E. et al.,Science 238: 491-497 (1987)]. Leukocyte integrin is expressed only on leukocytes and is involved in cell-cell interactions, and only a few known ligands are ICAM-1 and iC3b (ie, a fragment of complement component C3 that does not show immunoglobulin-like characteristics) -1 [Kishimoto, TK, etc.Leukocyte Typing III, McMi Chael, M., Springer Bellag, New York (1987); Springer, T.A., etc.Ann. Rev. Immunol. 5: 223-252 (1987); Anderson, D. C. et al.Ann. Rev.Med. 38175-194 (1987)]. The potential peptides within the ICAM-1 sequence recognized by LFA-1 by sequence analysis are shown in Table 9.

ICAM-1 is the first example of a member of the immunoglobulin supergene group that binds to integrins. Although both of these groups play an important role in cell adhesion, the interaction between them was not previously expected. In contrast, interactions within immunoglobulin supergenes are quite common. It is entirely possible that further examples of interactions between integrins and immunoglobulin groups will be discovered. LFA-1 recognizes a ligand different from ICAM-1 [Springer, T.A., etc.Ann. Rev. Immunol.5: 223-252 (1987)], leukocyte integrin Mac-1 recognizes a ligand different from C3bi of neutrophil-neutrophil adhesion [Anderson, D.C. et al.,Ann. Rev. Med. 38175-194 (1987)]. Furthermore, the purified MAG-containing vesicles (vesicles) bind to the MAG neurites and thus MAG must be capable of heterophilic interactions with heterologous receptors [Poltorak, M. et al.J. Cell. Biol. 105: 1893-1899 (1987)].
It has been suggested that the role of NCAM in nerve-nerve and nerve-muscle cell interactions is based on homophilic NCAM-NCAM interactions [Cunningham, B.A. et al.Science 236: 799-806 (1987)]. The critical role of MAG in the interaction between adjacent rotation loops of Schwuan cells that take up axons during myelin sheath formation may be based on interaction with heterologous receptors or homophilic MAG-MAG interaction. Homology with NCAM and frequent appearance of domain-domain interactions within immunoglobulin supergenes may allow ICAM-1 to be involved in homophilic interactions as well as ICAM-1-LFA-1 heterophilic interactions Causes sex. However, binding of B lymphoblasts co-expressing similar densities of LFA-1 and ICAM-1 to ICAM-1 in an artificial or cell monolayer is pretreatment of B-lymphoblasts with LFA-1 MAb Can be completely suppressed by, but adhesion is not affected by B-lymphoblast pretreatment with ICAM-1 MAb. Single layer pretreatment with ICAM-1 Mab completely destroys the binding [Dustin, M.L., et al.,J. Immunol137: 245-254 (1986); Marlin, S.D. et al.Cell,51: 813-819 (1987)]. These findings indicate that if an ICAM-1 homophilic interaction occurs at all, the effect must be considerably weaker than the heterophilic interaction with LFA-1.
The possibility that leukocyte integrins recognize ligands in a fundamentally different way is important in ligand binding and is consistent with the presence of a 180-residue sequence in their alpha subunit that is not present in RGD-recognizing integrins [Corbi, A. et al., EMBO J. et al. 6: 4023-4028 (1987)]. Although Mac-1 has been proposed to recognize the RGD sequence present in iC3b5086, there is no RGD sequence in ICAM-1 (FIG. 8). This is consistent with the inability of fibronectin peptide GRGDSP and control peptide GRGESP to suppress ICAM-1-LFA-1 adhesion [Marlin, S.D. et al.Cell.51: 813-819 (1987)]. However, related sequences such as PRGGS and RGEKE are present in ICAM-1 in the regions predicted for the loops between β-strands a and b of domain 2 and c and d of domain 2 (No. 1). 9), and therefore can be accepted for recognition. Interestingly, the homologous MAG molecule contains an RGD sequence between domains 1 and 2 [Poltorak, M. et al.J. Cell. Biol. 1051893-1899 (1987); Salzer, J.L.J. Cell. Biol. 104: 957-965 (1987)].
Example 19
Southern and Noisan blots
A Southern blot was performed with 5 μg of genetic DNA extracted from three cell lines: BL2, Burkitt lymphoma cell line (provided by Dr. Gilbert Lenoir); JY and Er-LCL EBV transformed B-lymphoma Like cell line.
Each DNA was digested with 5 × manufacturer recommended amounts of BamH1 and EcoRI endonuclease (New England Biolabs). Following electrophoresis on a 0.8% agarose gel, the DNA was transferred to a nylon membrane (Zeta Probe, Biorad). Filter prehybridized and α- (³²P) Hybridized by random priming according to standard procedures using ICAM cDNA from HL-60 labeled with dXTP's. Nousarn blot 20 μg total DNA or 6 μg poly (A)⁺Performed with RNA. The RNA was denatured, electrophoresed on a 1% agarose-formaldehyde gel, and electrophoresed to a zeta probe. Pre-hybridize each filter³²Hybridized as described above using P-labeled oligonucleotide probe (described above) HL-60 cDNA probe (Stauton, D.E. et al.,Embo J.6: 3695-3701 (1987)].
Southern blots using a 3 kb cDNA probe and genetic DNA digested with BamH1 and EcoR1 each show a single gene and single major hybrids of 20 and 8 kb indicating that most of the coding information is within 8 kb Sex fragment was shown. There was no evidence of restriction fragment polymorphism in the blots of the three cell lines.
Example 20
Expression of ICAM-1 gene
An “expression vector” can express DNA (or cDNA) cloned into a vector (based on the presence of appropriate transcriptional and / or translational control sequences), thereby producing a polypeptide or protein. It is a vector. Expression of the cloned sequence occurs when the expression vector is incorporated into a suitable host cell. If a prokaryotic expression vector is used, a suitable host cell will be any prokaryotic cell capable of expressing the cloned sequence. Similarly, when using eukaryotic expression vectors, a suitable host cell is any eukaryotic cell capable of expressing the cloned sequence. Importantly, since eukaryotic DNA can contain intervening sequences and such sequences cannot be processed accurately in prokaryotic cells, expressing ICAM-1 to produce a prokaryotic genomic expression vector library It is preferred to use cDNA from cells that can. Methods for preparing cDNA and generating genomic libraries have been disclosed by Maniatis, J. et al. [Molecular Cloning:A Laboratory ManualCold Spring Harbor Press, Cold Spring Harbor, NY (1982)].
A bank of host cells is created using the expression vector gene library described above (each of which contains a member of the library). Expression vectors can be incorporated into host cells by any of a variety of means (ie, transformation, transfection, protoplast fusion, electroporation, etc.). A bank of expression vector-containing cells is grown clonally and each member is assayed individually (using an immunoassay) to determine if they produce a protein that can bind to an anti-ICAM-1 antibody.
Expression vectors of cells producing proteins that can bind to anti-ICAM-1 antibodies are further analyzed to determine whether these vectors express (or contain) the entire ICAM-1 gene, only fragments of the ICAM-1 gene. It is determined whether it is expressed (or contained) or whether it expresses (or contains) a gene that is not ICAM-1 even though the product is immunologically related to ICAM-1. Although such analysis may be done in any convenient way, it is preferred to determine the nucleotide sequence of the DNA or cDNA that is cloned into the expression vector. Such a nucleotide sequence is examined to determine if it can encode a polypeptide having the same amino acid sequence as a trypsin digested fragment of ICAM-1 (Table 5).
An expression vector comprising a DNA or cDNA molecule encoding the ICAM-1 gene thus has (i) the ability to express a protein capable of binding to an anti-ICAM-1 antibody; and (ii) a trypsin fragment of ICAM-1 It can be recognized by the presence of a nucleotide sequence that can encode each. Cloning DNA molecules of such expression vectors can be removed from the expression vector and isolated in pure form.
Example 21
Functional activity of purified ICAM-1
In cells, ICAM-1 normally functions as a surface protein associated with the cell membrane. Thus, the function of purified ICAM-1 was tested after reconstituting the molecule by dissolving the protein in an artificial lipid membrane (liposomes or vesicles) in the protein in the detergent solubilized lipid and then removing the detergent by dialysis. . ICAM-1 purified from JY cells and eluted in detergent octylglycoside as described above is reconstituted in vesicles, and the ICAM-1-containing vesicles are fused to glass coverslips or plastic culture wells to bind to proteins. Enabled to detect cells.
Preparation of flat membrane and plastic bonded vesicles
Vesicles were prepared by the method of Gay et al.J. Immunol, 136: 2026 (1986)]. That is, egg phosphatidyl chlorin and cholesterol were dissolved in chloroform and mixed at a molar ratio of 7: 2. The lipid mixture was dried to a thin film while rotating under a stream of nitrogen gas and then lyophilized for 1 hour to remove all traces of chloroform. The lipid membrane was dissolved in 1% octylglycoside / 0.14M NaCl / 20 mM Tris (pH 7.2) to a final concentration of phosphatidylchlorin of 0.1 mM. Approximately 10 μg of purified ICAM-1 or human glycophorin as a control membrane glycoprotein (Sigma Chemical Co., St. Louis, MO) was added to each ml of dissolved lipid. The protein-lipid-detergent solution was dialyzed at 4 ° C. against 3 changes of 200 volumes of 20 mM Tris / 0.14 M NaCl, pH 7.2 and 1 change of HBSS.
The flat film was prepared by the method of Brian et al.Proc.Natl. Acad. Sci. 81: 6159 (1984)]. Glass cover slips (11 mm diameter) were boiled for 15 minutes in a 1: 6 dilution of 17 × Detergent (Linbro), washed overnight in distilled water, immersed in 70% ethanol and allowed to air dry. An 80 μl drop of vesicle suspension containing either ICAM-1 or cricophorin was placed in the well bottom of a 24-well Kurster plate and the glass cover slip prepared above was gently floated on top. After a 20-30 minute incubation at room temperature, each well was filled with HBSS and the coverslip was turned over so that the flat side was facing up. The wells were then washed thoroughly with HBSS to remove unbound vesicles. The flat membrane surface was not exposed to air at all.
During the experiment with a flat membrane fused to the glass surface, the vesicle containing ICAM-1 binds directly to the plastic surface of the multiwell tissue culture plate and retains the functional activity as revealed by specific cell binding. There was found. Such vesicles are hereinafter referred to as “plastic bound vesicles (PBV), because they do not measure the properties of lipid vesicles bound to plastic. Plastic bound vesicles directly apply 30 μl of vesicle suspension. In addition to the bottom of the wells in a 96 well tissue culture tray (Falcon), it was prepared by incubation and washing as described for the flat membrane.
Cell adhesion assay
Both cell adhesion assays using flat membranes or plastic binding vesicles were performed in essentially the same way, but the cell number and volume of the PBV assay was reduced to 1/5 that used in the flat membrane assay.
Normal control and leukocyte adhesion deficiency (LAD) patients who cannot express LFA-1 [Anderson, D.C., etc.J. Infect. Dis.152: 668 (1985)] in a peripheral blood mononuclear cell RPMI-1640 + 20% FCS containing 1 μg / ml concanavalin-A (Con-A).^FivePrepared by culturing at cell / ml for 3 days. Cells were then washed twice with RPMI; once with 5 mM methyl-alpha-b-manophilaside to remove residual lectin from the cell surface. Cells were grown in RPMI / 20% FCS containing 1 ng / ml recombinant IL-2 and used between 10 and 22 days after the start of culture.
Con A blast, T-lymphoma SKW-3, EBV transformed B-lymphoma-like cell line JY (LFA-1 positive) and LFA-1 deficient lymphoma-like cell line to detect cells that bind to flat membrane or RBV (BBN) [From Patient 1, Springer, TA, etc.J. Exper. Med. 160: 191-1918 (1986)] in 1 ml RPMI-1640 / 10% FCS⁷Cells were washed with 100 μCi Na⁵¹CrO_FourIncubated for 1 hour at 37 ° C., then washed 4 times with RPMI-1640 to remove unbound label and radiolabeled. In the monoclonal antibody blocking test, cells or plastic binding vesicles are pretreated with 20 μg / ml purified antibody in RPMI-1640 / 10% FCS for 30 minutes at 4 ° C. and then washed 4 times to remove unbound antibody. did. In experiments on the effect of divalent ions on cell binding, cells are²⁺, Mg²⁺Washed once with HBSS + 10% dialyzed FCS without CaCl and MgCl added to the determined concentration. In all experiments, cells and flat membranes or PBV were pre-equilibrated in the appropriate assay buffer at the appropriate temperature (4 ° C., 22 ° C. or 37 ° C.).
To measure cells that bind to purified ICAM-1⁵¹Cr-labeled cells (5 × 10 in flat membrane assay^FiveEBV transformants; 1 × 10 in PBV assay^FiveEBV transformant or SKW-3 cell, 2 × 10^FiveCon-A blasts) were centrifuged at 25 × g for 2 minutes on flat membranes or PBV and then incubated at 4 ° C., 22 ° C. or 37 ° C. for 1 hour. After incubation, unbound cells were removed by 8 cycles of pre-equilibrated buffer loading and aspiration at the appropriate temperature. Bound cells were quantified by solubilizing the well contents with 0.1N NaOH / 1% Triton X-100 and counting with a gamma counter. % Cell binding was determined by dividing cpm from the bound cells by the cpm of the input cells. In the flat membrane assay, the input cpm was collected against the ratio of the coverslip surface area compared to the surface area of the culture well.
In these assays, EBV-transformed B-lymphoma cells, SKW-3T-lymphoma cells, and Con-A T lymphoblasts specifically bound to ICAM-1 in the artificial membrane (FIGS. 11 and 12). ). Binding was specific because the cells bound very poorly to control flat membranes or vesicles containing equivalent amounts of other human cell surface glycoprotein glycophorins. Furthermore, LFA-1 positive EBV transformants and Con A blasts also bound, but their LFA-1 negative equivalents did not bind to any significant degree, and the binding was due to the presence of LFA-1 on the cells. Showed that it depends.
Both cell binding specificity and dependence on cellular LFA-1 were confirmed in a monoclonal antibody blocking test (FIG. 13). Binding of JY cells could be suppressed to 97% when ICAM-1-containing PBV was pretreated with anti-ICAM-1 monoclonal antibody RR1 / 1. Pretreatment of cells with the same antibody had little effect. Conversely, anti-LFA-monoclonal antibody RS1 / 18 inhibited binding by 96%, but only when cells were pretreated instead of PBV. Control antibody TS2 / 9 (different lymphocyte surface antigen) reactive with LFA-3 had no significant inhibitory effect when either cells or PBV were pretreated. This experiment shows that ICAM-1 itself without some amount of impurities in the artificial membrane mediates the observed cell adhesion and that adhesion is dependent on LFA-1 on the binding cells.
Cell binding to ICAM-1 in the artificial membrane also showed two other properties of the LFA-1-dependent adhesion system: temperature dependence and the need for divalent cations. As shown in FIG. 14, Con-A blasts bound ICAM-1 in PBV most effectively at 37 ° C., partially at 22 ° C., and very poorly at 4 ° C. As shown in FIG. 15, the binding is completely dependent on the presence of divalent cations. At physiological concentrations, Mg²⁺Showed the best cell binding by itself, but Ca²⁺Alone showed very low levels of binding. However, C²⁺1/10 the normal concentration of Mg combined with²⁺Had a synergistic effect and showed the best binding.
In summary, the specificity of cell binding to purified ICAM-1 contained in artificial membranes, specific inhibition by monoclonal antibodies, and the need for temperature and divalent cations are related to ICAM-1 being LFA-1-dependent It shows that it is a specific ligand of the system.
Example 22
Expression of ICAM-1 and HLA-DR in allergic and toxic patch test reactions
Five normal skin biopsies were performed for their ICAM-1 and HLA-DR expression. It was found that endothelial cells in certain blood vessels normally express ICAM-1, but keratinocytes from normal skin do not express ICAM-1. No staining of HLA-DR on any keratinocytes from normal skin biopsy was observed. The expression kinetics of ICAM-1 and class II antigens were examined in allergic and toxic skin injury biopsy cells. Half of the 6 subjects examined were found to have keratinocytes expressing ICAM-1 4 hours after application of the hapten (Table 10). The exposure time to the hapten increased the percentage of people expressing ICAM-1 on keratinocytes and also increased the intensity of staining showing more ICAM-1 expression per keratinocyte by 48 hours. In fact, at this point, a portion of all biopsy keratinocytes stained positive for ICAM-1. At 72 hours (24 hours after hapten removal), 7 of 8 subjects expressed ICAM-1 in keratinocytes, and ICAM-1 expression of one subject was between 48 and 72 hours I became weak.

Histologically, the stained image of ICAM-1 on keratinocytes from a biopsy taken 4 hours after application of the hapten was usually a small group. After 48 hours, ICAM-1 was expressed on most surfaces of keratinocytes and there was no difference between the center and periphery of the injury. The staining intensity decreased when keratinocytes approached the nail stratum corneum. This was found in biopsies taken from the center and periphery of the injury. Also at this time, the patch test was positive (wet, erythema, vesicle). There was no difference in ICAM-1 expression when different haptens were applied to susceptible individuals. In addition to keratinocytes, ICAM-1 was also expressed on several mononuclear cells and endothelial cells at the injury site.
The expression of HLA-DR on keratinocytes of allergic skin injury was less frequent than that of ICAM-1. None of the subjects studied had any damage due to keratinocytes that stained positive for HLA-DR by 24 hours after hapten application. In fact, only four biopsy samples had keratinocytes that expressed HLA-DR, and no biopsy had keratinocytes positive for HLA-DR but not positive for ICAM-1 (Table 10). ).
In contrast to allergic patch test injuries, toxic patch test injuries induced with mustard oil or sodium lauryl sulfate had keratinocytes with little ICAM-1 on their surface at all time points in the test ( Table 11). In fact, 48 hours after patch application, this is the optimal time for allergic patch test subjects, but one of 14 toxic patch test subjects expressed keratin during injury. Had cells. Also, in contrast to allergic patch test biopsies, HLA-DR was not expressed on keratinocytes in toxic patch test injury.
These data indicate that ICAM-1 is expressed in immune system inflammation and not in toxic system inflammation, thus whether ICAM-1 expression is due to rejection of the immunosuppressive therapeutic agent or urine toxicity It can be used to distinguish between immune and toxic inflammation such as acute kidney disease in wise transplant patients who are difficult to judge. Evaluation of renal biopsy and improved ICAM-1 expression will allow differentiation between immune system rejection and non-immune system toxic reactions.

Example 23
Expression of ICAM-1 and HLA-DR in benign skin disease
Cells from skin biopsies of injury from patients with various types of inflammatory skin diseases were examined for their expression of ICAM-1 and HLA-DR. One part of keratinocytes in biopsies of allergic contact eczema, pemphigus and lichen planus expressed ICAM-1. Lichen planus showed the strongest staining with an image comparable or somewhat stronger than the results seen in the 48-hour allergic patch test biopsy (Table 12). Similar to the results of the allergic patch test, the strongest ICAM-1 staining was seen at sites of high mononuclear cell infiltration. Furthermore, 8 of the 11 lichen planus biopsies tested were positive for HLA-DR expression on keratinocytes.
The expression of ICAM-1 on keratinocytes from skin biopsy of patients with rash and urticaria was low. Only 4 out of 7 patients tested with these diseases had keratinocytes expressing ICAM-1 at the injury site. HLA-DR expression was only in one patient and was associated with ICAM-1.
Endothelial cells and mononuclear cell infiltrates from all of the benign inflammatory skin diseases tested expressed ICAM-1 in varying degrees.

Example 24
ICAM-1 expression on keratinocytes of psoriatic skin disease
ICAM-1 expression during skin biopsies from 5 patients with psoriasis was examined periodically before and during treatment with PUVA treatment. Biopsies were obtained from 5 patients with classic psoriasis confirmed by histology. Biopsies were taken continuously prior to PUVA treatment and during the indicated time. PUVA was administered 3-4 times a week. Biopsies were taken around the psoriatic plaques of 5 patients and in addition to biopsies were taken from 4 clinically normal skins of these patients.
Each fresh skin biopsy sample was frozen and stored in liquid nitrogen. 6 micron stock sections were air dried overnight at room temperature, fixed in acetone for 10 minutes, stained immediately or stored at −80 ° C. until wrapped in aluminum foil and stained.
Staining was made by the following method. Incubate sections with monoclonal antibody and diaminobenzidine H₂O₂The substrate was stained by a three-step immunoperoxidase method [Stein, H. et al.Adv. Cancer Res. 42: 67-147, (1984)]. Tonsils and lymph nodes were used as positive controls for anti-ICAM-1 and HLA-DR staining. Tissue stained in the absence of primary antibody was a negative control.
Monoclonal antibodies against HLA-DR were purchased from Becton Dickinson (Mountain View, Calif.). The anti-ICAM-1 monoclonal antibody was R6-5-D6. Peroxidase-conjugated rabbit anti-mouse Ig and peroxidase-conjugated pig anti-rabbit Ig were purchased from DAKAPATTS, Copenhagen, Sweden. Diaminobenzidine-tetrahydrochloride was obtained from Sigma (St. Louis).
The results of the study showed that several vascular endothelial cells expressed ICAM-1 in both disease and normal skin, but the staining intensity and the number of vessels expressing ICAM-1 were within psoriatic skin lesions. Increased. Furthermore, the expression pattern of ICAM-1 in keratinocytes of untreated psoriatic skin lesions from 5 patients changed slightly from a small group of cell stains to a large number of keratinocytes stained. During the course of PUVA treatment, ICAM-1 expression in 2 patients (patients 2 and 3) showed a significant decrease preceding or simultaneously with clinical relief. Patients 1, 4 and 5 decreased or increased ICAM-1 expression during PUVA treatment, respectively, correlated with clinical relief or worsening. There was no ICAM-1 expression on keratinocytes from normal skin before and after PUVA treatment. This indicates that PUVA does not induce ICAM-1 on keratinocytes from normal skin.
What should be noted was that the mononuclear cell infiltration density correlated with the expression level of ICAM-1 on keratinocytes. This also reduces the number of injured mononuclear cells during PUVA treatment when ICAM-1 expression also weakens and mononuclear cells during PUVA treatment when ICAM-1 expression on keracene cells becomes more prominent Is related to both increasing the number of Endothelial cells and skin mononuclear cells are also ICAM-1 positive. In clinically normal skin, ICAM-1 expression was confirmed in endothelial cells without labeling of keratinocytes.
The expression of HLA-DR on keratinocytes was variable. There were no HLA-DR positive biopsies that were not ICAM-1 positive.
In summary, these results indicate that, prior to treatment, ICAM-1 expression is high on keratinocytes and correlates with mononuclear cell infiltration density. During PUVA treatment, a significant decrease in ICAM-1 staining is seen in parallel with clinical improvement. Histologically, skin infiltration had disappeared. When clinical deterioration was seen during treatment, the expression of ICAM-1 on keratinocytes as well as skin infiltration density increased. When clinical relief was seen during treatment, there was a simultaneous decrease in ICAM-1 staining on keratinocytes as well as a decrease in skin infiltration. That is, the expression of ICAM-1 on the keratinocytes corresponded to the mononuclear cell infiltration density of the skin. These data indicate that the clinical response to PUVA treatment results in an accelerated decrease in ICAM-1 expression on keratinocytes in parallel with a more gradual decline of mononuclear cells. This indicates that ICAM-1 expression on keratinocytes is responsive to the onset and persistence of skin infiltration, and that PUVA treatment down-regulates ICAM-1 to mitigate skin invasion and inflammatory responses Yes. The data also show that the expression of HLA-DR on keratinocytes during PUVA treatment was variable.
ICAM-1 expression on psoriatic injury kerase cells correlates with injury clinical severity and skin invasion size. That is, ICAM-1 plays a central role in psoriasis and suppression of its expression and / or suppression of its interaction with the CD18 complex on mononuclear cells would be an effective treatment for this lesion. Furthermore, monitoring ICAM-1 expression on keratinocytes would be an effective tool for assessing the diagnosis, prevention, and treatment course of psoriasis.

Example 25
Expression of ICAM-1 and HLA-DR in malignant skin disease
Unlike benign skin lesions, the expression of ICAM-1 on keratinocytes from malignant skin injury was varied (Table 14). Of the 23 skin T-cell lymphomas tested, ICAM-1-positive keratinocytes were identified in only 14 cases. Keratinocytes from biopsies of mycosis fungoides lesions tended to digest their ICAM-1 expression as the disease progressed to earlier stages. However, ICAM-1 expression was seen on mononuclear cell infiltration with a changing rate from most of the skin T cell lymphoma lesions. Of the remaining lymphomas tested, 4 out of 8 had keratinocytes expressing ICAM-1. Of the 29 patients with malignant skin disease tested, 5 had keratinocytes that expressed HLA-DR without expressing ICAM-1 (Table 14).

Example 26
Effect of anti-ICAM-1 antibody on proliferation of human peripheral blood mononuclear cells
Human peripheral blood mononuclear cells are induced and propagated by the presence and recognition of antigens or mitogens. Mitogen, Concanavalin A or T cell binding antibody OKT_ThreeCertain molecules such as cause non-specific proliferation of peripheral blood mononuclear cells.
Human peripheral blood mononuclear cells are heterogeneous in that they consist of a subpopulation of cells that can recognize specific antigens. When peripheral blood mononuclear cells capable of recognizing a specific specific antigen encounter that antigen, proliferation of the subpopulation of mononuclear cells is induced. Tetanus toxoid and keyhole limpet hemocyanin are examples of antigens recognized by a subpopulation of peripheral mononuclear cells, but are not recognized by all peripheral mononuclear cells in sensitized individuals.
The ability of the anti-ICAM-1 monoclonal antibody R6-5-D6 to suppress the proliferative response of human peripheral blood mononuclear cells in a system known to require cell-cell adhesion was tested.
Peripheral blood mononuclear cells were purified on a Ficoll-Paqua (Pharmacia) gradient as recommended by the manufacturer. After collecting the interface, the cells were washed 3 times with RPMI 1640 medium and 10% in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine and gentamicin (50 μg / ml) in a flat bottom 96 well microtiter plate.⁶Cultured at a concentration of cells / ml.
Antigen, T-cell mitogen, or concanavalin A (0.25 μg / ml); T-cell binding antibody OKT_Three(0.001 μg / ml); Keyhole limpet hemocyanin (10 g / ml) or tetanus toxoid (1: 100 dilution from source) in cells cultured as described above in anti-ICAM-1 antibody (R6 -5-D6; final concentration 5 g / ml) was added in the presence or absence. Cells will be 3.5 days (Concanavalin A test), 2.5 days (OKT) before the assay is complete._ThreeTest), or 5.5 days (keyhole limpet hemocyanin and tetanus toxoid test). 18 h before the end of the assay, 2.5 μCi^ThreeH-thymidine was added to the culture. Cell proliferation was assayed by measuring thymidine incorporation into DNA by peripheral blood mononuclear cells. Incorporated thymidine was collected and counted in a liquid scintillation counter [Merluzzi et al.J. Immunol.139: 166-168 (1987)]. The results of these experiments are shown in FIG. 16 (Concanavalin A test) and FIG. 17 (OKT)._ThreeTest), FIG. 18 (keyhole limpet hemocyanin test), and FIG. 19 (tetanus toxoid test).
Anti-ICAM-1 antibody provides a proliferative response to non-specific T-cell mitogen, ConA; non-specific T-cell associated antigen, OKT-3; and specific antigens, keyhole limpet hemocyanin and tetanus toxoid in mononuclear cells. It turned out to be suppressed. Suppression by anti-ICAM-1 antibody is comparable to that of anti-LFA-1 antibody, ICAM-1 is a functional ligand of LFA-1, and antagonist of ICAM-1 suppresses the specific defense system response It suggests that it will be.
Example 27
Effect of anti-ICAM-1 on mixed lymphocyte reaction
As previously mentioned, ICAM-1 is required for effective cellular interactions during immune responses mediated by LFA-1-dependent cell adhesion. Induction of ICAM-1 during an immune response or inflammatory disease allows leukocytes to interact or interact with endothelial cells.
When lymphocytes from two unrelated individuals are cultured in the presence of each other, blast transformation and cell proliferation of the lymphocytes are observed. This response to the presence of one population of leukocytes and the second population of leukocytes is known as the mixed lymphocyte reaction (MLR) and is similar to the response of lymphocytes to mitogen addition [Immunology The Science of Self-Nonself DiscriminationKlein, J. John Wiley & Sons, NY (1982), pp 453-458].
The effect of anti-ICAM monoclonal antibody on human MLR was tested. These experiments were performed as follows. Peripheral blood was collected by venipuncture from normal healthy donors. Blood was collected in heparinized tubes and diluted 1: 1 with Punk's G (GIBCO) balanced salt solution (BSS) at room temperature. The blood mixture (20 ml) was layered on a 15 ml Ficoll / Hypaque density gradient (Pharmacia, density 1.078, room temperature) and centrifuged at 1000 × g for 20 minutes. The interface was then collected and washed 3 times in Punk's G. Cells were counted on a hematocytometer and RPMI containing 0.5% gentamicin, 1 mM L-glutamine (GIBCO) and 5% heat inactivated (56 ° C., 30 minutes) human AB serum (Flow Laboratories) It was resuspended in -1640 medium (GIBCO) (hereinafter referred to as RPMI medium).
Mouse anti-ICAM-1 (R6-5-D6) was used in this experiment. All monoclonal antibodies (prepared from ascites by Jackson ImmunoResearch Laboratories, Boston, MA) were used as purified IgG preparations. Peripheral blood mononuclear cells (PBMC) were 6.25 × 10 6 in a Linbro round bottom microtiter plate (# 76-013-06).^FiveCultured in medium at cells / ml. Stimulator cells from separate donors were irradiated at 1000R and cultured at the same concentration as the responder cells. The total volume per culture was 0.2 ml. Controls included responder cells alone and stimulator cells alone. Culture plates at 37 ° C with 5% CO₂-Incubated in a humid air atmosphere for 5 days. Each well was treated with 0.5 μCi tritiated thymidine (^ThreeHT) (New England Nuclea) was pulsed for the last 18 hours of culture. In some cases, a two-way MLR was performed. The protocol was the same except that the second donor cells were not inactivated by irradiation.
Cells were harvested on glass fiber filters using an automated multiple sample harvester (Skatron, Norway) and rinsed with water and methanol. Filters were oven dried and counted in an Aquasol with a Begman (LS-3801) liquid scintillation counter. Results are shown as mean CPM ± standard error for 6 individual cultures.
Table 15 shows that the purified anti-ICAM-1 monoclonal antibody suppressed MLR in a dose-dependent manner with significant suppression apparently at 20 ng / ml. Purified mouse IgG showed little or no inhibitory effect. Inhibition of MLR by anti-ICAM-1 monoclonal antibodies occurs when antibodies are added within the first 24 hours of culture (Table 16).

In summary, the ability of antibodies to ICAM-1 to suppress MLR indicates that ICAM-1 monoclonal antibodies have therapeutic utility for acute transplant rejection. ICAM-1 monoclonal antibodies also have therapeutic utility in related immune-mediated disorders that depend on LFA-1 / ICAM-1 regulatory cell-cell interactions.
The above experiments show that the addition of monoclonal antibodies to ICAM-1 suppresses the mixed lymphocyte reaction (MLR) when added during the first 24 hours of the reaction. Furthermore, ICAM-1In vitroThe condition is improved for human peripheral blood mononuclear cells in culture.
Furthermore, it has been found that ICAM-1 is not expressed on quiescent human peripheral blood lymphocytes or mononuclear cells. ICAM-1 is conditioned by using conventional flow cytometric analysis on monocultured cells or co-cultured cells with irrelevant donor cells in a mixed lymphocyte reaction. This improved ICAM-1 status on mononuclear cells can be used as an indicator of inflammation, particularly when ICAM-1 is expressed on fresh human mononuclear cells with acute or chronic inflammation.
The specificity of ICAM-1 for activated mononuclear cells and the ability of antibodies to ICAM-1 to suppress MLR are diagnostic in related immune-mediated disorders where ICAM-1 monoclonal antibodies require acute transplant rejection and cell-cell interactions It shows that it can have top and therapeutic potential.
Example 28
Synergistic effect of mixed administration of anti-ICAM-1 and anti-LFA-1 antibodies
As shown in Example 27, MLR is suppressed by anti-ICAM-1 antibody. MLR can also be suppressed by anti-LFA-1 antibodies. To determine whether the combined administration of anti-ICAM-1 and anti-LFA-1 antibodies was enhanced or had a synergistic effect, an MLR assay (performed as described in Example 27) was performed on the two antibodies In the presence of various concentrations.
This MLR assay showed that the anti-ICAM-1 + anti-LFA-1 combination was significantly more effective at suppressing the MLR response at concentrations where the antibody alone did not dramatically suppress the MLR (Table 17). This result indicates that the treatment comprising co-administration of anti-ICAM-1 antibody (or fragment thereof) and anti-LFA-1 antibody (or fragment thereof) has the ability to provide improved anti-inflammatory treatment. Such improved therapies allow for the administration of lower antibody doses than other therapeutically effective methods, and are important when high concentrations of individual antibodies elicit an anti-idiotypic response Indicates.

Example 29
Additional effects of mixed administration of anti-ICAM-1 and other immunosuppressive drugs at the next best dose in MLR
As shown in Example 28, MLR is suppressed by a combination of anti-ICAM-1 antibody and anti-LFA-1 antibody. To see if mixed administration of anti-ICAM-1 with other immunosuppressive agents (such as dexamethasone, azethiopyrine, cyclosporin A or steroids (eg prednisone etc.) also has an improved effect, MLR assay To the suboptimal concentration of R6-5-D6 in combination with other immunosuppressive agents (ie, lower than the optimal concentration at which the agent is administered alone to the subject) as per the protocol of Example 27. Concentration).
The data show that the inhibitory effect of R6-5-D6 is at least added to the inhibitory effects of dexamethasone (Table 18), azethiopyrine (Table 19) and cyclosporin A (Table 20) at sub-optimal doses. Yes. This means that anti-ICAM-1 can be effective in reducing the required dosage of known immunosuppressive agents, ie, reducing its toxic side effects. To obtain such immunosuppression using an anti-ICAM-1 antibody (or fragment thereof), the antibody (or fragment thereof) and a single additional immunosuppressive agent or two or more additional immunosuppressive agents Administration with the combination is possible.

Example 30
Effect of anti-ICAM-1 antibody in suppressing rejection of transplanted allogeneic organs
In order to show the effect of anti-ICAM-1 antibody in suppressing rejection of allogeneic transplanted organs, allogeneic kidneys were applied to cynomolgus monkeys according to the method of Cosimi et al. [Transplant. Proc. 13: 499-503 (1981)]. However, a modification was made using valium and ketamine as anesthetics.
That is, kidney transplantation was performed essentially as follows. Atypical renal allografts were performed on 3-5 kg cynomolgus monkeys after induction of anesthesia with barium and ketamine, essentially as disclosed by Marquet [Marquet et al., Medical Primatology, Part II, Basel, Karger, p. 125. (1972)]. End-lateral anastpmoses of the donor kidney on the aorta or aortic patch were constructed using 7-0 Prolene sutures. The donor ureter was spatula treated and embedded in the bladder by external methods (Taguchi, Y. et al., Dausset et al., Advances in Transplantation, Baltimore, Williams and Wilkins, p393 (1968)). Renal function was assessed by serum creatinine measurements every week or every two weeks. In addition, frequent allograft biopsies were taken for histopathology and complete dissection was performed on all dead receptors. For most receptors, bilateral nephrectomy was performed at the time of transplantation and subsequent uremic death was considered the end point of allograft survival. For some receptors, unilateral live nephrectomy and contralateral ureter ligation were performed at the time of transplantation. When allograft rejection occurred, the ligature on the same ureter was removed, giving us an opportunity to continue normal kidney function regeneration and immunological monitoring of the recipient animal.
Monoclonal antibody R6-5-D6 was administered daily for 12 days, starting at a dose of 1-2 mg / kg / day from 2 days prior to export. Creatinine serum levels were periodically tested to monitor rejection. The effect of anti-ICAM-1 antibody on immune system rejection of allogeneic kidney is shown in Table 21.

The above results indicate that R6-5-D6 is effective in extending the life span of monkeys that have received allogeneic transplantation.
Example 31
Effect of anti-ICAM-1 antibody in suppressing acute rejection of transplanted organs
To demonstrate that anti-ICAM-1 antibodies are effective in an acute model of transplant rejection, R6-5-D6 was also tested in an on-treatment or acute renal rejection model. In this model, monkey kidneys were transplanted (using the protocol of Example 30) and 15 mg / kg of cyclosporin A (CyA) was administered intramuscularly until stable renal function was obtained around the tissue. The CyA dose was then reduced every 2 weeks in 2.5 mg / kg increments until rejection occurred as indicated by elevated blood creatinine levels. At this point, R6-5-D6 was administered for 10 days and survival time was monitored. It is important to note that in this protocol, the dose of CyA remains sub-optimal because it does not change as soon as the acute rejection occurs. In this model, histological controls (N = 5) without antibody assistance survive 5-14 days from the onset of rejection. So far, 6 animals have been tested with R6-5-D6 in this protocol (Table 22). Two of these animals are still alive (M12: 31 days and M5: 47 days after administration of R6-5-D6). Two animals lived 38 and 55 days after initiation of R6-5-D6 treatment, and two animals died from causes other than acute rejection (one died from CyA toxicity and one under anesthesia R6 Died during administration of -5-D6). This model more closely approximates the clinical situation using R6-5-D6 from the beginning.

Example 32
Gene construction and expression of terminally truncated derivatives of ICAM-1
In its native state, ICAM-1 is a cell membrane-bound protein that contains the extracellular domain transmembrane domain and the cytoplasmic domain of five immunoglobulin-like domains. It was desirable to construct a functional derivative of ICAM-1 that lacks the transmembrane domain and / or cytoplasmic domain in that it can express solubles secreted from ICAM-1. These functional derivatives were constructed by oligonucleotide-related mutagenesis of the ICAM-1 gene and subsequent expression in monkey cells after input with the mutated gene.
Mutations in the ICAM-1 gene giving amino acid substitutions and / or truncated derivatives were generated by the method of Kunkel, T. [Proc. Natl. Acad. Sci. (USA) 82: 488-492 (1985). ]]. The ICAM-1 cDNA prepared as described above was digested with restriction endonuclease Sal1 and Kpm1, and the resulting 1.8 kb DNA fragment was subcloned into plasmid vector CDM8 [Seed, B. et al., Proc. Natl. Acad. Sci. (USA) 84: 3365-3369 (1987)]. Next, E. Coli (BW313 / P3)dut ^-,ung ^-The strain was transformed with the above construct, designated pCD1.8C. Single strand uracil-containing templates were rescued by infecting transformants with helper phage R408 (Stratorgene R). The mutant ICAM-1 cDNA is then second strand synthesized with an oligonucleotide having a mismatched base and subsequentlyung ⁺It was produced by initiating transformation with the resulting heteroduplex of the host (MC1061 / P3). Mutants were isolated by screening for newly created endonuclease restriction sites into which the mutant oligonucleotide was introduced. Mutant ICAM-1 protein is prepared using standard DEAE-dextran method [Selden, RF, etc., Current Protocols in Molecular Biology (edited by Ausebel, FM, etc.), 9.2.1-9.2.6 (1987)]. It was expressed by transfer of Cos-7 cells with the mutant DNA in the eukaryotic expression vector CDM8.
A truncated functional derivative of ICAM-1 was prepared that lacks the transmembrane domain and cytoplasmic domain but contains all five immunoglobulin-like domains. A 30 bp mutant oligonucleotide (CTC TCC CCC CGG TTC TAG ATT GTC ATC ATC) was used to replace the amino acid tyrosine (Y) and glutamic acid (E) codons at positions 452 and 453 with phenylalanine (F) and translation stop codon, respectively. (TAG) was transformed. The mutant is isolated by its specific Xba1 restriction site and Y⁴⁵²Displayed as E / F, TAG.
Three mutant subclones (# 2, # 7, and # 8) were transferred to COS cells to express the mutant protein. Three days after transfer with these three mutant subclones, culture supernatants and cell lysates were analyzed by immunoprecipitation with anti-ICAM-1 monoclonal antibody RR1 / 1 and SDS-PAGE. ICAM-1 precipitated from the culture supernatant of cells transfected with mutant subclones # 2 and # 8, but not from detergent lysates of these cells. The molecular weight of ICAM-1 found in the culture supernatant was about 6 kd lower than that of the ICAM-1 membrane, which is consistent with the size expected from the mutant DNA. That is, this functional derivative of ICAM-1 is secreted as a soluble protein. In contrast, ICAM-1 was not immunoprecipitated from the culture supernatant of cells transfected with control native ICAM-1, indicating that the ICAM-1 membrane was not expressed from COS cells. Furthermore, ICAM-1 did not immunoprecipitate from culture supernatants or cell lysates from negative control mock-transfected cells.
End-truncated ICAM-1 secreted from transfected cells was purified by immunoaffinity chromatography with an ICAM-1-specific antibody (R6-5-D6) and tested for sensory activity in cell binding assays. After purification in the presence of the detergent octyl glucoside, the preparation containing native ICAM-1 or truncated truncation was diluted to a final concentration of 0.25% octyl glucoside (below the critical micelle concentration of the detergent) Concentration). These preparations of ICAM-1 were bound to the surface of a plastic 96-well plate (Nunc) to prepare ICAM-1 bound to a solid phase. After washing away unbound, about 75-80% and about 83-85% of SKW-3 cells containing LFA-1 on the surface are specific for ICAM-1 native and truncated, respectively. Combined. These data indicate that secreted truncated ICAM-1-functional derivatives retain both immunological reactivity and the ability to mediate ICAM-1-dependent adhesion characteristic of native ICAM-1. Show.
A functional derivative of ICAM-1 lacking only the cytoplasmic domain was prepared in a similar manner. Using a 25 bp oligonucleotide (TC AGC ACG TAC CTC TAG AAC CGC CA), the codon at amino acid 476 (Y) was changed to a TAG translation stop codon. This variant is Y⁴⁷⁶/ TAG is displayed. Immunoprecipitation analysis and SDS-PAGE of Cos cells transfected with this mutant detected a membrane conjugate of ICAM-1 having a molecular weight about 3 kd smaller than that of negative ICAM-1. Indirect immunofluorescence analysis of the mutant-transfected Cos cells showed a point-stained image similar to native ICAM-1 expressed on LPS stimulated human endothelial cells. Moreover, the cells transfected with the mutant DNA specifically produced purified LFA-1 on the plastic surface in the same manner as the Cos cells transfected with native ICAM-1 DNA (Table 23).

Example 33
Mapping of ICAM-1 functional domains
A study of ICAM-1 found that the molecule has a domain of 7. Five of these domains are extracellular (domain 5 is closest to the cell surface, domain 1 is furthest from the cell surface), one domain is transmembrane domain, and one domain is cytoplasmic ( That is, it exists in the cell). In order to see which domains contribute to the ability of ICAM-1 to bind to LFA-1, an epitope mapping test was used. To perform such tests, different deletion mutants were generated and characterized for their ability to bind LFA-1. Separately, tests were performed with anti-ICAM antibodies known to interfere with the ability of ICAM-1 to bind to LFA-1. Suitable examples of such antibodies include RR1 / 1 [Rothlein, R. et al., J. Immunol. 137: 1270-1274 (1986)], R6.5 (Springer, TA et al., US patent application Ser. No. 07 / 250,446. No.), LB-2 [Clark, EA et al., Leukocyte Typing I (edited by A. Bernard et al.), Springer-Verlag, 339-346 (1984)], or CL203 [Staunton, DE et al., Cell. 56: 849-853 (1989)].
ICAM-1 deletion mutants can be prepared by any of a variety of methods. However, such mutants may be obtained by site-directed mutagenesis or by other recombinant means (such as by constructing an ICAM-1-expressing gene sequence lacking a sequence encoding a specific protein region). Preferably it is produced. Suitable procedures for producing such variants are well known in the art. Using such a procedure, three ICAM-1 deletion mutants were prepared. The first variant lacks amino acid residues F185-P284 (ie, lack of domain 3). The second variant lacks amino acid residues P284-R451 (ie, lack of domains 4 and 5). The third mutant lacks the amino acid residue after Y476 (ie, lack of the cytoplasmic domain). The results of such tests indicate that domains 1, 2 and 3 are mainly involved in ICAM-1 interaction with anti-ICAM-1 antibody and LFA-1.
Example 34
Effect of ICAM-1 mutations on LFA-1 binding
The ability of ICAM-1 to react and bind to LFA-1 is mediated by ICAM-1 amino acid residues present in domain 1 of the ICAM-1 molecule (Figures 8, 9 and 10). However, such reactions are facilitated by the contribution of amino acids present in domains 2 and 3 of ICAM-1. Thus, among the preferred functional derivatives of the present invention are soluble fragments of ICAM-1 molecules containing ICAM-1 domains 1, 2 and 3. More preferred are soluble fragments of ICAM-1 molecules containing ICAM-1 domains 1 and 2. Most preferred is a soluble fragment of ICAM-1 containing domain 1 of ICAM-1. Several amino acid residues within the first ICAM-1 domain are included in the reaction of ICAM-1 and LFA-1. Substitution of these amino acids with other amino acids alters the ability of ICAM-1 to bind to LFA-1. These amino acid residues and substitution products thereof are shown in FIG. FIG. 25 shows the effect of such mutations on the ability of the resulting mutant ICAM-1 molecule to bind to LFA-1. In FIGS. 23-25, each residue is described with respect to the single letter code for the amino acid, followed by the position of that residue in the ICAM-1 molecule. Thus, for example, “E90” refers to the glutamic acid residue at position 90 of ICAM-1. Similarly, “E90V” indicates a dipeptide consisting of a glutamic acid residue at position 90 and a valine residue at position 91. The replacement sequence is shown to the right of the slash (“/”) mark. The V4, R13, Q27, Q58 and D60S61 residues of ICAM-1 are included in LFA-1 binding.
Substitution of these amino acids changes the binding ability of ICAM-1 to LFA-1. For example, replacement of V4 with G results in the formation of a mutant ICAM-1 molecule that binds less to LFA-1 (FIG. 25). Substitution of the R13 residue of ICAM-1 with E results in the formation of a mutant molecule that has substantially reduced ability to bind LFA-1 (FIG. 25). Substitution of the Q58 residue of ICAM-1 with H gives a mutant molecule that has substantially the ability to bind to normal LFA-1 (FIG. 25). Substitution of the D60S residue of ICAM-1 with KL gives a mutant molecule with substantially small ability to bind LFA-1 (FIG. 25).
A glycosylation site in the second domain is also included in LFA-1 binding (FIG. 23). Replacement of N103 with K or A155N with SV results in the formation of a mutant ICAM-1 molecule that cannot substantially bind LFA-1. In contrast, replacement of glycosylation site N175 with A did not appear to substantially affect the ability of the mutant ICAM-1 to bind LFA-1.
The mutation in the third ICAM-1 domain did not appear to change appreciably the ICAM-1-LFA-1 binding (FIG. 24).
Example 35
Multimeric forms of ICAM-1 with increased biological half-affinity and clearance capacity
A quinola molecule is constructed by linking domains 1 and 2 of ICAM-1 to the hinge region of its long immunoglobulin chain. A preferred construct attaches the C-terminus of ICAM-1 domain 2 to a segment of an immunoglobulin long chain just N-terminal to the hinge region, allowing the segment flexibility conferred by the hinge region. ICAM-1 domains 1 and 2 will thus replace the Fab fragment of the antibody. Binding of IgGs to long chains and production of animal cells will result in the production of chimeric molecules. Generation of molecules containing long chains derived from IgA or IgM will result in the production of higher multimeric molecules containing 2-12 ICAM-1 molecules. Co-expression of a single chain gene in an animal cell producing an ICAM-1 long-chain chimera molecule consists of IgA molecules containing 4-6 ICAM-1 molecules and IgM containing about 10 ICAM-1 molecules. A suitable assembly of IgA and IgM multimers will be given which will mainly give the case. These chimeric molecules have several advantages. First, Ig molecules can be designed to stay longer in the circulatory system, thereby improving biological half-life.
Furthermore, the multimerism of these processed molecules is necessary for administration to allow these molecules to react with rhinovirus as well as cell surface LFA-1 with higher binding activity, depending on the therapeutic context, thus giving an effective dosage. Greatly reducing the amount of recombinant protein. IgA and IgM are hyperglycosylated molecules normally present in mucosal secretions such as in the nose. Its high hydrophilicity helps retain microorganisms and viruses that bind in the mucosa, preventing binding to cells and preventing crossing of epithelial cell membrane barriers. That is, these molecules have increased therapeutic efficacy. IgM and in particular IgA are stable in the mucosal environment and increase the stability of the ICAM-1 construct. When such an ICAM-1 functional derivative is administered into the blood stream, this derivative also increases the biological half-life. IgA does not fix complement and therefore would be ideal in applications where it is harmful. If an IgG heavy chain chimera is desired, it may be possible to mutate the region involved during binding to complement as well as reaction with Fc receptors.
Example 36
Expression of ICAM-1 mutant
Oligonucleotide-specific mutagenesis
The coding region of ICAM-1 cDNA in the 1.8 kb Sal1-Kpm1 fragment was subcloned into the expression vector CDMB [Seed, B. et al., Proc. Natl. Acad. Sci. (USA) 84: 3365-3369 (1987). ]. Kunkel, T. [Proc. Natl. Acad. Sci. (USA) 82: 488-492 (1985)] and a modification of Staunton D. et al. (Staunton DE et al., Cell 52: 925-933 (1988)). This construct (pCD1.8) was used to prepare a single strand uracil-containing template for use in oligonucleotide-specific mutagenesis.
In short, E. Coli strain XS127 was transformed with pCD1.8. Single colonies were grown to near saturation in 1 ml Luria Broth (LB) medium (Difco) containing 13 μg / ml ampicillin and 8 μg / ml tetracycline. 100 μl of the culture was infected with R408 helper phage (Strategene) at a multiplicity of infection (MOI) of 10 and 10 ml of LB medium containing ampicillin and tetracycline was added at 37 ° C. for 16 hours. After centrifugation at 10,000 rpm for 1 minute and 0.22 μm filtration of the supernatant, the phage suspension was infected with E. Coli BW313 / P3, which was then LB agar (Difco) supplemented with ampicillin and tetracycline. It was applied on a plate. Colonies were picked, grown to near saturation in 1 ml LB medium containing ampicillin and tetracycline, and infected with helper phage at MOI10. The culture volume was then increased to 250 ml and the cells were cultured overnight. Single strand DNA was isolated by standard phage extraction.
The mutant oligonucleotide was phosphorylated and used in the second strand synthesis reaction with the pCD1.8 template [Staunton D.E. et al., Cell 52: 925-933 (1988)].
Import
COS cells were seeded in 10 cm tissue culture plates to 50% confluence in 16-24 hours. The COS cells were then washed once with TBS and 4 ml RPMI containing 10% Nu serum (Collaborative), 5 μg / ml chloroquine, 3 μg mutant plasmid and 200 μg / ml DEAE-dextran sulfate. For 4 hours. The cells were then washed with 10% DMSO / PBS then PBS and cultured in culture medium for 16 hours. The culture medium was replaced with fresh medium. After 48 hours, post-transfer COS cells were suspended by trypsin / EDTA (Gibco) treatment and subdivided into 2, 10 cm plates for HRV binding as well as 24-well tissue culture plates. At 72 hours, cells were harvested from 10 cm plates with 5 mM EDTA / HBSS and processed for adhesion to LFA-1 coated plastic and immunofluorescence analysis.
LFA-1 and HRV binding
LFA-1 was purified from SKW-3 by immunoaffinity chromatography on TS2 / 4LFA-1 mAb Sepharose, and 2 mM MgCl.₂And eluted at pH 11.5 in the presence of 1% octyl glucoside. Add LFA-1 (10 μg / 200 μl / 6 cm plate) to 2 mM MgCl in a microbiological petri dish.₂The octyl glucoside was diluted to 0.1% in PBS containing phosphate buffered saline and incubated at 4 ° C. overnight. Each plate was blocked with 1% BSA (bovine serum albumin) and 2 mM MgCl.₂, 0.2% BSA, 0.025% azide, and 50 μg / ml gentamicin in PBS.
5% FCS (fetal bovine serum), 2 mM MgCl₂In PBS (buffer) containing 0.025% azide⁵¹Cr-labeled COS cells were incubated for 1 hour at 25 ° C. in the presence or absence of 5 μg / ml RR1 / 1 and R6.5 in LFA-1-coated microtiter plates. Non-adherent cells were removed by washing 3 times with buffer. Adherent cells were eluted to 10 mM by addition of EDTA and γ-calculated.
Result
Anti-ICAM-1 antibodies such as RR1 / 1, R6.5, LB-2, or CL203 have been identified. If these antibodies can inhibit ICAM-1 function, these antibodies must be able to bind to specific sites on the ICAM-1 molecule, which is also important for ICAM-1 function. That is, whether or not the missing domain is functionally important by preparing the aforementioned ICAM-1 deletion mutant and measuring the extent to which the anti-ICAM-1 antibody can bind to the deletion. Can do.
ICAM-1 is a complex membrane protein and its extracellular domain is predicted to consist of 5 Ig-like C-domains. To identify the domains involved in binding LFA-1, domain 3 and domains 4 and 5 (carboxyl terminus) are deleted by oligonucleotide-specific mutagenesis and functionally tested after expression in COS cells did. In addition, the entire cytoplasmic domain was deleted to confirm its potential impact on the ICAM-1 response. As expected, the cytoplasmic domain deficient, Y476 /^*Does not show a loss of reactivity of RR1 / 1, R6.5, LB-2 and CL203, whereas the domain 3 deletion F184-R451 resulted in a decrease and loss of CL203 reactivity (FIG. 20). . That is, the CL203 epitope appears to be in domain 4, while RR1 / 1, R6.5, and LB-2 appear to be in two amino-terminal domains.
All three deletion mutants showed wild-type levels of adhesion to LFA-1 (FIG. 21). Amino acid substitutions with predicted β-turns in domains 1, 2 and 3 were also prepared and tested for post-expression functionality in COS cells. The R6.5 epitope is localized to the sequence E111GGA of domain 2 and may contain E39 in domain 1, although RR1 / 1 and LB-2 are both dependent on R13 of domain 1 (FIG. 22). Furthermore, RR1 / 1 binding was reduced by mutations in the sequence D71GOS. Mutations that reduce the N-linked glycosylation sites of N103 and N163 reduced RR1 / 1, R6.5 and LB-2, LFA-1 HRV binding. These mutations appear to process such that ICAM-1 dimers are generated.
Other mutations in domain 2 or 3 did not result in altered LFA-1 adhesion (FIGS. 23 and 24). Both domain 1 amino acids, R13 and D60, are involved by binding to LFA-1 (FIG. 25).
That is, LFA-1 and HRV binding appears to be a function of the amino terminal Ig-like domain of ICAM-1. FIG. 26 shows the sequence of the ICAM-1 terminal domain.
Although the present invention has been described with respect to specific embodiments thereof, it will be understood that further modifications are possible and the present application generally follows the principles of the present invention and the technology to which the present invention pertains. Including all modifications, uses or applications, including avoidance from the description herein, which may be applied to essential features such as included in known or customary implementations within and claimed. .
[Brief description of the drawings]
FIG. 1 schematically shows adhesion between normal cells and LFA-1-deficient cells.
FIG. 2 schematically shows the normal cell / normal cell adhesion process.
FIG. 3 shows the kinetics of cell aggregation in the absence (x) or presence (o) of 50 ng / ml PMA.
FIG. 4 shows LFA-1^-Cells and LFA-1⁺Shows aggregation between cells. As shown in the figure, carboxyfluorescein diacetate labeled EBV-transformed cells (10^Four) 10^FiveUnlabeled allogeneic cells (black frame) or JY cells (white frame) were mixed in the presence of PMA. After 1.5 hours, aggregated or free labeled cells were counted using the quantitative assay of Example 2. The percentage of labeled cells in the aggregate is shown. One representative test of two is shown.
FIG. 5 shows immunoprecipitation of ICAM-1 and LFA-1 from JY cells. Antibodies capable of binding ICAM-1 to JY cell Triton X-100 lysate (lanes 1 and 2) or control lysis buffer (lanes 3 and 4) or antibodies capable of binding to LFA-1 (lanes 1 and 3). Immunoprecipitation was performed in lanes 2 and 4). Panel A shows the results under reducing conditions and Panel B shows the results obtained under non-reducing conditions. Molecular weight standards are shown in lane S.
FIG. 6 shows the kinetics of the effects of IL-1 and gamma interferon on ICAM-1 expression on human skin fibroblasts. Human skin fibroblasts are 8 × 10^FourCell / 0.32cm²Grown to density of wells. IL-1 (10 μ / ml, black circle) or recombinant gamma-interferon (10 μ / ml, white square) was added, and the indirect binding assay was performed by cooling to 4 ° C. for the time indicated in the figure. The standard deviation did not exceed 10%.
FIG. 7 shows the concentration dependence of IL-1 and gamma interferon effects on ICAM-1. Human skin fibroblasts are 8 × 10^FourCell / 0.32cm²/ Grown to well density. IL-2 (white circles), recombinant human IL-1 (white squares), recombinant mouse IL-1 (black squares) and recombinant beta interferon (white triangles) for 4 hours (IL) -1) or 16 hours (beta and gamma interferon). The results shown show the average of 4 measurements and the standard deviation does not exceed 10%.
FIG. 8 shows the nucleotide and amino acid sequence of ICAM-1 cDMA. The first ATG is at position 58. The translation sequence corresponding to the ICAM-1 tryptic peptide is underlined. The hydrophobic putative signal peptide and transmembrane sequences are underlined. N-linked glycosylation sites are boxed. The polyadenylation signal AATAAA at position 2976 is overlined. The sequence shown is for the HL-60 cDNA clone. Endothelial cDNA sequenced over most of its length and showed only small differences.
FIG. 9 shows the correlation to the ICAM-1 homology domain and immunoglobulin supergenes. (A) Sequence of five homologous domains (D_1-5): Two or more identical residues in a row are enclosed. Residues contained more than once in the NCAM domain, and set C₂And C₁Residues contained in the domain of are arranged by ICAM-1 internal repeats. The position of the predicted β-strand in the domain of ICAM-1 is marked with a bar and lower case on the sequence, and the known position of the β-strand in the immunoglobulin c domain is marked with an upper case under the bar and on the sequence. is doing. The position of the putative disulfide bridge within the ICAM-1 domain is marked by SS. (BD) sequence of protein domains homologous to ICAM-1: Each protein is first sequenced by searching the NBRF database using the FASTP program. Each protein sequence is MAG, NCAN, T cell receptor α subunit V domain, IgM μ chain and α-1-B-glycoprotein.
FIG. 10 is a comparative view of secondary structures of ICAM-1 and MAG.
FIG. 11 shows LFA-1 positive EBV-transformed B-lymphoblastoid cells bound to ICAM-1 in a flat membrane.
FIG. 12 shows LFA-1-positive T-lymphoblasts and T-lymphocytes bound to ICAM-1 in plastic binding vesicles.
FIG. 13 shows inhibition of binding by pretreatment with JY B-lymphoblastoma cells or vesicle monoclonal antibodies bound to ICAM-1 in plastic binding vesicles.
FIG. 14 shows the effect of temperature on the binding of T-lymphoblasts to ICAM-1 in plastic binding vesicles.
FIG. 15 shows the need for a divalent cation in binding T-lymphoblasts to ICAM-1 in plastic binding vesicles.
FIG. 16 shows the effect of anti-adhesive antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of the T-cell associated antigen OKT3. “OKT3” indicates the addition of antigen.
FIG. 17 shows the effect of anti-adhesive antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of concanavalin A, a non-specific T-cell mitogen. “CONA” indicates the addition of concanavalin A.
FIG. 18 shows the effect of anti-adhesive antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of the keyhole limpet hemocyanin antigen. “KLH” indicates the addition of keyhole limpet hemocyanin to cells.
FIG. 19 shows the effect of anti-adhesive antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of the tetanus toxoid antigen. “AGN” indicates the addition of tetanus toxoid antigen to cells.
FIG. 20 shows the binding of monoclonal antibodies RR1 / 1, R6.5, LB2, and CL203 to ICAM-1 deletion mutants.
FIG. 21 shows the binding of an ICAM-1 deletion mutant to LFA-1.
FIG. 22 shows the epitopes recognized by the anti-ICAM-1 monoclonal antibodies RR1 / 1, R6.5, LB2, and CL203.
FIG. 23 shows the binding ability of the ICAM-1 domain 2 mutant to LFA-1.
FIG. 24 shows the binding ability of the ICAM-1 domain 3 mutant to LFA-1.
FIG. 25 shows the binding ability of ICAM-1 domain 1 to LFA-1.
FIG. 26 shows the sequence of the ICAM amino terminal domain.

Claims (14)

A functional derivative of ICAM-1 which is a mutant or analog of ICAM-1, and lacks at least either the cytoplasmic domain or transmembrane domain of ICAM-1, E90 / Q, R49KV / EKL, K39KE / Said derivative containing a substitution selected from the group consisting of ERQ, G15S / SA and Q1T / KA and retaining the ability to bind to LFA-1. The ICAM-1 according to claim 1, comprising domain 1 (residues 1 to 88 of the following sequence), domains 1 and 2 (residues 1 to 185), or domains 1, 2 and 3 (residues 1 to 284). Functional derivatives of

The functional derivative of ICAM-1 according to claim 1 or 2, wherein residues V4, R13, Q27, Q58, D60 and S61 of ICAM-1 having the sequence according to claim 2 are not mutated. The functional derivative of ICAM-1 according to claim 1 or 2, wherein sugar chain forming sites N103 and A155N are not mutated. The functional derivative of ICAM-1 according to claim 1 or 2, wherein the functional derivative contains a mutation in domain 3 of ICAM-1. The functional derivative of ICAM-1 according to claim 1, wherein said functional derivative contains ICAM-1 domains 1 and 2 linked to the N-terminus of the immunoglobulin heavy chain to the immunoglobulin hinge region. The functional derivative according to claim 6 , wherein the immunoglobulin heavy chain is of the IgG class. 2. The functional derivative of ICAM-1 according to claim 1, wherein the functional derivative comprises ICAM-1 domains 1 and 2 bound to an IgA or IgM class immunoglobulin heavy chain. A pharmaceutical composition for treating inflammation caused by a response of a specific or non-specific defense system, comprising the functional derivative of ICAM-1 according to any one of claims 1 to 8 . A pharmaceutical comprising the functional derivative of ICAM-1 according to any one of claims 1 to 8 , for inhibiting metastasis of hematopoietic tumor cells that require a functional member of the LFA-1 family to migrate Composition. A pharmaceutical composition for suppressing the growth of LFA-1-expressing tumor cells, comprising the toxin-derived functional derivative of ICAM-1 according to any one of claims 1 to 8 . An anti-inflammatory pharmaceutical composition comprising (a) the functional derivative of ICAM-1 according to any one of claims 1 to 8 and (b) a conventional carrier or excipient. A recombinant DNA molecule capable of expressing the functional derivative of ICAM-1 according to any one of claims 1-8 . A method for producing a functional derivative of ICAM-1,
Culturing a host microorganism transformed with a recombinant vector encoding the functional derivative of ICAM-1 according to any one of claims 1 to 8 , under conditions where the derivative is expressed and secreted into the medium; And thereafter isolating the derivative,
Including said method.

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