JP5890017B2 - 抗原特異的t細胞寛容を誘導する抗体およびその使用 - Google Patents
抗原特異的t細胞寛容を誘導する抗体およびその使用 Download PDFInfo
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Classifications
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C07K—PEPTIDES
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
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Description
抗体またはその断片を生産する方法は、(a)ヒトICAM−1蛋白質(Gene Bank Accession No.NP_000192)または蛋白質の断片、あるいはヒトICAM−1を発現する細胞(例えば、活性化されたヒト末梢血液単核球および/またはDu145(ATCC HTB−81))で動物を免疫化する段階と、(b)免疫化された動物から脾臓細胞(splenocyte)を抽出する段階と、(c)動物の脾臓細胞を骨髄腫(myeloma)細胞株と融合させる段階と、(d)ハイブリドーマ細胞をスクリーニングし、ICAM−1とLFA−1との間の相互作用を妨げないものの、移植された器官の拒絶反応を抑制することだけでなく、樹状細胞の機能および分化を調節することも可能な、好適なハイブリドーマ細胞を選別する段階とを含む。
前記抗体またはその断片は、インビトロ培養によるか、あるいは前記抗体またはその断片を生産する細胞を動物に投与することによって得られる。前記抗体またはその断片は、前記抗体またはその断片を生産する細胞が腹膜内に投与された動物の腹水から得られる。前記抗体またはその断片は、イオン交換クロマトグラフィーまたは親和性カラムクロマトグラフィーによって培養上澄液または腹水から精製することができる。
ヒト末梢血液単核細胞を、フィコール−パック(GE Healthcare)密度勾配遠心法によって健康な供与者から分離した。ICAM−1/Fc蛋白質を製造するために、フィトヘマグルチニン(phytohemagglutinin、PHA)活性化されたヒト末梢血液単核細胞からmRNAを抽出し、ヒトICAM−1の細胞外ドメインに相応するcDNA断片の5’および3’末端にそれぞれNheIおよびEcoRIサイトを導入し、重合酵素連鎖反応(PCR)で増幅した。次のプライマーを用いて重合酵素連鎖反応(PCR)を行った。
5’プライマー:GCT AGC GCA ACC TCA GCC TCG CTA TGG CTC(配列番号1)
3’プライマー:GAA TTC ATC TCA TAC CGG GGG GAG AGC AC(配列番号2)
ICAM−1およびヒトIgG Fcの融合構造体を製造するために、上記断片を、ヒトIgG1 Fc領域を含む、EcoRIおよびXhoI−制限酵素処理されたpSecTagプラスミド(Invitrogen)内に連結させた。HEK293細胞(ATCC CRL−1573)に前記プラスミドを一時的に形質移入させ、蛋白質Gカラムを用いてICAM−1/Fc蛋白質を培養上澄液から精製した。
ICAM−1は、5つの免疫グロブリン様(Ig様)ドメイン、膜貫通ドメインおよび細胞質ドメインで構成された構造を有する(Cell.1992、68:71−81)。具体的なICAM−1 Ig様ドメインへのMD−3の結合サイトの位置を知るために、ヒトおよびマウスICAM−1cDNAクローンを用いて、ヒトドメイン1/マウスの膜貫通および細胞質ドメインを含むマウスドメイン2〜5をコーディングするcDNA(h1m2345)、ならびに、ヒトドメイン1〜2/マウスドメイン3〜5をコーディングするcDNA(h12m345)の2種類のキメラcDNAを製造した。これらのキメラ突然変異体を製作するためにオーバーラッピングPCR技術を用いた。まず、次のプライマーを用いてヒトドメイン1(h1)を含むcDNAを増幅した:5’プライマーGAA TTC ATG GCT CCC AGC AGC CCC CGG CCC GCG CT(配列番号3);3’プライマーAGG TCT CAG CTC CAC CCG TTC TGG AGT CCA GTA CAC GGT GAG GAA G(配列番号4)。マウスドメイン2−5、膜貫通ドメインおよび細胞質ドメイン(m2345)の製造のためのプライマーは次の通りである:5’プライマーCTG GAC TCC AGA ACG G GTG GAG CTG AGA CCT CTG CCA GCC TGG CAG(配列番号5);3’プライマーGGA TCC GGG AGG TGG GGC TTG TCC CTT GAG TTT TAT GGC(配列番号6)。これら2種類のcDNAを混合し、配列番号3および配列番号6のプライマーを用いて再増幅し、最終産物(h1m2345)をpcDNA3.1ベクターにクローニングした。h12m345をコーディングする変異体は、次のプライマーを用いて、h1m2345に対するものと類似の方式でクローニングした:h12に対する5’プライマーGAA TTC ATG GCT CCC AGC AGC CCC CGG CCC GCG CT(配列番号7);h12に対する3’プライマーGGT AGC TGG AAG ATC AAA GGT CTG GAG CTG GTA GGG GGC CGA GGT(配列番号8);m345に対する5’プライマーCAG CTC CAG ACC TTT GAT CTT CCA GCT ACC ATC CCA AAG CTC GAC ACC(配列番号9);m345に対する3’プライマーGGA TCC GGG AGG TGG GGC TTG TCC CTT GAG TTT TAT GGC(配列番号10)。
末梢血液単核細胞を、フィコール−ハイパック密度勾配遠心法によって健康な供与者の血液から分離した。PBSで2回洗浄した後に、細胞を1x108cells/mlの濃度でMACSバッファー(Milteny−Biotech)中に再懸濁させ、次に、氷中で15分間、抗CD14マグネチックビーズ(20μl/107cells;Miltenyi Biotec、Bergisch Gladbach、Germany)と共にインキュベートし、次に、MACS(Milteny−Biotech)を用いて磁性分離した。精製されたCD14+単核球を、10%(v/v)FBSが含まれているRPMI(Invitrogen)内に再懸濁し、大食細胞コロニー刺激因子(GM−CSF、1000U/ml)およびIL−4(1000U/ml)と共に6日間培養した。培養0日目および3日目に培地にMD−3または同アイソタイプの対照抗体(10mg/ml)を追加し、6日目にLPSを培養培地に追加した。1日経過後に、培養した細胞を収集し、MHCクラスI、MHCクラスII、CD80、CD86、およびCD40の発現を、フローサイトメトリーにより平均蛍光強度(MFI)として測定した(図2A)。培養培地内のIL−12p70、IL−6、IFN−γ、およびTNF−αの量を測定するためにELISAも行った(図2B)。CD14+単核球をGM−CSFおよびIL−4の存在下で培養すると、未成熟樹状細胞に分化することができ、LPSの処理は、未成熟樹状細胞の成熟を誘導して、結果的に、MHCおよび共刺激分子の表面発現の上方調節ならびにサイトカインの産生をもたらした。しかし、LPS刺激に先立ち、MD−3抗体で前処理した場合には、遮断(ブロッキング)抗体で樹状細胞を処理した場合とは異なり、樹状細胞における表面分子発現の上方調節およびサイトカインの生成が顕著に抑制された(図5)。したがって、このような結果は、MD−3抗体が単核球由来の樹状細胞の成熟を抑制する能力を有することを示すものである。
抗ICAM−1抗体のインビボ(in vivo)効果を調査するために、Ito,M.et al.,Blood.2002、100:3175−82に記載の方法により、免疫欠乏マウスにヒト造血幹細胞を移植してヒト化マウスを製造した。
移植拒絶反応を抑制するMD−3抗体の効果を実証するために、膵島異種移植モデルを用いた。ブタ膵島を得るために、温虚血時間なしで膵臓全摘術を行い、先に開示された修正Ricordi方法(Cell Transplant 2010、19:299−311)により膵島を分離した。精製された膵島を、10%ブタ血清、10mMニコチンアミド、および1%ペニシリン−ストレプトマイシンが添加された199培地(Gibco、BRL、Life Technologies Ltd、Paisley、Scotland、UK)で37℃にて一晩培養し、その後、ヒト化マウスの腎臓被膜下で培養した。携帯用血糖測定器(Accu−Chek、Roche Diagnostics、ソウル、韓国)を用いて血中グリコース水準を測定するために、眼窩副鼻腔(retro−orbital sinus)から週ごとに末梢血液試料を得て、血清内のブタC−ペプチド水準は放射線免疫分析(radioimmunoassay、Linco、St.Charles、MO、USA)で測定した。ブタC−ペプチド>0.5ng/mlの場合、成功した移植と定義された。移植拒絶反応は、第1日目のブタC−ペプチド<0.1ng/mlの場合と定義し、移植片の組織学的分析(histological analysis)により確認した。対照群において、組織生存期間の中央値は21日間であり、抗ICAM−1遮断抗体R6−5−D6の投与は、生存期間の中央値を40日間まで延長させた(図4)。しかし、移植された膵島は、結局、2つの群のいずれでも拒絶反応が現れた。これと対照的に、MD−3処理されたマウスは、実験期間全体を通して移植拒絶反応が現れなかった。
図5に示されているように、対照群では、インスリン陽性膵島細胞は完全に破壊され、CD3+T細胞およびCD68+大食細胞の主要な浸潤細胞により置き換えられた。しかし、MD−3処理群では、膵島近傍における無視できる程度の単核細胞の浸潤と共に、インスリン陽性膵島細胞の大きな群が明確に見られた。
抗IL−2または抗IFN−γ捕獲抗体でコーティングされたプレートを、滅菌リン酸緩衝食塩水(phosphate buffered saline、PBS;200μl/well)で4回洗い、10%のヒト血清が添加されたRPMI1640培地を用いて常温で30分間ブロッキングした。前記培地を除去した後、ヒト化マウス由来の3×105個の脾細胞を、5×104個のブタ膵島細胞と共に、10%のヒト血清が添加されたRPMI1640培地中で、37℃で40時間、5%二酸化炭素(CO2)インキュベータで培養した。
抗原特異的T細胞寛容を評価するために、キーホールリンペットヘモシアニン(KLH;0.1mg/ml)、または、3人の供与者から集めたT細胞枯渇化γ線照射ヒト末梢血液単核細胞(PBMCs;7×105/well)も刺激剤として使用した。
1つの陰性対照(NC)として、膵島移植していないヒト化マウスの脾臓細胞をブタ膵島により刺激し、抗膵島反応に使用した。これと対比させて、移植を受けたマウスの脾臓細胞を刺激抗原非存在下で培養したものを、抗同種血液単核球(allo−PBMCs)および抗KLHの反応に使用した。40時間培養後、細胞を除去し、プレートをPBS溶液(200μl/well)で5回洗った。アルカリホスファターゼコンジュゲート化検出用抗体を、0.5%ウシ胎仔血清が含まれているPBS100μl中で、IL−2またはIFN−γについてそれぞれ1:200または1:1000に希釈して加え、常温で2時間インキュベートした。前記プレートをPBSで5回洗い、BCIP/NBP基質100μlを加えた。水道水で洗うことによって発色を停止させた。結果として得られたスポットを、コンピュータ支援によるAID EliSpot Reader System(AID、Strassberg、Germany)で数えた。対照マウス由来の全脾臓細胞をブタ膵島で刺激した時、有意な数のIL−2またはIFN−γ分泌T細胞が検出された(図6左段)。これと対照的に、MD−3で処理したマウスでは、IL−2またはIFN−γ分泌T細胞が検出されておらず、これは、異種移植抗原に対するT細胞反応が完全に抑制されたことを示す。しかし、この系において、ヒト同種血液単核球(allo−PBMCs)に対するT細胞の反応は対照と同程度であった(図6中段)。このような結果は、T細胞の無反応は、ブタ膵島抗原に特異的な寛容の誘導を反映していたことを示唆する。
この抗原特異的T細胞寛容の誘導は、無関係な抗原によるインビボ負荷によってさらに証明された。移植後28日目に、MD−3が完全に除去された時、マウスを溶解性(soluble)KLH抗原で免疫化した。2週間後、KLH特異的T細胞の数をELISPOT分析により測定した。図6(右段)に示されているように、MD−3処理したマウスから得られたT細胞は、IFN−γおよびIL−2を生産する細胞の数という観点において、KLHに対する強い反応を示した。このような結果は、MD−3の投与が、移植膵島抗原に対して特異的なT細胞寛容を誘導したことを示唆している。
Claims (12)
- 受託番号KCLRF−BP−00264で寄託されたハイブリドーマから生産される、単クローン抗体MD−3。
- 重鎖および軽鎖を含む、ヒトICAM−1に結合する単クローン抗体であって、
前記重鎖は、受託番号KCLRF−BP−00264で寄託されたハイブリドーマによって生産される抗体の重鎖の3つの相補性決定領域(CDR)を含み、
前記軽鎖は、受託番号KCLRF−BP−00264で寄託されたハイブリドーマによって生産される抗体の軽鎖の3つの相補性決定領域(CDR)を含む、抗体。 - 受託番号KCLRF−BP−00264で寄託されたハイブリドーマから生産されるMD−3抗体由来のキメラ形態またはヒト化形態である、請求項2に記載の抗体。
- 受託番号KCLRF−BP−00264で寄託された、ハイブリドーマ細胞。
- 請求項1〜3のいずれか1項に記載の単クローン抗体を含む、薬学組成物。
- 前記単クローン抗体は、単離された形態である、請求項5に記載の薬学組成物。
- 請求項1〜3のいずれか1項に記載の単クローン抗体を有効成分として含む薬学組成物であって、ICAM−1ドメイン2への結合および抗原特異的T細胞寛容の誘導により樹状細胞の分化および機能を調節するための薬学組成物。
- 請求項1〜3のいずれか1項に記載の単クローン抗体を有効成分として含む、T細胞媒介免疫疾患を予防または治療するための薬学組成物。
- 前記T細胞媒介免疫疾患は、移植拒絶、移植片対宿主疾患または自己免疫疾患である、請求項8に記載の薬学組成物。
- ICAM−1ドメイン2への結合および抗原特異的T細胞寛容の誘導により樹状細胞の分化および機能を調節することに用いるための、請求項1〜3のいずれか1項に記載の単クローン抗体。
- T細胞媒介免疫疾患を予防または治療することに用いるための、請求項1〜3のいずれか1項に記載の単クローン抗体。
- 前記T細胞媒介免疫疾患は、移植拒絶、移植片対宿主疾患または自己免疫疾患である、請求項11に記載の抗体。
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