JP5552070B2 - Hiv/エイズの受動免疫治療用ループス抗体 - Google Patents
Hiv/エイズの受動免疫治療用ループス抗体 Download PDFInfo
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Description
(a)VL及びVHドメインを含む単鎖Fv構築物又は
(b)軽鎖サブユニットを含む、全身性エリテマトーデスに罹患した個体の発現された抗体レパートリーを、複数の前記個体からプールされたリンパ球のmRNAを得た後、前記レパートリーにおいて前記単鎖Fv構築物又は前記軽鎖サブユニットに対応するcDNAを逆転写酵素ポリメラーゼ反応によって得て、ファージ粒子の表面上で又は細菌発現宿主において可溶性形態で、前記抗体断片の発現を可能にするファージミドベクターにより前記cDNAをクローン化することによって調製するステップ、固定化されたgp120、合成gp120(421-436)ペプチド、固定化されたHIV、gp120のオリゴマー、又は残基414‐439の範囲の任意の長さの合成gp120ペプチドに結合するクローンをスクリーニングすることによって、目的の抗体断片クローンを前記ファージ粒子又は細菌発現宿主から単離するステップ、目的の前記抗体断片クローンを可溶性形態で発現し、精製し、さらに前記クローンのgp120(421−436)ペプチドとの結合能を確認するステップ、gp120(421−436)ペプチドに結合して複数の主要なHIV分離株を中和する、目的の前記抗体断片クローンの能力をアッセイするステップ、及び前記HIV中和アッセイから抗体断片を選択するステップを含む前記製造方法。
HIVgp120に対するポリクローナル抗体は、ループス及び混合結合組織病の患者の血清中に存在し、検出された(1,2)。ループス患者の血清中に見出される抗体は、gp120の残基421-436で構成されている比較的保存された決定基(以後gp120(421-436)とする)に結合するので、HIV感染に対する防御因子として興味がある。しかしながら、実験動物で免疫原としてgp120(421-436)に対応する合成ペプチドを使った研究では、このペプチドに対する抗体がHIV-1中和活性を示さないと報告された(3)。その結果、HIV-1感染の受動免疫治療用にこのような抗体を開発することにはほとんど興味が失われた。
前述の検討事項から見て、本発明者は、HIVに対する抗体の標的として残基421-436からなる決定基を検討した。表2はこのエピトープのアミノ酸配列が多様なHIV-1株で保存されている程度を示す。各位置のコンセンサスアミノ酸の頻度は残基424、429及び432を除いて80%より高い。gp120のこの領域は、抗体の最も良く保存されている可能性のある標的のひとつである。
コンセンサスが由来する菌株の番号を示す(すべての株はLos Alamosデータベースで入手可能)。頻度は:(示された残基を発現する株の#×100/株の全#)
値は50%(IC50%)及び90%(IC90)中和するμg抗体断片/mlで示す。括弧の中はそれぞれ、方程式:%HIV中和=100%/[1+10(logIC50.抗体濃度)×傾き]に適合したカーブの傾き(Hill Slope)及び2乗相関係数である。カーブは開始点を通らせた(ゼロ中和)。傾き値は変化するパラメーターとして使用した。ntはテストなし。
本明細書に開示されているように、HERVポリペプチドを認識する抗体は、今日の微生物に対して防御機能を果たし得る。この予測に対する理論的な根拠は、HERV配列の発現が進化の過程で宿主−微生物関係の不可欠な構成要素になったということである。この理論において、本発明者は、宿主生物が防御免疫応答をすることができるポリペプチドをコードする微生物の重要な核酸配列が、破壊メカニズムとして宿主ゲノムの中に組み込まれると考えた。宿主ゲノムに組み込まれると、発現されたHERV配列は自己抗原として宿主免疫系によって処理され、発達途上の免疫系において、HERV抗原(及び重要な微生物抗原エピトープ)に対する寛容が、自己抗原に対する免疫応答を制限する通常の寛容メカニズムの過程で発達する。これらのメカニズムは、自己免疫疾患を妨げる方向に向けられる、さまざまな抗原に対するT及びB細胞のクローナルサイレンシング(clonal silencing)及び失敗の事象からなる。したがって、自己免疫疾患のない生物の生理学的環境下では、HERVの存在は、微生物によって、宿主細胞の免疫応答を損ない、感染をより簡単に起こすために使われるメカニズムとして考えられる。
本明細書に記載した抗体は、医薬品として患者に一般的に投与される。
1.Bermas, B.L., Petri, M., Berzofsky, J.A., Waisman, A., Shearer, G.M. and Mozes, E. Binding of glycoprotein120 and peptides from the HIV-1 envelope by autoantibodies in mice with experimentally induced systemic lupus erythematosus and in patients with the disease. AIDS Res Hum Retroviruses. 10:1071-1077, 1994.
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HIV感染の治療の進歩にもかかわらず、効果的な免疫療法及びHIV用ワクチンの開発が相変らず急務である。アジドチミジンやレトロウイルスタンパク質分解酵素抑制剤などの薬剤はウイルス量(viral burden)を減少させる。しかしながら、これらの薬剤に耐性のウイルス変異株が生じ得、投薬中止は再感染を起こし得、しかも重大な副作用がある。最近、適応可能な免疫応答の効果的な武器、すなわち抗体及び細胞溶解性T細胞の両方がHIVに対する防御を達成するのに重要であるというコンセンサスが生まれた(1,2)。細胞傷害性T細胞応答はHIVに感染した対象の減少したウイルス量に一時的に一致する。しかしながら、細胞溶解性T細胞応答に依存するワクチン接種のストラテジーでは、HIVの回避変異体の発生が認められている(3、4)。細胞溶解性T細胞は感染した宿主細胞を溶解するが、細胞フリーのビリオンを不活性化しないので、無菌化免疫の可能性は示さない。体液免疫系がHIVに対して防御できるということは、SHIVマカク属サルモデルにおいて侵入抑制剤としての機能を果たし感染を抑えるモノクローナル中和抗体、例えばgp120のCD4結合部位(CD4b)に対するb12抗体、gp120のマンノース依存エピトープに対する2G12抗体及びgp41に対する2F5抗体の同定によって示唆される(5-7)。これらの抗体はHIV-1株の多くを中和するがすべてではなく、動物防御実験においてカクテル療法としての使用が奨められる(8)。
1.Devico, A.L., Fouts, T.R., Shata, M.T., Kamin-Lewis, R., Lewis, G.K. and Hone, D.M. Development of an oral prime-boost strategy to elicit broadly neutralizing antibodies against HIV-1. Vaccine. 20:1968-1974, 2002.
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受動免疫療法には、充分に特徴付けられた抗体の再生可能な均質の供給源が必要とされる。ヒト由来の抗体をクローン化する今までの方法では、末梢血(又は手術によって得られたリンパ系組織)由来のリンパ球を、例えばエプスタインバーウイルスでの形質転換により不死化した後、ミエローマ細胞株と融合させる。生じたハイブリドーマ細胞株が所望の抗体を産生するか否か、例えばELISAにより結合を測定することでスクリーニングする。
ループス患者由来の抗gp120抗体断片を単離する前記の技術を使用した。以下のファージ表示ライブラリーを調製した(図7、文献5):(a)pCANTAB5his6ベクターでクローン化されたヒトループスL鎖(3患者より)及び(b)pHEN2(イギリス、MRC、タンパク質工学のセンターによって快く提供されたベクター、国際公開WO9201047-A、GenBank登録1926701)のヒトループス単鎖Fv構築物(2患者より)。FvライブラリーをVL-リンカー-VH[リンカー:SS(GGGGS)2GGSA]構築物としてクローン化し、赤血球の低張溶解後、末梢血白血球(血液100mlから)において、全RNAを単離し、cDNA複製物をフォワードプライマーを使用して調製し、完全長L鎖と、VH、Vκ及びVλドメインのcDNAをPCRで調製した(残基1-124;1-123;1-107及び1-107にそれぞれ対応する;カバット(Kabat)の番号付け)。使用したプライマーは以下の通りである:
(a) ヒト完全長L鎖:
VLκ バック (Sfi I 部位下線あり)-
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGACATCCAGATGACCCAGTCTCC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGATGTTGTGATGACTCAGTCTCC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAATTGTGTTGACGCAGTCTCC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGACATCGTGATGACCCAGTCTCC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAACGACACTCACGCAGTCTCC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAATTGTGCTGACTCAGTCTCC;
Cκ フォワード(Not I 部位下線あり) -- CCATCCTGCGGCCGCACACTCTCCCCTGTTGAAGCTCTT;
(b) ヒト単鎖 Fv:
VLκ バック- バックプライマー参照, 完全長L鎖; VLκ フォワード (Xho I 部位下線あり)-
GCCTGAACCGCCTCCACCACTCGAGCGTTTGATTTCCACCTTGGTCCC,
GCCTGAACCGCCTCCACCACTCGAGCGTTTGATCTCCAGCTTGGTCCC,
GCCTGAACCGCCTCCACCACTCGAGCGTTTGATATCCACTTTGGTCCC,
GCCTGAACCGCCTCCACCACTCGAGCGTTTGATCTCCACCTTGGTCCC,
GCCTGAACCGCCTCCACCACTCGAGCGTTTAATCTCCAGTCGTGTCCC;
VLλ バック (Sfi I 部位下線あり)-
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGTCTGTGTTGACGCAGCCGCC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGTCTGCCCTGACTCAGCCTGC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCTCCTATGTGCTGACTCAGCCACC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCTCTTCTGAGCTGACTCAGGACCC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCACGTTATACTGACTCAACCGCC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGCTGTGCTCACTCAGCCGTC,
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCAATTTTATGCTGACTCAGCCCCA;
VLλ フォワード (Xho I 下線あり) -
GCCTGAACCGCCTCCACCACTCGAGCCTAGGACGGTGACCTTGGTCCC,
GCCTGAACCGCCTCCACCACTCGAGCCTAGGACGGTCAGCTTGGT CCC,
GCCTGAACCGCCTCCACCACTCGAGCCTAAAACGGTGAGCTGGGTCCC;
CLλ フォワード - TGAAGATTCTGTAGGGGCCACTGTCTT;
VH バック (ApaL 部位下線あり)-
CATGACCACAGTGCACTTCAGGTGCAGCTGGTGCAGTCTGG,CATGACCACAGTGCACTTCAGGTCAACTTAAGGGAGTCTGG,
CATGACCACAGTGCACTTGAGGTGCAGCTGGTGGAGTCTGG,CATGACCACAGTGCACTTCAGGTGCAGCTGCAGGAGTCGGG,
CATGACCACAGTGCACTTCAGGTGCAGCTGTTGCAGTCTGC,CATGACCACAGTGCACTTCAGGTACAGCTGCAGCAGTCAGG;
VH フォワード (Not I 部位下線あり)-
GAGTCATTCTGCGGCCGCGGGGAAGACSGATGGGCCCTTGGT,GAGTCATTCTGCGGCCGCGGGGAAAAGGGTTGGGGCGGATGC;
R−置換変異;S-サイレント変異。*、アミノ酸の数。生殖細胞系列対応物はhttp://www.ncbi.nim.nih.gov/igblastから同定した。CDRはカバットデータベースで比較して同定した。突然変異の計数はV遺伝子の3’末端に限定した。FR1残基1-7はPCRバックプライマーによってコードされているので考慮しなかった。ファミリー及びサブグループの指定はhttp://immuno.bme.nwu.edu/による。5’及び3’方向で決定されたcDNA配列は同一であった。生殖細胞系列V及びJ遺伝子のアラインメントはV-(D)-J組換えによる広い多様性を示唆した。この理由のため生殖細胞系列D遺伝子は指定できなかった。Fv JL413はVH遺伝子3’末端及びJ遺伝子5’末端に、それぞれ20及び17の欠失を含んでいた。
1.Pantoliano, M.W., Bird, R.E., Johnson, S., Asel, E.D., Dodd, S.W., Wood, J.F. and Hardman, K.D. Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli. Biochemistry. 22:10117-10125, 1991.
2.Masat, L., Wabl, M. and Johnson, J.P. A simpler sort of antibody. Proc Natl Acad Sci USA. 91:893-896, 1994.
3.Sun, M., Li, L., Gao, Q.S. and Paul, S. Antigen recognition by an antibody light chain. J Biol Chem. 269:734-738, 1994.
4.Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J., Griffiths, A.D. and Winter, G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 222:581-597, 1991.
5.Paul, S., Tramontano, A., Gololobov, G., Zhou, Y.X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. Phosphonate ester probes for proteolytic antibodies. J Biol Chem. 276:28314-28320, 2001.
6.Tyutyulkova, S., Gao, Q.S., Thompson, A., Rennard, A. and Paul, S. Efficient vasoactive intestinal polypeptide hydrolyzing autoantibody light chains selected by phage display. Biochim Biophys Acta. 1316:217-223, 1996.
7.Karle, S., Nishiyama, Y., Zhou, Y.X., Luo, J., Planque, S., Hanson, C. and Paul, S. Carrier-dependent specificity of antibodies to a conserved peptide determinant of gp120. Vaccine. 21:1213-121, 2003.
8.Nossal, G.J. B lymphocyte physiology: the beginning and the end. Ciba Found Symp. 204:220-230, 1997.
9.Karle, S., Planque, S., Nishiyama, Y., Taguchi, H., Zhou, Y.X., Salas, M., Lake, D., Thiagarajan, P., Arnett, F., Hanson, C.V. and Paul, S. Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library. AIDS. 18:329-331, 2004.
10.Lake, D.F., Kawamura, T., Tomiyama, T., Robinson, W.E., Matsumoto, Y., Masuho, Y. and Hersh, E.M. Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro. AIDS. 6:17-24, 1992.
11.Neurath, R.A., Strick, N. and Lee, E.S. B cell epitope mapping of human immunodeficiency virus envelope glycoproteins with long (19- to 36-residue) synthetic peptides. J Gen Virol. 71:85-95, 1990.
12.Morrow, W.J., Williams, W.M., Whalley, A.S., Ryskamp, T., Newman, R., Kang, C.Y., Chamat, S., Kohler, H. and Kieber-Emmons, T. Synthetic peptides from a conserved region of gp120 induce broadly reactive anti-HIV responses. Immunology. 75:557-564, 1992.
抗原結合の目的のため、Vドメインは最小の機能ユニットであると考えられる。正しい特異性を有する抗HIV抗体断片が得られると、これらの抗体断片を標準の抗体工学処理法で改善することができる。治療グレード抗体を改変する実行可能性は、免疫されていないヒト患者から調製されたファージライブラリーを使った腫瘍壊死因子に対するヒトFv構築物の開発によって支持される。完全長IgGとして再クローン化されたこの構築物はリウマチ様関節炎の治療によいと最近認められた(1)。
ループスFvクローンの中和能力は、HIV免疫治療の候補として提案されている一価のFab断片及び二価のIgG抗体に匹敵する。可能性のある更なる進歩は一価のFvクローンを二価のIgGとして再クローニングすることによって実現される。一価のFabのIgGバージョンは結合活性を増強することにより400倍に増加した中和される可能性を提示すると既に報告された(2)。一価のFvの十価の発現はHIV-1結合活性をさらに増加するはずである。
既に述べたように、抗原の結合親和性の強さは抗体の中和能の重要な決定因子である。最大の結合親和性を発現するB細胞受容体(Igα及びIgβサブユニットに複合された表面抗体)に対する抗原の選択的結合は、B細胞の増殖を促進する。従って、結合親和性を増強するVドメイン突然変異が選択される。親和性成熟と称される過程である。この過程はインビトロで以下のようにシュミレートされる。突然変異体を、変異原性プライマーを使ったCDRに導入して、変異分子をファージの表面に発現させる。抗原結合を、最も大きな結合親和性を有するファージを分別するのに用いる。この工程を数回繰り返し、更なる突然変異体が各サイクルで導入され、続いて抗原結合によるファージ分離を行う。1010-1011M-1(Ka)の大きさの結合親和性を有する抗原特異的Fvクローンが、未免疫のヒトドナーによって発現されたFvレパートリーを出発物質として使用して、得られた。VL及びVHドメインの6つのCDRは約100アミノ酸を含む。20の天然アミノ酸のそれぞれを用いてこれらの残基で包括的に変異させた抗体の研究は、結果として生じる突然変異ライブラリーの大きなサイズのため(〜10020クローン)非現実的である。CDRH3での抗原接触は抗原-抗体相互作用に対する特異性を付与すると考えられるので、VHドメインのCDR3が突然変異を導入するのにしばしば選択される。幾つかのグループは、VH CDR3の構造の最適化は抗原結合特性を改善すると報告している(7-10)。このストラテジーによって改善されたHIV-1認識の例を以下に示す。
Fvクローンの他に、gp120に対する正しい特異性を提示するループスライブラリーから得られたL鎖クローンを、工学的方法による改善に利用可能である。抗体Vドメインは、VL及びVHドメインにより形成された生来の結合部位と比較して減少した結合親和性を有するにもかかわらず、互いに関係なく抗原を認識できる。抗HIV L鎖を含む個々のVLドメインの結合活性は、適切なVHライブラリーから適合するVHドメインを検索することによって改善される。このアプローチの可能性は以下の検討事項によって示唆される:(a)VL及びVHドメインは、独立に抗原と結合することができ(12、13)、VHドメインは全抗原結合特異性に主要な寄与を提供する(14)。この一例は、VIP認識L鎖をそのパートナーVHドメインと対合することによって改善された抗原VIPの認識である(15)。原則としてこのようなアプローチは天然の抗原特異的抗体よりも優れた抗体を得るのに用いることができるであろう。インビトロでの親和性改善の繰り返されるラウンドに対する障害は何もない。相対的に、B細胞発達を支配する生物学的な力によってインビボでの抗体特異性及び親和性に上限が強いられる。一方、2つのVドメインを別々に操る能力は、有用な目標に活用され得る。
必要であればFvのVドメインの配向性を変える。Fv結合を詳細に調べているいくつかのグループは、どちらの配向であっても(VH-VL又はVL-VH)抗原に結合するFvの能力に、有意な違いを見つけなかった(17、18)。簡単にいえば、オリゴヌクレオチドプライマーを合成して、5’位置に連結し得るように、Sfi IとXho I 制限酵素認識部位を有するVHをPCR増幅する。同様に合成して、Apa LI部位及びNot I部位にリンカーの3’を連結させるためにVLを増幅する。両方の配向性のFvを精製してgp120への結合とHIV中和をテストする。
既に述べたように、Fv構築物は分子間凝集を起こすことができる(19-21)。このような効果を測定するため、Fvをゲルろ過カラムで分析する。各多量体種に対応するピークを、標準タンパク質の滞留時間とで比較することにより特定し、単量体及び凝集した状態で存在するFvの割合を計算する。ELISA調査を可溶性Fv濃度の関数として行い、その結果を凝集現象の濃度依存性と比較する。
1.van de Putte, L.B., Rau, R., Breedveld, F.C., Kalden, J.R., Malaise, M.G., van Riel, P.L., Schattenkirchner, M., Emery, P., Burmester, G.R., Zeidler, H., Moutsopoulos, H.M., Beck, K. and Kupper, H. Efficacy and safety of the fully human anti-tumour necrosis factor alpha monoclonal antibody adalimumab (D2E7) in DMARD refractory patients with rheumatoid arthritis: a 12 week, phase II study. Ann Rheum Dis. 62:1168-1177, 2003.
2.Kessler, J.A., McKenna, P.M., Emini, E.A., Chan, C.P., Patel, M.D., Gupta, S.K., Mark, G.E., Barbas, C.F., Burton, D.R. and Conley, A.J. Recombinant human monoclonal antibody IgG1b12 neutralizes diverse human immunodeficiency virus type 1 primary isolates. AIDS Res Hum Retroviruses. 13:575-582, 1997.
3.Coloma, M.J., Hastings, A., Wims, L.A. and Morrison, S.L. Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. J Immunol Methods. 152:89-104, 1992.
4.Shin, S.U. and Morrison, S.L. Production and properties of chimeric antibody molecules. Methods Enzymol. 178:459-76, 1989.
5.Paul, S. Protein engineering. In Walker, J. (ed) Molecular Biotechniques. Totowa: Humana Press, pp. 547-566, 1998.
6.Brinkmann, U., Buchner, J. and Pastan, I. Independent domain of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections. Proc Nat Acad Sci USA. 89:3075-3079, 1992.
7.Yang, W.P., Green, K., Pinz-Sweeney, S., Briones, A.T., Burton, D.R. and Barbas, C.F. CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J Mol Biol. 254:392-403, 1995.
8.Barbas, C.F., Bain, J.D., Hoekstra, D.M. and Lerner, R.A. Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Proc Natl Acad Sci USA. 89:4457-4461, 1992.
9.Hoogenboom, H.R. and Winter, G. By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol. 227:381-388, 1992.
10.Barbas, C.F. Hu, D., Dunlop, N., Sawyer, L., Cababa, D., Hendry, R.M., Nara, P.L. and Burton, D.R. In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity. Proc Natl Acad Sci USA. 91:3809-3813, 1994.
11.Luo, G.X., Kohlstaedt, L.A., Charles, C.H., Gorfain, E., Morantte, I., Williams, J.H. and Fang, F. Humanization of an anti-ICAM-1 antibody with over 50-fold affinity and functional improvement. J Immunol Methods. 275:31-40, 2003.
12.Ward, E.S., Gussow, D., Griffiths, A.D., Jones, P.T. and Winter, G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature. 341:544-546, 1989.
13.Sun, M., Li, L., Gao, Q.S. and Paul, S. Antigen recognition by an antibody light chain. J Biol Chem. 269:734-738, 1994.
14.Davies, D.R. and Chacko, S. Antibody structure. Acc Chem Res. 26:421-427, 1993.
15.Sun, M., Gao, Q.S., Kirnarskiy, L., Rees, A. and Paul, S. Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain. J Mol Biol. 271:374-385, 1997.
16.Karle, S., Nishiyama, Y., Zhou, Y.X., Luo, J., Planque, S., Hanson, C. and Paul, S. Carrier-dependent specificity of antibodies to a conserved peptide determinant of gp120. Vaccine. 21:1213-1218, 2003.
17.Hamilton, S., Odili, J., Gundogdu, O., Wilson, G.D. and Kupsch, J.M. Improved production by domain inversion of single-chain Fv antibody fragment against high molecular weight proteoglycan for the radioimmunotargeting of melanoma. Hybrid Hybridomics, 20:351-360, 2001.
18.Lawrence, L.J., Kortt, A.A., Iliades, P., Tulloch, P.A. and Hudson, P.J. Orientation of antigen binding sites in dimeric and trimeric single chain Fv antibody fragments. FEBS Lett. 425:479-84, 1998.
19.Pluckthun, A. and Skerra, A. Expression of functional antibody Fv and Fab fragments in Escherichia coli. Methods Enzymol. 178:497-515, 1998.
20.Skerra, A. and Pluckthun, A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 240:1038-1041, 1988.
21.Worn, A. and Pluckthun, A. Stability Engineering of antibody single-chain Fv fragments. J Mol Biol. 305:989-1010, 2001.
22.Tang, Y., Jiang, N., Parakh, C. and Hilvert, D. Selection of linkers for a catalytic single-chain antibody using phage display technology. J Biol Chem. 271:15682-15686, 1996.
ループス患者における、gp120の残基421-436に対して相同性を有する発現可能な内在性配列の同一性を決定することは、gp120を認識する抗体の合成を促進するHERV成分を同定すること及び新規なワクチン候補を開発することを含む幾つかの目的に有用である。この目的に用いられるストラテジーの例を以下に示す。
残基422-432に相同的な内在性レトロウイルス配列に対する増加した発現及び/又は増加した免疫反応性は、gp120を認識する抗体の合成を促進すると信じられる。これは、残基421-432がループス抗体認識に対するコアエピトープを形成することを示唆する先の研究及びgp120残基422-432に対して部分的な相同性を有するゲノムHERV-L配列を同定する新しいデータベース分析に基づいている。残基422-432と公知のヒトタンパク質との間の配列相同性は明らかになっていないが、gp120の他の領域はいくつかのタンパク質に相同的である(1-3)。
gp120残基422-432に対応するPCR産生物の同一性が確認されたら、健康な個体とループス患者のmRNAレベルの比較測定は、同時定量できるRT-PCRによることが好ましい(つまり発現がループス患者に限定されない場合)。簡単にいえば、蛍光標識したプライマーを用い、各PCRサイクルで蛍光の増加が生じ(増幅された産生物の加水分解によって測定される)、この蛍光増加は産生分子の数に直接比例し、従って、鋳型のアンプリコンの数の直接的尺度となる。少なくとも各20の健康な及びループス患者から得られた全RNAをRNaseフリーDNase Iで処理し、鋳型とする。プライマー配列は上記で得られたcDNA配列情報に基づく。初めの数サイクルは比較的低い融解温度(Tm)でプライマーセットを使用して実施する。PCRの第二段階では、蛍光プライマーをより高い融解温度でアニールするように設計する。信頼性のあるシグナルを得るために必要なPCRサイクルの数を標準化する。β-アクチン又はサイクロフィリンmRNAに類似のmRNAの増幅を並行して行う。
更なる実験手技が先の研究の結果により決定される。例えば上で同定されたcDNAを既に特徴づけされた完全長HERVタンパク質に対応させることができよう。別の筋書きは、残基422-432に相同な比較的短いHERV成分が非HERVタンパク質をコードする遺伝子に挿入されるということである。例として、以下、ある一般的な方法について簡単に説明する。gp120に相同であると同定される遺伝子断片を、市販で入手可能なキットを使って[32P]dCTPで放射能標識し、ハイブリダイゼーションプローブとして適用して、Clontechから入手可能なλファージミドのヒト白血球発現ライブラリー(585人の白人からプールされたヒトRNAから得られ、>3k塩基の長いcDNA挿入物を含み、完全長遺伝子にほとんど対応する)等のcDNAライブラリーをスクリーニングする。所望の遺伝子がループス患者にのみ発現されることが分かったら、(8)にあるように、ループス患者のPBMCに由来する新たなcDNAライブラリーを構築する。標準的なハイブリダイゼーション方法を、プローブとアニーリングするクローンを同定して配列決定するのに適用でき、gp120残基422-432に相同なペプチド決定基をコードする完全長遺伝子を同定するのに役立つ。オープンリーディングフレームの存在の確認に続いて(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)、完全長に対応するcDNAを適切な発現ベクター(例えば適切な翻訳後プロセッシングを確実にするバキュロウイルス系;his6タグを導入して迅速な精製を可能にする)で再クローニングする。
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Claims (3)
- 残基421-436を含む高度に保存されたエピトープgp120に結合し、それによってHIVの遺伝学的に分岐した株を中和することのできる単離された抗体又はその断片であって、
配列番号46に示すアミノ酸配列を含むVLドメイン及び配列番号47に示すアミノ酸配列を含むVHドメイン、又は
配列番号44に示すアミノ酸配列を含むVLドメイン及び配列番号45に示すアミノ酸配列を含むVHドメイン
を含む、前記抗体又はその断片。 - 前記断片が一本鎖Fvである、請求項1に記載の抗体又はその断片。
- 残基421-436を含む高度に保存されたエピトープgp120に結合し、それによってHIVの遺伝学的に分岐した株を中和することのできる、配列番号43に示すアミノ酸配列を含むVLドメインを含む単離された抗体のL鎖。
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CA2604683C (en) | 2005-04-12 | 2019-04-30 | Duke University | Method of inducing neutralizing antibodies to human immunodeficiency virus |
AU2008239628B2 (en) | 2007-04-13 | 2014-01-30 | Duke University | Method of inducing neutralizing antibodies to human immunodeficiency virus |
WO2009023043A1 (en) | 2007-04-23 | 2009-02-19 | Sudhir Paul | Immunoglobulins directed to bacterial viral and endogenous polypeptides |
JP2013505201A (ja) | 2009-04-03 | 2013-02-14 | デューク ユニバーシティー | 広域反応性中和抗hiv抗体を誘導するための配合物 |
US10076567B2 (en) | 2013-09-27 | 2018-09-18 | Duke University | MPER-liposome conjugates and uses thereof |
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US5952462A (en) * | 1983-11-29 | 1999-09-14 | Igen International Inc. | Transition state analogs |
US5807715A (en) * | 1984-08-27 | 1998-09-15 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin |
US5445960A (en) * | 1988-03-31 | 1995-08-29 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | Monoclonal antibodies specific for HIV and hybridomas for their production |
US5695927A (en) * | 1988-03-31 | 1997-12-09 | The University Of Arizona, Department Of Internal Medicine, Section Of Hematology And Oncology | Monoclonal antibodies specific for HIV and the hybridomas for production thereof |
US6309880B1 (en) * | 1989-04-25 | 2001-10-30 | Tanox, Inc. | Antibodies specific for CD4-binding domain of HIV-1 |
DE69233254T2 (de) * | 1991-06-14 | 2004-09-16 | Genentech, Inc., South San Francisco | Humanisierter Heregulin Antikörper |
US5605793A (en) * | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
WO1997003696A1 (en) * | 1995-07-21 | 1997-02-06 | University Of Nebraska Board Of Regents | COMPOSITIONS AND METHODS FOR CATALYZING HYDROLYSIS OF HIV gp120 |
US6855804B2 (en) * | 1998-03-23 | 2005-02-15 | Board Of Regents, The University Of Texas System | Covalently reactive transition state analogs and methods of use thereof |
US6235714B1 (en) * | 1998-03-23 | 2001-05-22 | Sudhir Paul | Methods for identifying inducers and inhibitors of proteolytic antibodies, compositions and their uses |
US6406863B1 (en) * | 2000-06-23 | 2002-06-18 | Genetastix Corporation | High throughput generation and screening of fully human antibody repertoire in yeast |
US8980646B2 (en) | 2003-03-26 | 2015-03-17 | Sudhir Paul | Proteolytic and covalent antibodies |
EP1610808A4 (en) | 2003-03-26 | 2011-04-06 | Sudhir Paul | COVALENT BINDING OF LIGANDS TO NUCLEOPHILIC PROTEINS UNDER GUIDANCE BY NONCOVALENT BINDING |
JP2007524603A (ja) | 2003-03-27 | 2007-08-30 | ザ ユニバーシティー オブ テキサス | Hiv/エイズの受動免疫治療用ループス抗体 |
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WO2004087738A3 (en) | 2007-08-23 |
US20070105218A1 (en) | 2007-05-10 |
EP1613643A4 (en) | 2009-10-28 |
EP1613643A2 (en) | 2006-01-11 |
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JP2011137013A (ja) | 2011-07-14 |
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