JP2022541303A - Wrsタンパク質に特異的に結合する抗体及びその用途 - Google Patents
Wrsタンパク質に特異的に結合する抗体及びその用途 Download PDFInfo
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Abstract
Description
a)個体から試料を取得する段階;
b)前記試料において抗体又は抗体の断片でWRSタンパク質の発現量を測定する段階;及び
c)前記b)段階で測定したタンパク質の発現量が増加する場合、癌であると判断する段階;を含む癌診断方法を提供することにある。
a)個体から試料を取得する段階;
b)前記試料において抗体又は抗体の断片でWRSタンパク質の発現量を測定する段階;及び
c)前記b)段階で測定したタンパク質の発現量が増加する場合、感染疾患又は感染合併症であると判断する段階;を含む感染疾患又は感染合併症診断方法を提供することにある。
a)個体から試料を取得する段階;
b)前記試料において抗体又は抗体の断片でWRSタンパク質の発現量を測定する段階;及び
c)前記b)段階で測定したタンパク質の発現量が増加する場合、癌であると判断する段階;を含む癌診断方法を提供する。
a)個体から試料を取得する段階;
b)前記試料において抗体又は抗体の断片でWRSタンパク質の発現量を測定する段階;及び
c)前記b)段階で測定したタンパク質の発現量が増加する場合、感染疾患又は感染合併症であると判断する段階;を含む感染疾患又は感染合併症診断方法を提供する。
a)個体から試料を取得する段階;
b)前記試料において抗体又は抗体の断片でWRSタンパク質の発現量を測定する段階;及び
c)前記b)段階で測定したタンパク質の発現量が増加する場合、癌であると判断する段階;を含む癌診断方法を提供する。
i)個体から試料を取得する段階;
ii)前記試料からWRSタンパク質の発現水準を測定する段階;
iii)前記ii)段階で測定されたタンパク質が全て発現された場合、癌であると判断する段階;及び
iv)前記判断された個体に癌を治療するための治療薬物(抗癌剤など)を投与したり、放射線を照射したり、手術を行うことによって癌を治療する段階。
a)個体から試料を取得する段階;
b)前記試料において抗体又は抗体の断片でWRSタンパク質の発現量を測定する段階;及び
c)前記b)段階で測定したタンパク質の発現量が増加する場合、感染疾患又は感染合併症であると判断する段階;を含む感染疾患又は感染合併症診断方法を提供する。
i)個体から試料を取得する段階;
ii)前記試料からWRSタンパク質の発現水準を測定する段階;
iii)前記ii)段階で測定されたタンパク質が全て発現された場合、感染疾患又は感染合併症であると判断する段階;及び
iv)前記判断された個体に感染疾患又は感染合併症を治療するための治療薬物を投与したり、手術を行うことによって感染疾患又は感染合併症を治療する段階。
(1)ハイブリドーマ細胞の製作
1)動物免疫化と細胞融合
-免疫原の準備:1.5mg~2mgのWRSタンパク質(純度>75%、濃度>0.4mg/ml)
-動物免疫化:Balb/cマウスに免疫原を接種し、抗体生産を誘発した。
-細胞融合:マウスから得たB細胞とマウスの骨髄腫細胞(myeloma cell)を電気融合(electro-fusion)し、少なくとも10,000個のハイブリドーマ細胞を得た。
-1次選別:間接ELISAを通じて抗原に結合する抗体を生産するハイブリドーマ細胞を選別した。
-2次選別:1次選別で得た陽性クローンを用いて、ウェスタンブロッティングで抗原に結合するハイブリドーマ細胞株1種を選別し、選別されたハイブリドーマ細胞株で生産される抗体を4D10G6と命名した。
-アイソタイピング(Isotyping):選別作業中、最も結果が良いクローン5個を選び、アイソタイピングを進行した。
-サブクローニング、細胞増量、凍結保管:結果が良いクローンに対してサブクローニング、細胞増量、凍結保管を進行した。
-抗体生産:選別作業中、最も結果が良いハイブリドーマ細胞株において少なくとも2mgの抗体が生産されるように抗体を生産した。
1)マウスが3日以上の適応期間を経ると、プリスタンアジュバント(Pristane adjuvant)を100μl/mouceで投与した。ハイブリドーマ細胞株は、プリスタンアジュバントを投与してから5日~7日が経過したときに注入できるように培養した。
1)生産した腹水は、-70℃で取り出し、4℃で溶かし、精製する抗体のサブタイプ(subtype)を確認し、使用するビードを決定した。使用されるビードの量は、腹水の0.5volumeであった。
総RNAは、TRIzol試薬の技術マニュアルによってハイブリドーマ細胞から分離した。PrimeScript 1st Strand cDNA Synthesis Kitの技術マニュアルにより、汎用プライマーを用いて総RNAをcDNAに逆転写させた。重鎖可変領域(VH)及び軽鎖可変領域(VL)の抗体の断片は、RACD(rapid amplification of cDNA ends)によって増幅した。増幅された抗体の断片を標準クローニングベクターに別途にクローニングした。コロニーPCRは、正確な大きさの挿入物を有するクローンをスクリーニングするために行われた。正確な大きさのインサートを有する5個以上のコロニーがそれぞれの断片に対して配列化された。異なるクローンの配列を整列し、これらのクローンのコンセンサス配列を提供した。
前記実施例1で製造したモノクローナル抗体が認識するポリペプチド領域を確認するために、471個のアミノ酸からなる配列番号1のWRSタンパク質(1-471)及びタンパク質の断片(48-471、1-104、1-154及び45-154)を準備した。
前記実施例1で製造されたモノクローナル抗体及び商用抗体2種(Abnova、抗-WRS抗体(Cat# H00007453-M02)及びNovus biological、抗-WRS抗体(Cat# NBP2-32186))の結合親和性を評価するために、配列番号1の全長(full-length)WRSタンパク質に対する間接ELISAアッセイを行った。
前記実施例1で製造されたモノクローナル抗体及び商用抗体2種(Abnova、抗-WRS抗体(Cat# H00007453-M02)及びNovus biological、抗-WRS抗体(Cat# NBP2-32186))の結合特異性を評価するために、HCT116細胞の溶解物20μgに対して下記の条件によるそれぞれの1次抗体及び2次抗体を処理し、通常の方法によってウェスタンブロットを行った:
*1次抗体(常温、1時間)
4D10G6:1μg/ml
Abnova Ab:1:5,000dilution
Novus Ab:1:10,000dilution
*2次抗体(常温、1時間)
抗マウス(Anti-mouse)HRP(Millipore、AP181P):1:10,000dilution:Abnova、4D10G6
抗ウサギ(Anti-rabbit)HRP(Millipore、AP187P):1:10,000dilution:Novus
前記実施例1で製造されたモノクローナル抗体が、WRS以外に細胞外に分泌される更に他のARS(Aminoacyl-tRNA synthetase)タンパク質であるCRS(Cysteinyl-tRNA synthetase)、AIMP1(Aminoacyl tRNA synthase complex-interacting multifunctional protein 1)、GRS(Glycyl tRNA synthetase)及びKRS(Lysyl tRNA synthetase)に対して交差反応性を示すかどうかを確認するために、次のような方法によって間接ELISAアッセイを行った:
Claims (18)
- WRS(tryptophanyl-tRNA synthetase)タンパク質において配列番号2で表されるアミノ酸配列のポリペプチドに特異的に結合する抗体又は抗体の断片。
- 前記抗体は、配列番号3で表されるアミノ酸配列を含む相補性決定部位(CDR)L1、配列番号4で表されるアミノ酸配列を含む相補性決定部位(CDR)L2、及び配列番号5で表されるアミノ酸配列を含む相補性決定部位(CDR)L3を含む抗体軽鎖可変領域(VL)と、配列番号6で表されるアミノ酸配列を含む相補性決定部位(CDR)H1、配列番号7で表されるアミノ酸配列を含む相補性決定部位(CDR)H2、及び配列番号8で表されるアミノ酸配列を含む相補性決定部位(CDR)H3を含む抗体重鎖可変領域(VH)と、を含む、請求項1に記載の抗体又は抗体の断片。
- 前記抗体又は抗体の断片は、配列番号9で表されるアミノ酸配列を含む軽鎖可変領域;及び配列番号10で表されるアミノ酸配列を含む重鎖可変領域;を含むことを特徴とする、請求項1に記載の抗体又は抗体の断片。
- 前記抗体は、モノクローナル抗体であることを特徴とする、請求項1に記載の抗体又は抗体の断片。
- 前記抗体は、IgG、IgA、IgM、IgE及びIgDからなる群から選ばれることを特徴とする、請求項1に記載の抗体又は抗体の断片。
- 前記抗体の断片は、ダイアボディ、Fab、Fab'、F(ab)2、F(ab')2、Fv及びscFvからなる群から選ばれることを特徴とする、請求項1に記載の抗体又は抗体の断片。
- 請求項1~6のいずれか1項に記載の抗体又は抗体の断片をコードするポリヌクレオチド。
- 請求項7に記載のポリヌクレオチドを含むベクター。
- 請求項8に記載のベクターで形質転換された細胞。
- 請求項9に記載の細胞を、ポリヌクレオチドが発現される条件下で培養し、軽鎖及び重鎖可変領域を含むポリペプチドを生産する段階、及び前記細胞又はこれを培養した培養培地から前記ポリペプチドを回収する段階を含むWRSに結合する抗体又は抗体の断片生産方法。
- 請求項1~6のいずれか1項に記載の抗体又は抗体の断片を含む癌診断用組成物。
- 請求項1~6のいずれか1項に記載の抗体又は抗体の断片を含む感染疾患又は感染合併症診断用組成物。
- 前記感染疾患は感染性炎症疾患であることを特徴とする、請求項12に記載の診断用組成物。
- 前記感染性炎症疾患は、敗血症又は敗血症性ショックであることを特徴とする、請求項13に記載の診断用組成物。
- 癌診断用製剤を製造するための請求項1~6のいずれか1項に記載の抗体又は抗体の断片の使用。
- a)個体から試料を取得する段階;
b)前記試料において請求項1~6のいずれか1項に記載の抗体又は抗体の断片でWRSタンパク質の発現量を測定する段階;及び
c)前記b)段階で測定したタンパク質の発現量が増加する場合、癌であると判断する段階;を含む癌診断方法。 - 感染疾患又は感染合併症診断用製剤を製造するための請求項1~6のいずれか1項に記載の抗体又は抗体の断片の使用。
- a)個体から試料を取得する段階;
b)前記試料において請求項1~6のいずれか1項に記載の抗体又は抗体の断片でWRSタンパク質の発現量を測定する段階;及び
c)前記b)段階で測定したタンパク質の発現量が増加する場合、感染疾患又は感染合併症であると判断する段階;を含む感染疾患又は感染合併症診断方法。
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