JP2019522966A - 患者由来腫瘍スフェロイドを用いた抗体スクリーニング方法 - Google Patents
患者由来腫瘍スフェロイドを用いた抗体スクリーニング方法 Download PDFInfo
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Abstract
Description
以下、本発明を実施例を挙げて詳述する。これらの実施例は単に本発明をより具体的に説明するためのものであり、本発明の範囲がこれらの実施例に制限されないことは当業者において通常の知識を有する者にとって自明である。
抗−NRP1抗体断片の同定のための細胞パンニング(cell panning)に必要な細胞を選別するために、事業団(サムスンメディカルセンターの難治癌研究事業団)で保有している患者由来細胞のNRP1発現レベルを、RNA−Seqの後、RPKM(Reads Per Killobase Million)方法により分析し(図12)、cultureの有無とFACS(Fluorocence Activated Cell Sorting)方法により選別して、患者由来細胞を細胞パンニングに用いた。
3回目の細胞パンニングで回収されたファージ粒子を、宿主細胞(ER2537)感染により培養培地でコロニーとして確認した。このコロニーを取り、200μlのSB/アンピシリン培養培地の入った96−ウェルプレートに接種した後、37℃で2−3時間培養した。
実施例1における3回目の細胞パンニングで回収した候補抗体を、実施例1で使用した患者由来細胞が皮下移植されたマウスに腫瘍内投与(intra−tumoral injection)により注入した。
ファージミドの基本構成は図4で確認することができる。上記の実施例で使用された宿主細胞ER2537の場合、phage pIIIの前に位置した転写抑制コドン(amber codon(UAG))を抑制するため、scFv単独発現が不可能である。したがって、非抑制宿主(non−suppressor strain)である発現菌株(TOP10F´)を用いて、ファージミドを発現菌株中に形質導入した。その後、DNA配列分析により、各ファージミドが突然変異なしに導入された発現菌株であることを確認した。この発現菌株をコロニーとして取った後、LB/アンピシリン培養培地3mlに接種してから、37℃で一晩培養した。その後、一晩培養させた培養液3mlを400mlの培養培地(SB/アンピシリン)に移し、O.D600で0.5−0.7になるまで培養した。最終濃度1mMのIPTGを添加し、30℃で一晩培養した。培養液は、遠心分離後に、TES溶液40mlを用いて発現宿主を溶解させた後、0.2 x TES60mlを投入して、周辺質中のファージ粒子を回収した。回収した上清液は、0.45μmのフィルターで濾過した。濾過された溶液中に存在するscFvタンパク質は、His−tag精製のために、Ni−NTA bead(Qiagen)1mlが添加されて常温で1時間結合された後、重力カラム(gravity column、Bio−rad)にパッキングされ、200mMのイミダゾール溶液を用いて回収した。各クローンの発現および精製の後、SDS−PAGEおよびクーマシー染色(coomassie blue staining)により、scFvの大きさが約28kDaであることを確認した(図5)。
ヒトNRP1タンパク質に対する結合能をELISAにより確認した後、実際に細胞膜に存在するNRP1に結合するかを調べるために、NRP1の発現が高い患者由来細胞を用いてFACS分析をした。各scFv当たり5X105個の患者由来細胞と4℃で約1時間結合させた後、FACS溶液1mlで3回洗浄した。その後、赤色蛍光(PE;phycoerithrin)が結合されたHA抗体1μgを処理し、4℃で30分間結合させた。次いで、FACS溶液1mlで3回洗浄した後、FACS CaliburTMシステムを用いて分析した。
3種の抗−NRP1抗体断片の場合、細胞中への透過性を細胞免疫蛍光染色法により確認した。チャンバースライドにPD−リシン溶液を入れ、常温で1−2時間コーティングさせた。その後、溶液を除去した後、スライドを乾燥させた。次に、5X104個の患者由来細胞が入っているNBA溶液200μlをスライドに処理した後、37℃で4−5時間培養してスライドに固定させた。次に、NBA溶液を除去し、4%のパラホルムアルデヒドを入れ、4℃で10分間固定させた。その後、PBSで洗浄過程を3回経た後、0.1%のTriton X−100を処理した細胞の透過度増進作業を行った。その後、NRP1タンパク質の染色のために、抗−ヒトNRP1抗体(R&D)と同時に抗−NRP1抗体断片を処理し、37℃で15/30/60分に分けて結合させた。PBSで洗浄過程を3回経た後、非特異的な結合を阻止するために、1%のBSA溶液で室温で1時間程度ブロッキングした。2次抗体としては、NRP1タンパク質が見られるように緑色蛍光(Alexa−Fluor 488)が標識された塩素抗−マウス抗体(Invitrogen)を処理し、抗−NRP1抗体断片を見るための抗−HA抗体(Santacruz biotechnology)を処理した後、常温で1時間結合させた。最後に、核を染色するためのDAPI染色を行った後、最終洗浄してから、ガラス蓋をスライド上に固定させ、共焦点レーザー走査顕微鏡を用いて観察した。
抗−NRP1抗体断片をIgG形態に形態転換させるために、NRP1抗体断片の重鎖配列と軽鎖配列の遺伝子をExpi 293F発現システム(life technologies)を用いて形質注入した。培養液にある抗−NRP1 IgG抗体を得るために、AKTAタンパク質精製システムとAmicon遠心分離フィルターを用いて精製した。生産量は、IRCR−101(IgG形態に転換した3H10)は120mg/L、1A03は66mg/L、4F12は15mg/Lであった。精製した抗−NRP1 IgG抗体の純度を確認するために、高速液体クロマトグラフィーを導入した。IgGの大きさが150kDであるため、マーカーピークが16.388分に現れる物質に該当する。3種の抗−NRP1 IgG抗体がこのピークで検出され、純度は99.5、99.4、99.5%であった(図13)。生産された3種の抗−NRP1 IgG抗体のエンドトキシン(endotoxin)数値を調べるために、Limulus Amebocyte Lysate(LAL)QCL−1000TMキットを用いた。分析結果、3種の抗体は0.5−3.1EU/mg程度であって、治療用タンパク質のエンドトキシン正常数値に該当した(図14)。
MNRP1685Aの結合ドメインがVEGFドメインであるため、MNRP1685Aを陽性対照群として使用した。96ウェルプレートに、各ウェル当たりhNRP1タンパク質をコーティングさせた後、500nMのIRCR−101とMNRP1685Aを25℃で1時間結合させ、PBSTで洗浄した後、ビオチンを結合させたVEGF、Sema3Aを常温で15分間結合させた。
pHrodoR Red Microscale Labeling Kit(Thermo #p35363)を用いて、3種の抗−NRP1 IgG抗体が癌細胞と正常細胞に内在化される様相を比較した。前記キットの原理は、抗体に発色試料をコンジュゲート(conjugate)させ、該抗体が細胞外にある際には発色されず、細胞内に入り込んで周囲環境が酸性化すると発色することになることであり、この原理を用いて、該抗体の細胞内在化を確認することができる。3種の抗−NRP1 IgG抗体にコンジュゲートさせ、患者由来癌細胞と正常細胞であるHUVEC細胞を用いて内在化様相を比較した結果、患者由来癌細胞では、20分が経過した時から内在化された抗体が観察され始めた(図17)。
膠芽細胞癌細胞株であるU87MGと患者由来細胞を用いて、3種の抗−NRP1 IgG抗体が癌細胞の移動を抑制するかを確認した。各抗体処理後、37℃で24時間培養させてから確認し結果、IRCR−101、1A03の場合、両方の細胞で50%以上の癌細胞移動を抑制しており、4F12は、患者由来細胞で40%程度の癌細胞移動を抑制した(図20)。
膠芽細胞癌患者由来細胞を用いて2種の皮下投与モデル(subcutaneous model)を製作し、5mg/kgのIRCR−101を週3回静脈注射で注入した後、腫瘍サイズを測定した。対照群に比べて30−40%の腫瘍サイズを抑制し、免疫蛍光(Immunofluorescence)により、IRCR−101による細胞死(apoptosis)が増加することをTUNEL assayで確認した(図23)。
IRCR−101の副作用(side−effect)を調べるために、既存のNRP1抗体(MNRP1685A抗体、特許(WO2011143408A1)に記載の配列合成により自体製作)とそのとともに雄および雌のMonkey TMA(Tissue microArray)を行った。分析結果、殆どの正常臓器組織で、IRCR−101が既存のNRP1抗体よりも結合が低いか、殆どないことを確認した。したがって、正常組織に対して結合力が低いか、殆どないことから、臨床試験時にIRCR−101の副作用は低いと予測される(図27)。
Claims (9)
- (i)抗原を発現する患者由来細胞を、抗体またはその抗原結合断片を含むライブラリーで処理し、抗原に結合する抗体またはその抗原結合断片をスクリーニングするステップと、
(ii)前記スクリーニングされた抗体またはその抗原結合断片を、抗原が発現されない患者由来細胞と反応させるステップと、
(iii)前記ステップ(i)で選別された抗体またはその抗原結合断片のうち、ステップ(ii)の患者由来細胞に結合する抗体またはその抗原結合断片を分離・除去するステップと、
を含む、抗原に結合する抗体またはその抗原結合断片をスクリーニングする方法。 - (i)抗原を発現する患者由来細胞を、抗体またはその抗原結合断片を含むライブラリーで処理し、抗原に結合する抗体またはその抗原結合断片を1次スクリーニングするステップと、
(ii)前記1次スクリーニングされた抗体またはその抗原結合断片ライブラリーを、抗原が発現されない患者由来細胞に処理するステップと、
(iii)前記抗原を発現する患者由来細胞が移植されている動物モデルに、前記ステップ(i)で選別された抗体またはその抗原結合断片のうち、ステップ(ii)の患者由来細胞に結合する抗体またはその抗原結合断片を分離・除去した抗体またはその抗原結合断片を投与し、抗原に結合する抗体またはその抗原結合断片を2次スクリーニングするステップと、
(iv)前記2次スクリーニングされた抗体またはその抗原結合断片のうち、抗原以外の抗原に結合する抗体またはその抗原結合断片を分離・除去するステップと、
を含む、抗原に結合する抗体またはその抗原結合断片をスクリーニングする方法。 - 前記抗体またはその抗原結合断片は、細胞内に内在化される抗体またはその抗原結合断片であることを特徴とする請求項1または請求項2に記載の方法。
- 前記抗原を発現する患者由来細胞は、下記ステップを経て収得されることを特徴とする請求項1または請求項2に記載の方法:
(a)分離された癌患者由来の癌組織を粉砕した後、前記粉砕物から細胞分画を収得するステップ; および
(b)前記収得された細胞分画をタンパク質分解酵素で処理した後、濾過および遠心分離し、懸濁して単細胞化するステップ。 - 前記抗原を発現する患者由来細胞が移植されている動物モデルは、免疫欠乏マウスであることを特徴とする請求項2に記載の方法。
- 前記免疫欠乏マウスは、ヌードマウス、NOD(non−obese diabetic)マウス、SCID(Severe combined immunodeficiency)マウス、NOD−SCIDマウス、またはNOG(NOD/SCID I12rg−/−)マウスであることを特徴とする請求項5に記載の方法。
- 前記方法によって選別された抗体またはその結合断片をIgG形態で製作するステップをさらに含むことを特徴とする請求項1または請求項2に記載の方法。
- 請求項1または請求項2に記載の方法によってスクリーニングされた抗体またはその抗原結合断片。
- 請求項8に記載の抗体またはその抗原結合断片を含む、癌の予防または治療用組成物。
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CA3207763A1 (en) * | 2021-02-10 | 2022-08-18 | Zongda Wang | Anti-vegf antibody and use thereof |
WO2024091058A1 (ko) * | 2022-10-28 | 2024-05-02 | 주식회사 와이바이오로직스 | 세포 패닝 방법 |
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JP6895460B2 (ja) | 2021-06-30 |
CA3026236C (en) | 2021-01-26 |
JP2021097696A (ja) | 2021-07-01 |
AU2017273169B2 (en) | 2020-07-30 |
US11199536B2 (en) | 2021-12-14 |
KR101993892B1 (ko) | 2019-06-28 |
EP3480598A4 (en) | 2020-01-22 |
CN109416364A (zh) | 2019-03-01 |
US20190227052A1 (en) | 2019-07-25 |
KR20170137655A (ko) | 2017-12-13 |
EP3480598A2 (en) | 2019-05-08 |
SG11201810779PA (en) | 2019-01-30 |
AU2017273169A1 (en) | 2019-01-17 |
CN109416364B (zh) | 2022-04-12 |
CA3026236A1 (en) | 2017-12-07 |
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