JP7317712B2 - ネオアンチゲン特異的なt細胞受容体配列を単離する方法 - Google Patents
ネオアンチゲン特異的なt細胞受容体配列を単離する方法 Download PDFInfo
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Description
本特許出願は、2017年3月31日に出願された米国仮特許出願番号62/479,398の利益を主張するものであり、それは参照により組み込まれる。
本発明はアメリカ国立衛生研究所、全米がん研究所により与えられた、プロジェクト番号ZIABC010985の下における政府支援により行われた。政府は本発明において一定の権利を有する。
本明細書に全体として参照により組み込まれるのは、本明細書と同時に提出され、下記:2018年3月22日付けの”737921 SeqListing_ST25.txt”と名付けられた1つの5,530バイトのASCII(テキスト)ファイル、より同定される、コンピューター読み取り可能なヌクレオチド/アミノ酸の配列表である。
がん抗原(例えばネオアンチゲン)特異的なT細胞受容体(TCR)を発現するように遺伝的に加工されている細胞を使用する養子細胞療法(ACT)は、一部のがん患者においてポシティブな臨床応答を奏することができる。それにも関わらず、がんと他の疾患を幅広く治療するためにTCRが加工された細胞を成功裏に使用するためには障害が残っている。例えばがん抗原(例えばネオアンチゲン)を特異的に認識するTCRの同定及び/又は患者からの単離は困難であるかもしれない。従ってがん反応性(例えばネオアンチゲン反応性)のTCRを得る改善された方法に対する需要がある。
1態様において本発明は、対を成すTCRアルファとベータ鎖の配列、又はその抗原結合部分を単離する方法を提供する。
YX1CX2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22(配列番号5)
のアミノ酸配列モチーフをコードする、任意のcDNA配列(複数可)の同定により実施することができ、ここでX1からX9の各々は任意の天然由来のアミノ酸であり、X10からX21の各々はアミノ酸がないか又は任意の天然由来のアミノ酸であり、及び、X22はフェニルアラニン又はトリプトファンである。配列番号5のアミノ酸配列モチーフは、Vセグメントによりコードされるアミノ酸配列のC末端の近傍に位置する保存されたアミノ酸配列モチーフである。
本発明の目的のために、T細胞の様々な用量がそれぞれ与えられる1組の哺乳動物間で、哺乳動物にある特定の用量のそのようなT細胞が投与された際に、本発明のTCR、又はその抗原結合部分を発現するT細胞により、標的細胞が溶解され又はIFN-γが分泌される程度を比較することを含むアッセイが、哺乳動物に投与する開始用量を決定するために用いられ得る。ある用量の投与により標的細胞が溶解する又はIFN-γが分泌される程度は、本技術分野で既知の方法でアッセイできる。
実施例1~5において述べられた実験のために、下記の材料及び方法が採用された。
全ての患者材料は、全米がん研究所の審査委員会が承認した臨床治験(治験登録ID:NCT01174121)から得られた。ネオアンチゲンとネオアンチゲン反応性のTIL集団を同定する方法は、国際公開番号2016/053338に述べられている。簡単に言えば、腫瘍断片を切り出し、IL-2を含む培地(6000IU/mL)中で3~6週間培養した(Dudley et al., J. Immunother., 26: 332-342 (2003))。細胞数が増えたTIL培養物を、ネオアンチゲン認識についてスクリーニングした。増えたTIL培養物をネオアンチゲン認識についてスクリーニングするために、全エキソーム及びRNA配列決定(RNA-seq)により、腫瘍内の非同義変異を同定した。非同義変異を包含するタンデムミニ遺伝子(TMG)ライブラリーを合成した。TMGを発現している自家の樹状細胞(DC)をスクリーニングし、TILにより認識されるネオアンチゲン(複数可)を同定した(Lu et al., Clin. Cancer Res., 20: 3401-3410 (2014))(図1参照)。
単一細胞(single cell)のRNA配列決定(RNA-seq)データから、ネオアンチゲン特異的なTCR配列を同定する方法を、図2に示す模式図で概説する。ネオアンチゲン反応性のTIL培養物を同定した後に、1×106個のTILを、1×106個のTMGをパルスされた樹状細胞(DC)と共に4時間共培養した。共培養の後にT細胞を再懸濁し、大々的に洗浄した。その後、製造業者の指示(フリューダイム(サンフランシスコ、カリフォルニア))とクローンテック(マウンテンビュー、カリフォルニア)に従って、T細胞を単一細胞の分取(single-cell sorting)にかけ、RNA配列決定試料を調製した。96個全ての単一細胞のRNA配列決定試料を、NEXTERA XT DNAライブラリー調製キット(イルミナ(サンディエゴ、カリフォルニア州))を用いてバーコード化し、その後、試薬キットV3(2×250塩基対(b.p.))を使用したILLUMINA MISEQシステムを用いて配列決定した。
詳細なプロトコールは、Morganら、Science, 314: 126-129 (2006)に述べられており、本明細書に述べられた幾つかの小さな改変を伴っている。改変されたマウス定常領域を有し、フリンSGSGP2Aリンカー(RAKRSGSGATNFSLLKQAGDVEENPGP)(配列番号1)により連結された、全長TCRαとTCRβ配列を合成し、MSGV8レトロウイルス発現ベクター(Wargoら, Cancer Immunol. Immunother., 58: 383-394 (2009))にクローン化した。MSGV8-TCRプラスミド(1.5μg)と0.75μgのVSV-G(RD114)プラスミドを、(各6穴の中の)1×106個の293GP細胞の中に、LIPOFECTAMINE2000トランスフェクション試薬(サーモフィッシャーサイエンティフィック)を用いて共トランスフェクトした。48時間後に上清を回収し、1分当たり3000回転(rpm)で10分間回転し、破片を除去した。2000gで2時間遠心分離することにより、レトロウイルスの上清を、RETRONECTIN試薬(タカラ、大津、日本)で被覆された6穴プレート上に搭載した。
この実施例は、TIL4090培養物から、ネオアンチゲン特異的なTCRの対を成すアルファとベータ鎖配列を単離する方法を示す。
この実施例は、実施例1で単離されたTCRアルファとベータ鎖の配列が形質導入されたT細胞は、実施例1の患者のがんにより発現されたネオアンチゲンを特異的に認識することを示す。
この実施例は、TIL4095培養物から、ネオアンチゲン特異的なTCRの対を成すアルファとベータ鎖配列を単離する方法を示す。
この実施例は、実施例3で単離されたTCRアルファとベータ鎖配列を形質導入されたT細胞が、実施例3の患者のがんにより発現されたネオアンチゲンを特異的に認識することを示す。
この実施例は、TIL4112培養物から、ネオアンチゲン特異的なTCRの対を成すアルファとベータ鎖の配列を単離する方法を示す。
この実施例は、TIL4171培養物から、ネオアンチゲン特異的なTCRの対を成すアルファとベータ鎖配列を単離する方法を示す。
Claims (14)
- 対を成すT細胞受容体(TCR)アルファ及びベータ鎖の配列、又はその抗原結合部分の配列を単離する方法であって、
(a)生体試料から、がん特異的な変異によりコードされる変異したアミノ酸配列に対して抗原特異性を有するT細胞を単離する工程;
(b)単離されたT細胞を、変異したアミノ酸配列を提示する抗原提示細胞(APC)と共培養する工程であって、それによりT細胞は1つ以上のT細胞活性化マーカーを発現する工程;
(c)共培養されたT細胞を分取して分離した単一のT細胞試料とする工程;
(d)各々の分離した単一のT細胞試料からmRNAを単離する工程;
(e)各々の分離した単一のT細胞試料からmRNAを配列決定する工程であって、ここで該配列決定は:
(i)mRNAからcDNAを作製し、該cDNAを増幅する工程;
(ii)増幅された該cDNAの多様な断片を作製し、該多様な断片にタグを付す工程;
(iii)タグが付された該cDNAの多様な断片を増幅する工程;及び
(iv)増幅され、タグが付された該cDNAの多様な断片を配列決定する工程であって;ここで配列決定は、cDNAの多様な断片の各々の配列を同定する工程;
(f)cDNAの多様な断片の各々の配列を、1つ以上のT細胞活性化マーカーの既知の配列に対してアラインメントして、どの単一のT細胞試料が、1つ以上のT細胞活性化マーカーを発現する単一のT細胞を含むかを同定する工程;
(g)cDNAの多様な断片の各々の配列を、参照TCR配列データベースに対してアラインメントして、(f)で1つ以上のT細胞活性化マーカーを発現すると同定された、各々の分離した単一のT細胞試料のcDNAの多様な断片の、TCRアルファ鎖の可変(V)セグメント配列とTCRベータ鎖のVセグメント配列を同定する工程、
(h)(g)で同定されたTCRアルファ鎖のVセグメント配列を含むcDNAの多様な断片内と、(g)で同定されたTCRベータ鎖のVセグメント配列を含むcDNAの多様な断片内の、TCR相補性決定領域3(CDR3)配列を同定する工程;
(i)同じアルファ鎖のCDR3アミノ酸配列を共有するcDNAの多様な断片の数と、同じベータ鎖のCDR3アミノ酸配列を共有するcDNAの多様な断片の数を計測する工程;
(j)同じアルファ鎖のCDR3配列をコードする最も数が多いcDNAの多様な断片、同じベータ鎖のCDR3配列をコードする最も数が多いcDNAの多様な断片、及び任意選択で、同じアルファ鎖のCDR3配列をコードする2番目に数が多いcDNAの多様な断片を採取し、TCRアルファとベータ鎖のCDR3配列を同定する工程であって、ここで2番目に数が多いcDNAの多様な断片によりコードされるアルファ鎖のCDR3配列は、最も数が多いcDNAの多様な断片によりコードされるアルファ鎖のCDR3配列とは異なる工程;
(k)(j)で採取された最も数が多いcDNAの多様な断片のTCRアルファ鎖のVセグメント配列、(j)で採取された最も数が多いcDNAの多様な断片のTCRベータ鎖のVセグメント配列、及び任意選択で、(j)で採取された2番目に数が多いcDNAの多様な断片のTCRアルファ鎖のVセグメント配列を同定し、TCRアルファとベータ鎖のVセグメント配列を同定する工程;並びに
(l)(k)で同定されたTCRアルファ鎖のVセグメント配列と(j)で採取されたTCRアルファ鎖のCDR3配列を含むTCRアルファ鎖、及び
(k)で同定されたTCRベータ鎖のVセグメント配列と(j)で採取されたTCRベータ鎖のCDR3配列を含むTCRベータ鎖、
をコードする1つ以上のヌクレオチド配列をアセンブリングし、
任意選択で、(k)で同定された2番目に数が多いcDNAの多様な断片のTCRアルファ鎖のVセグメント配列と(j)で採取された2番目に数が多いcDNAの多様な断片のTCRアルファ鎖のCDR3配列を含む第2のTCRアルファ鎖、及び、
(k)で同定されたTCRベータ鎖のVセグメント配列と(j)で採取されたTCRベータ鎖のCDR3配列を含むTCRベータ鎖、
をコードする1つ以上の第2のヌクレオチド配列をアセンブリングし、
単離され、対を成すTCRアルファ及びベータ鎖配列、又はその抗原結合部分の配列を作製する工程、
を含む方法。 - 1つ以上のT細胞活性化マーカーが、インターフェロン(IFN)-γ、インターロイキン(IL)-2、腫瘍壊死因子アルファ(TNF-α)、プログラム細胞死1(PD-1)、リンパ球活性化遺伝子3(LAG-3)、T細胞免疫グロブリン及びムチンドメイン3(TIM-3)、4-1BB、OX40、CD107a、グランザイムB、顆粒球/単球コロニー刺激因子(GM-CSF)、IL-4、IL-5、IL-9、IL-10、IL-17、及びIL-22の1つ以上を含む、請求項1に記載の方法。
- 各々の分離した単一のT細胞試料に由来するmRNAを、各々の分離した単一のT細胞試料に対する異なるタグによりラベル化する工程をさらに含む、請求項1又は2に記載の方法。
- (h)が、アルファとベータ鎖のVセグメントによりコードされるアミノ酸配列のC末端近傍に位置する、保存されたアミノ酸残基をコードするcDNA配列を同定することにより、TCR CDR3配列を同定する工程を含む、請求項1~3のいずれか1項に記載の方法。
- (k)が、(j)で採取された最も数が多いcDNAの多様な断片のTCRアルファ鎖の定常(C)領域配列と、(j)で採取された最も数が多いcDNAの多様な断片のTCRベータ鎖のC領域配列を同定する工程、をさらに含む請求項1~4のいずれか1項に記載の方法。
- (l)が、(k)で同定されたTCRアルファ鎖のVセグメント配列、(k)で同定されたTCRアルファ鎖のC領域配列、及び(j)で採取されたTCRアルファ鎖のCDR3配列を含むTCRアルファ鎖をアセンブリングする工程、並びに、(k)で同定されたTCRベータ鎖のVセグメント配列、(k)で同定されたTCRベータ鎖のC領域配列、及び(j)で採取されたTCRベータ鎖のCDR3配列を含むTCRベータ鎖をアセンブリングする工程、を含む請求項5に記載の方法。
- (l)が、(k)で同定されたTCRアルファ鎖のVセグメント配列、外因性のTCRアルファ鎖のC領域配列、及び(j)で採取されたTCRアルファ鎖のCDR3配列を含むTCRアルファ鎖をアセンブリングする工程、並びに、(k)で同定されたTCRベータ鎖のVセグメント配列、外因性のTCRベータ鎖のC領域配列、及び(j)で採取されたTCRベータ鎖のCDR3配列を含むTCRベータ鎖をアセンブリングする工程、を含む請求項1~4のいずれか1項に記載の方法。
- 使用者のコンピューター装置において、(f)で同定された単一のT細胞のcDNAの多様な断片の配列を受け取る工程をさらに含む、請求項1~7のいずれか1項に記載の方法であって;
ここで(g)は、cDNAの多様な断片の各々の配列を、参照TCR配列データベースに対してコンピューター化されたアラインメントをして、(f)で同定された単一のT細胞のcDNAの多様な断片の、TCRアルファ鎖の可変(V)セグメント配列とTCRベータ鎖のVセグメント配列を同定する工程を含み;
ここで(h)は、(g)で同定されたTCRアルファ鎖のVセグメント配列を含むcDNAの多様な断片内と(g)で同定されたTCRベータ鎖のVセグメント配列を含むcDNAの多様な断片内の、TCR CDR3配列のコンピューター化された同定を行う工程を含み;
ここで(i)は、同じアルファ鎖のCDR3アミノ酸配列を共有するcDNAの多様な断片の数と、同じベータ鎖のCDR3アミノ酸配列を共有するcDNAの多様な断片の数のコンピューター化された計測を行う工程を含み;
ここで(j)は、同じアルファ鎖のCDR3配列をコードする最も数が多いcDNAの多様な断片、同じベータ鎖のCDR3配列をコードする最も数が多いcDNAの多様な断片、及び任意選択で、同じアルファ鎖のCDR3配列をコードする2番目に数が多いcDNAの多様な断片、のコンピューター化された採取を行い、TCRアルファとベータ鎖のCDR3配列を同定する工程であって、ここで2番目に数が多いcDNAの多様な断片によりコードされるアルファ鎖のCDR3配列は、最も数が多いcDNAの多様な断片によりコードされるアルファ鎖のCDR3配列とは異なる工程を含み;及び
ここで(k)は、(j)で採取された最も数が多いcDNAの多様な断片のTCRアルファ鎖のVセグメント配列、(j)で採取された最も数が多いcDNAの多様な断片のTCRベータ鎖のVセグメント配列、及び任意選択で、(j)で採取された2番目に数が多いcDNAの多様な断片のTCRアルファ鎖のVセグメント配列のコンピューター化された同定を行い、TCRアルファとベータ鎖のVセグメント配列を同定する工程を含む、
方法。 - 対を成すTCRアルファとベータ鎖又はその抗原結合部分を発現する細胞集団を調製する方法であって、該方法は、
請求項1~8のいずれか1項の方法により、対を成すTCRアルファとベータ鎖の配列又はその抗原結合部分の配列を単離する工程、及び
単離された、対を成すTCRアルファとベータ鎖の配列又はその抗原結合部分の配列をコードするヌクレオチドを、宿主細胞内に導入し、対を成すTCRアルファとベータ鎖又はその抗原結合部分を発現する細胞を得る工程、
を含む方法。 - 対を成すTCRアルファとベータ鎖又はその抗原結合部分を発現する宿主細胞の数を増大させる工程をさらに含む、請求項9に記載の方法。
- がん特異的な変異によりコードされる、変異したアミノ酸配列に対して抗原特異性を有するT細胞受容体(TCR)アルファとベータ鎖のVセグメント配列とTCRのCDR3配列を自動的に同定する方法であって、該方法は、
(a)使用者のコンピューター装置において、cDNAの多様な断片の配列を受け取る工程であって、ここで該cDNAは単一のT細胞により、該T細胞と変異したアミノ酸配列を提示する抗原提示細胞(APC)との共培養に続いて作製されるmRNAによりコードされ、それによって該T細胞は1つ以上のT細胞活性化マーカーを発現する工程;
(b)cDNAの多様な断片の各々の配列を、参照TCR配列データベースに対してコンピューター化されたアラインメントをして、cDNAの多様な断片のTCRアルファ鎖の可変(V)セグメント配列とTCRベータ鎖のVセグメント配列を同定する工程;
(c)(b)において同定されたTCRアルファ鎖のVセグメント配列を含むcDNAの多様な断片内の、及び、(b)において同定されたTCRベータ鎖のVセグメント配列を含むcDNAの多様な断片内の、TCR相補性決定領域3(CDR3)配列のコンピューター化された同定を行う工程;
(d)同じアルファ鎖のCDR3アミノ酸配列を共有するcDNAの多様な断片の数と、同じベータ鎖のCDR3アミノ酸配列を共有するcDNAの多様な断片の数、のコンピューター化された計測を行う工程;
(e)同じアルファ鎖のCDR3配列をコードする最も数が多いcDNAの多様な断片、同じベータ鎖のCDR3配列をコードする最も数が多いcDNAの多様な断片、及び任意選択で、同じアルファ鎖のCDR3配列をコードする2番目に数が多いcDNAの多様な断片のコンピューター化された採取を行い、TCRアルファとベータ鎖のCDR3配列を同定する工程であって、ここで2番目に数が多いcDNAの多様な断片によりコードされるアルファ鎖のCDR3配列は、最も数が多いcDNAの多様な断片によりコードされるアルファ鎖のCDR3配列と異なる工程;並びに
(f)(e)で採取された最も数が多いcDNAの多様な断片のTCRアルファ鎖のVセグメント配列、(e)で採取された最も数が多いcDNAの多様な断片のTCRベータ鎖のVセグメント配列、及び任意選択で、(e)で採取された2番目に数が多いcDNAの多様な断片のTCRアルファ鎖のVセグメント配列のコンピューター化された同定を行い、TCRアルファとベータ鎖のVセグメント配列を同定する工程;
を含む方法。 - (c)が、アルファとベータ鎖のVセグメントによりコードされるアミノ酸配列のC末端の近傍に位置する、保存されたアミノ酸残基をコードするcDNA配列を同定することにより、TCRのCD3配列を同定する工程を含む、請求項11に記載の方法。
- 保存されたアミノ酸残基が
YX1CX2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22(配列番号5)
のアミノ酸配列を含み、
ここで:
X1-X9の各々は任意の天然由来のアミノ酸であり、
X10-X21の各々はアミノ酸がないか又は任意の天然由来のアミノ酸であり、及び、
X22はフェニルアラニン又はトリプトファンである、
請求項12に記載の方法。 - (f)は、(e)で採取された最も数が多いcDNAの多様な断片のTCRアルファ鎖の定常(C)領域配列と、(e)で採取された最も数が多いcDNAの多様な断片のTCRベータ鎖のC領域配列、のコンピューター化された同定を行う工程をさらに含む、請求項11~13のいずれか1項に記載の方法。
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