JP2016508143A - ヒト血清アルブミン結合化合物およびその融合タンパク質 - Google Patents
ヒト血清アルブミン結合化合物およびその融合タンパク質 Download PDFInfo
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Abstract
Description
方法
1)ヒト血清アルブミンタンパク質に対するライセートELISA
Schlatterらに記載されている(Schlatterら著、(2012)、mAbs、4(4)、p.497−50))Fynomer(登録商標)ファージライブラリーを使用して、ヒト血清アルブミンに特異的なFyn−SH3由来の結合タンパク質を、抗原としてヒト血清アルブミン(Sigma−Aldrich、カタログ番号A3782)並びに齧歯動物種からの血清アルブミン(ラット血清アルブミン、Sigma−Aldrich、カタログ番号A6414)を使用し、及び選択技術として標準ファージディスプレイを使用して単離した(Grabulovski D.ら著、(2007)、J Biol Chem 282、p.3196−3204、Viti,F.ら著、(2000)、Methods Enzymol.326、480−505)。
Fyn SH3由来アルブミン結合ポリペプチドを、TG1大腸菌(E.coli)細菌の細胞質ゾル中で発現させ、並びにGrabulovskiらに記載されている通りに(Grabulovskiら著、(2007)、JBC、282、p.3196−3204)精製した。
Biacore T200装置(GE Healthcare)を使用して、親和性測定を行った。マウス、ラット若しくはヒトに由来する血清アルブミン、及びFyn SH3由来アルブミン結合ポリペプチドの間の相互作用分析については、シリーズS CM5チップ(GE Healthcare)を、アミンカップリングキット(GE Healthcare)を使用して固定化されたアルブミンタンパク質と共に使用した。異なる種(マウス、ラット又はヒト)からの血清アルブミンタンパク質を、チップの異なるフローセル上に固定化し(2000〜3000RU)、一方ブランクを固定化したフローセルは、参照フローセルとして働いた。泳動緩衝液は、pH7.4で0.05%のTween 20を含有するPBSであった。相互作用を30μl/分のフロー及び25℃にて測定し、異なる濃度のFyn SH3由来アルブミン結合ポリペプチドを注入した。相互作用の全ての動力学データを、Biacore T200評価ソフトウェアを使用して評価した。
1)ELISA陽性のヒト血清アルブミンに結合するFyn SH3由来ポリペプチドのアミノ酸配列を、配列表に添付されているように配列番号4〜40で提示する。加えて、Fyn SH3由来のポリペプチド(配列番号4〜32)はまた、ライセートELISA及び/又はBiacore親和性測定によって確認されたように、マウス血清アルブミンへの結合も示した。
方法
アルブミンに結合するFyn−SH3由来ポリペプチドの薬物動態プロファイルをBALB/cマウス(Charles River)において検討し、WT Fyn−SH3分子と比較した。Fynomer(登録商標)C1(配列番号4)、Fynomer(登録商標)17H(配列番号5)及びWT Fyn−SH3(配列番号3)を、ヨウ素−125(Perkin Elmer、カタログ番号NEZ033A001MC)及びクロラミンT(Sigma−Aldrich、カタログ番号31224)を使用して放射標識した。標識反応を室温で2分間行い、その後、PD MiniTrap G−25カラム(GE Healthcare、カタログ番号28−9180−07)を使用して標識試薬を除去した。3匹のBALB/cマウスに、13.5μgの放射標識化Fynomer(登録商標)C1(配列番号4)、Fynomer(登録商標)17H(配列番号5)又はWT Fyn−SH3(配列番号3)のいずれかを静脈注射した。10分後、2.5、4、6、9、25、35時間後に、血液をEDTA被覆マイクロベット(microvettes)(Sarstedt)に採取し、9300gで10分間遠心分離した。放射活性を、Supermix Perkin Elmerシンチレーション流体との混合及び1450 MicroBeta Triluxシンチレーションカウンターで各試料のβ放射の定量化によって計数し、血清レベルを計算した(結果は、血液の注射された用量(ID)/mlの%として表された)。異なる時点において血清中で決定されたFynomer(登録商標)C1、Fynomer(登録商標)17H及びWT Fyn−SH3の血清レベル及び得られた排出相の勾配k(片対数目盛でプロットされた)から、式t1/2=In2/−kを使用して半減期を計算した。
表3に示すように、Fynomer(登録商標)C1(配列番号4)及びFynomer(登録商標)17H(配列番号5)は、WT Fyn−SH3(配列番号3)と比べると、著しく良好な終末相半減期を示した。半減期計算に使用された時間点は、Fynomer(登録商標)C1及びFynomer(登録商標)17Hが2.5〜35時間であり、WT Fyn−SH3が2.5〜25時間であった。
方法:
1)アルブミンに結合するFyn−SH3融合タンパク質の発現及び精製
Fynomer(登録商標)17H(配列番号5)を、15個のアミノ酸のリンカー(リンカー配列番号52)を介して、BITE(登録商標53)に特異的なCD3−PSMAに遺伝子的に融合して、三重特異性(trispecific)抗アルブミン/PSMA/CDタンパク質COVA406(配列番号54)を産生した。BITE(登録商標)タンパク質(配列番号53)及びC末端ペンタ−his−タグを運ぶ本発明の融合タンパク質COVA406(配列番号54)を、FreeStyle CHO−S細胞に一時的にトランスフェクトし、血清フリー/動物成分フリーの培地中で6日間発現させた。AKTA浄化装置(GE Healthcare)を備えたタンパク質L親和性クロマトグラフィー(Thermo Scientific、カタログ番号89928)によって、上澄み液からタンパク質を精製した。280nmにおける吸光度測定によって濃度を決定した。収量を表4に示す。両タンパク質のSDS PAGEを図2に示す。
ポリペプチドCOVA406(配列番号54、最終濃度300nM)を、PBS/1%BSA/0.2%アジドナトリウム中で、(i)1×105個のジャーカット(Jurkat)E6−1細胞(CD3陽性細胞)、(ii)1×105個の22Rv1前立腺癌細胞(PSMA陽性細胞)又は(iii)1×105個のLS174T結腸直腸腺癌細胞(PSMA及びCD3陰性、ATCCカタログ番号CL−188)のいずれかを含有する細胞懸濁液100μlと混合した。陰性対照として、COVA406の代わりに同じ細胞をPBS/1%BSA/0.2%アジドナトリウム中でインキュベートした(PBS対照)。氷上で60分のインキュベーションの後に、細胞を洗浄し、10μg/mlのマウス抗テトラ−HIS抗体(Qiagen、カタログ番号34670)とのインキュベーションによって、続いて、10μg/mLの濃度においての抗マウスIgG−Alexa488結合体(Invitrogen)とのインキュベーションによって、結合したタンパク質を検出した。最終的に、細胞を3回洗浄し、次いで染色した細胞を、Guava easyCyte(商標)(Millipore)フローサイトメーターで分析した。
ポリペプチドCOVA406(配列番号54)を、Dreierら著、(2002)、Int.J.Cancer:100、690−697から採用したプロトコルを使用して、再指向T細胞媒介性細胞傷害アッセイで試験した。ヒトPBMCをエフェクター細胞として使用した。実験前日に、標準的手法を使用するFicoll Plaque plus(GE Healthcare)及び密度勾配遠心分離によって、新鮮なバッフィーコート調製物からPBMCを単離した。次いで、単離したPBMCを、10%のFCS、RPMI中、4×106個細胞/mlの細胞濃度で、37℃、5%のCO2にて一晩インキュベートした。細胞致死実験については、PBMCを遠心分離し、10%のFCS、RPMI中に、2.5×107個細胞/mlの濃度で再懸濁させた。細胞を、10μMのカルセインAMの最終濃度で37℃、5%CO2にて30分間インキュベートすることによって、標的細胞をカルセインAMで標識付けした。続いて、細胞を薬15mLの培地で洗浄することによって、過剰の染料を除去した。最後に、標的細胞の数を1×106個細胞/mlに調整した。標的腫瘍細胞は、22Rv1細胞(PSMA陽性、ATCCカタログ番号CRL−2505)又はHT29直腸癌細胞(PSMA陰性、DSMZカタログ番号ACC−299)のいずれかであった。エフェクター分子を、10%のFCS、RPMIで希釈し、1200ng/mLの最大濃度とした。1/10希釈の希釈系列を調製した。最後に、標的細胞懸濁液、エフェクター細胞及び異なる濃度のポリペプチドCOVA406(配列番号54)を次いで等量で混合した。合計で50000個の標的細胞を、ウェル毎に添加し、エフェクターの標的細胞に対する比は25/1であった。エフェクター分子の最終の最大濃度は、400ng/μlであった。細胞溶解を、37℃及び5%のCO2における5時間のインキュベーション後に測定した。インキュベーション後に、細胞懸濁液を遠心分離し、蛍光読み取り機を使用しての細胞懸濁液中のカルセインAMの蛍光の検出によって細胞溶解を定量化した。再指向細胞溶解の量を最大溶解対照(1%のTriton X−100の添加によって溶解した細胞)及び自然溶解(エフェクター分子の不在下でPMBCとインキュベートされた標的細胞)に正規化した。細胞溶解の百分率を以下の式に従って計算した:
溶解の%=(((試料の蛍光)−(自然溶解対照の蛍光))/((最大溶解対照の蛍光)−(自然溶解対照の蛍光)))×100
C57BL/6マウス(Charles River)中のCOVA406の薬物動態プロファイルを検討し、親BITE(登録商標)分子と比較した。5匹のC57BL/6マウスに、それぞれ48μgのCOVA406(配列番号54)又はBITE(登録商標)(配列番号53)を静脈内注射した。10分及び30分、1、3、5、7、9、12、24、28、33及び48時間後に、血液をEDTA被覆マイクロベット(microvettes)(Sarstedt)に採取し、9300gで10分間遠心分離し、COVA406又はBITE(登録商標)の血清レベルをELISAによって決定した。簡単に言うと、ブラックマキシソープ(maxisorp)マイクロタイタープレート(Nunc)を、10μg/mlのCD3に由来するペプチド(配列:QDGNEEMGGITQTPYKVSISGTTVILT;配列番号55)(Fc−融合と表わされる)で被覆し、4℃で一晩インキュベートした。PBS中4%のミルク(Rapilait、Migros、Swizerland)でブロックした後に、血清試料を適切な希釈で加え、2%のマウス血清(Sigma)及び4%のミルクの最終緩衝液濃度を得た。1時間のインキュベーション後に、ウェルをPBSで洗浄し、結合したCOVA406又はBITE(登録商標)を、ペンタ−His−ビオチン(Qiagen)続いてストレプトアビジン−HRP結合体(Sigma)で検出した。このアッセイは、QuantaRed蛍光基質(Pierce)で展開した。反応を3分のインキュベーション後に停止させ、蛍光強度を544nm(励起)及び590nm(放射)で測定した。COVA406及びBITE(登録商標)の血清レベルを、COVA406及びBITE(登録商標)(それぞれ333−0.5ng/mlで希釈)の標準曲線を使用して決定した。異なる時点において血清中で決定されたCOVA406及びBITE(登録商標)の濃度及び得られた排出相の勾配k(片対数目盛でプロットされた)から、式t1/2=In2/−kを使用して半減期を計算した。半減期の計算で使用された時間点は、COVA406は1〜48時間であり、BITE(登録商標)は1〜12時間であった。
COVA406(配列番号54)は、BITE(登録商標)分子(配列番号53)と同様な収量で発現した(表4)。
材料及び方法については、欧州特許出願公開第2054432号明細書及び「Grabulovski、Dragan、The SH3 domain of fyn kinase as a scaffold for the generation of new binding proteins.ETH Dissertation Nr 17216(2007年5月).http://dx.doi.org/10.3929/ethz−a−005407897」を参照されたい。
Claims (15)
- ヒト血清アルブミンに結合し、
(a)GVTLFVALYDY(X1)(X2)(X3)(X4)(X5)(X6)D(X7)SFHKGEKFQIL(X8)(X9)(X10)(X11)(X12)G(X13)(X14)W(X15)(X16)RSLTTG(X17)(X18)G(X19)IPSNYVAPVDSIQ(配列番号1)であって、式中、
(X1)が、A、V、I、L、M、G、P、S、T、N、Q、C、R、H、K、D又はEであり;
(X2)が、R、H、K、A、V、I、L、M、G、P、S、T、N、Q又はCであり;
(X3)が、R、H、K、S、T、N、Q、C、F、Y、W、A、V、I、L、M、G又はPであり;
(X4)が、S、T、N、Q、C、A、V、I、L、M、G、P、R、H、K、F、Y、W、D又はEであり;
(X5)が、S、T、N、Q、C、D、E、F、Y、W、A、V、I、L、M、G、P、R、H又はKであり;
(X6)が、F、Y、W、A、V、I、L、M、G、P、R、H、K、S、T、N、Q又はCであり;
(X7)が、A、V、I、L、M、G、P、R、H又はKであり;
(X8)が、S、T、N、Q、C、D又はEであり;
(X9)が、S、T、N、Q、C、D、E、A、V、I、L、M、G、P、F、Y又はWであり;
(X10)が、A、V、I、L、M、G又はPであり;
(X11)が、F、Y、W、R、H又はKであり;
(X12)が、S、T、N、Q、C、F、Y又はWであり;
(X13)が、F、Y、W、R、H、K、S、T、N、Q、C、D、E、A、V、I、L、M、G又はPであり;
(X14)が、F、Y、W、A、V、I、L、M、G又はPであり;
(X15)が、D、E、A、V、I、L、M、G又はPであり;
(X16)が、A、V、I、L、M、G又はPであり;
(X17)が、D、E、A、V、I、L、M、G、P、R、H又はKであり;
(X18)が、S、T、N、Q、C、A、V、I、L、M、G又はPであり;
(X19)が、F、Y、W、S、T、N、Q又はCである、アミノ酸配列と、
(b)(a)の前記アミノ酸配列と少なくとも90%同一であるアミノ酸配列と、からなる群から選択されるアミノ酸配列を含む又はこれらからなるポリペプチドであって、
前記同一性の決定が、アミノ酸位置(X1)〜(X19)を除外する、ポリペプチド。 - 請求項1に記載のポリペプチドであって、前記ポリペプチドが、
(a)GVTLFVALYDY(X1)(X2)(X3)(X4)(X5)(X6)D(X7)SFHKGEKFQIL(X8)(X9)(X10)(X11)(X12)G(X13)(X14)W(X15)(X16)RSLTTG(X17)(X18)G(X19)IPSNYVAPVDSIQ(配列番号2)であり、式中、
(X1)が、V、T、L、H、Q、E、R、G又はMであり;
(X2)が、S、A、R、K、N、M、L又はTであり;
(X3)が、N、S、R、H、Q、F、M、K又はVであり;
(X4)が、T、Y、N、L、H、R、Q、W、M又はEであり;
(X5)が、G、S、A、E、W、L、R又はKであり;
(X6)が、F、Y、P、W、R又はQであり;
(X7)が、L又はRであり;
(X8)が、Q又はDであり;
(X9)が、N、D、A、Q、W、M又はSであり;
(X10)が、L、I又はVであり;
(X11)が、W又はRであり;
(X12)が、T又はFであり;
(X13)が、D、W、N、K、S、E又はAであり;
(X14)が、W又はGであり;
(X15)が、E又はVであり;
(X16)が、A又はVであり;
(X17)が、E、L又はRであり;
(X18)が、T、S又はMであり;
(X19)が、Y又はSである、アミノ酸配列と、
(b)(a)の前記アミノ酸配列と少なくとも90%同一であるアミノ酸配列と、からなる群から選択されるアミノ酸配列を含む又はこれらからなり、
前記同一性の決定が、アミノ酸位置(X1)〜(X19)を除外する、ポリペプチド。 - 請求項1に記載のポリペプチドであって、前記ポリペプチドが、齧歯類血清アルブミンにさらに結合し、
(a)GVTLFVALYDY(X1)(X2)(X3)(X4)(X5)(X6)D(X7)SFHKGEKFQIL(X8)(X9)(X10)(X11)(X12)G(X13)(X14)W(X15)(X16)RSLTTG(X17)(X18)G(X19)IPSNYVAPVDSIQ(配列番号41)であり、式中、
(X1)が、A、V、I、L、M、G、P、S、T、N、Q、C、R、H、K、D又はEであり;
(X2)が、R、H、K、A、V、I、L、M、G、P、S、T、N、Q又はCであり;
(X3)が、R、H、K、S、T、N、Q、C、F、Y、W、A、V、I、L、M、G又はPであり;
(X4)が、S、T、N、Q、C、A、V、I、L、M、G、P、R、H、K、F、Y又はWであり;
(X5)が、S、T、N、Q、C、D、E、F、Y、W、A、V、I、L、M、G、P、R、H又はKであり;
(X6)が、F、Y、W、A、V、I、L、M、G、P、R、H、K、S、T、N、Q又はCであり;
(X7)が、A、V、I、L、M、G、P、R、H又はKであり;
(X8)が、S、T、N、Q、C、D又はEであり;
(X9)が、S、T、N、Q、C、D又はEであり;
(X10)が、A、V、I、L、M、G又はPであり;
(X11)が、F、Y又はWであり;
(X12)が、S、T、N、Q又はCであり;
(X13)が、F、Y、W、R、H、K、S、T、N、Q、C、D、E、A、V、I、L、M、G又はPであり;
(X14)が、F、Y、W、A、V、I、L、M、G又はPであり;
(X15)が、D、E、A、V、I、L、M、G又はPであり;
(X16)が、A、V、I、L、M、G又はPであり;
(X17)が、D、E、A、V、I、L、M、G、P、R、H又はKであり;
(X18)が、S、T、N、Q、C、A、V、I、L、M、G又はPであり;
(X19)が、F、Y、W、S、T、N、Q又はCであるアミノ酸配列と、
(b)(a)の前記アミノ酸配列と少なくとも90%同一であるアミノ酸配列と、からなる群から選択されるアミノ酸配列を含む又はこれらからなり、
前記同一性の決定が、アミノ酸位置(X1)〜(X19)を除外する、ポリペプチド。 - 請求項3に記載のポリペプチドであって、前記ポリペプチドが、齧歯類血清アルブミンにさらに結合し、
(a)GVTLFVALYDY(X1)(X2)(X3)(X4)(X5)(X6)D(X7)SFHKGEKFQIL(X8)(X9)(X10)(X11)(X12)G(X13)(X14)W(X15)(X16)RSLTTG(X17)(X18)G(X19)IPSNYVAPVDSIQ(配列番号42)であり、式中、
(X1)が、V、T、L、H、Q、E、R、G又はMであり;
(X2)が、S、A、R、K、N、M、L又はTであり;
(X3)が、N、S、R、H、Q、F、M、K又はVであり;
(X4)が、T、Y、N、L、H、R、Q、W又はMであり;
(X5)が、G、S、A、E、W、L又はRであり;
(X6)が、F、Y、P、W、R又はQであり;
(X7)が、L又はRであり;
(X8)が、Q又はDであり;
(X9)が、N又はDであり;
(X10)が、L又はIであり;
(X11)が、Wであり;
(X12)が、Tであり;
(X13)が、D、W、N、K、S、E又はAであり;
(X14)が、W又はGであり;
(X15)が、E又はVであり;
(X16)が、A又はVであり;
(X17)が、E、L又はRであり;
(X18)が、T、S又はMであり;
(X19)が、Y又はSである、アミノ酸配列と、
(b)(a)の前記アミノ酸配列と少なくとも90%同一であるアミノ酸配列と、からなる群から選択されるアミノ酸配列を含む又はこれらからなり、
前記同一性の決定が、アミノ酸位置(X1)〜(X19)を除外する、ポリペプチド。 - 請求項4に記載のポリペプチドであって、前記アミノ酸配列が、配列番号5、4又は6〜32のアミノ酸配列から選択される、ポリペプチド。
- 薬学的に及び/又は診断学的に活性なタンパク質又はペプチドに融合された、請求項1〜5のいずれか一項に記載のポリペプチドを含む融合タンパク質。
- 請求項6に記載の融合タンパク質であって、前記ポリペプチドが、前記薬学的に及び/又は診断学的に活性なタンパク質又はペプチドに直接融合される、融合タンパク質。
- 請求項6に記載の融合タンパク質であって、前記ポリペプチドが、リンカーを介して、前記薬学的に及び/又は診断学的に活性なタンパク質又はペプチドに融合される、融合タンパク質。
- 請求項6〜8のいずれか一項に記載の融合タンパク質であって、前記薬学的に及び/又は診断学的に活性なタンパク質又はペプチドが、組換えタンパク質、抗体、血液因子、ホルモン、抗凝血剤、血清溶解剤、サイトカイン、ケモカイン、及びインターフェロンからなる群から選択される、融合タンパク質。
- 請求項9に記載の融合タンパク質であって、前記抗体が、二重特性T細胞動員抗体である、融合タンパク質。
- 請求項1〜5のいずれか一項に記載の前記ポリペプチド又は請求項6〜10のいずれか一項に記載の前記融合タンパク質をコード化する、核酸分子。
- 請求項11に記載の前記核酸分子を含む、ベクター。
- 請求項11に記載の前記核酸分子又は請求項12に記載の前記ベクターを含む、単離された細胞。
- 請求項1〜5のいずれか一項に記載の前記ポリペプチド又は請求項6〜10のいずれか一項に記載の前記融合タンパク質を産生する方法であって、
(a)請求項13に記載の前記単離された細胞を培養する工程と、
(b)前記産生されたポリペプチド又は融合タンパク質を単離する工程と、を含む、方法。 - 請求項6〜10のいずれか一項に記載の前記融合タンパク質を含む、医薬組成物及び/又は診断用組成物。
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US11028166B2 (en) | 2017-02-16 | 2021-06-08 | Sonnet Bio Therapeutics | Albumin binding domain fusion proteins |
CN107353346A (zh) * | 2017-08-09 | 2017-11-17 | 芜湖英特菲尔生物制品产业研究院有限公司 | 一种由猪白蛋白与猪干扰素γ组成的融合蛋白及其制备方法和一种重组猪长效干扰素γ |
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