JP6976351B2 - 弱毒化細菌に基づくタンパク質送達 - Google Patents
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Description
− Asp−Asp−Asp−Asp−Lys:エンテロキナーゼ(軽鎖)/エンテロペプチダーゼ
− Leu−Glu−Val−Leu−Phe−Gln/Gly−Pro:PreScissionプロテアーゼ/ヒトライノウイルスプロテアーゼ(HRV 3C)
− TEVプロテアーゼ(タバコエッチウイルス)により認識される、Glu−Asn−Leu−Tyr−Phe−Gln−Ser、及びGlu−X−X−Tyr−X−Gln−Gly/Ser(ここでのXは任意のアミノ酸である)に基づく修飾モチーフ
−Glu−Thr−Val−Arg−Phe−Gln−Ser:TVMVプロテアーゼ
− Ile−(Glu又はAsp)−Gly−Arg:第Xa因子プロテアーゼ
− Leu−Val−Pro−Arg/Gly−Se:トロンビン。
プロモーター、
前記プロモーターに作用可能に連結されている、細菌エフェクタータンパク質からの送達シグナルをコードする第1のヌクレオチド配列、
前記第1のヌクレオチド配列の3’末端とインフレームで融合している異種タンパク質をコードする第2のヌクレオチド配列
を含む、ヌクレオチド分子、例えばベクターで、形質転換される。
細菌エフェクタータンパク質からの送達シグナル又はその断片をコードする第1のヌクレオチド配列、
前記第1のヌクレオチド配列の3’末端とインフレームで融合している異種タンパク質をコードする第2のヌクレオチド配列
を含む、ヌクレオチド分子、例えばベクターで、形質転換される。
エフェクターをコードしている遺伝子のコード領域において、コードされているエフェクタータンパク質の触媒活性を消失させるような変異を生じさせることもできる。エフェクタータンパク質の「触媒活性」は、エフェクタータンパク質の抗標的細胞機能、すなわち毒性を通常は意味する。そのような活性は、エフェクタータンパク質の触媒ドメインにおける触媒モチーフにより支配される。エフェクタータンパク質の触媒ドメイン及び/又は触媒モチーフを同定する手法は、当業者に周知である。例えば、27、28を参照されたい。
細菌エフェクタータンパク質からの送達シグナル又はその断片をコードする第1のヌクレオチド配列、
前記第1のヌクレオチド配列の3’末端とインフレームで融合している異種タンパク質をコードする第2のヌクレオチド配列
を含む。
a)プロテアーゼが、細菌エフェクタータンパク質からの送達シグナルと異種タンパク質としてのプロテアーゼとの融合タンパク質を発現する本明細書に記載の組換え弱毒化グラム陰性菌株により真核細胞に移行される方法、又は
b)プロテアーゼが、真核細胞において構成的に若しくは一過性に発現される方法
である。
i)本明細書に記載の組換え弱毒化グラム陰性菌株を培養すること、
ii)がん細胞をi)の組換え弱毒化グラム陰性菌株と接触させること(ここで、細菌エフェクタータンパク質からの送達シグナルと異種タンパク質とを含む融合タンパク質が、組換え弱毒化グラム陰性菌株により発現され、がん細胞内に移行される)により前記組換え弱毒化グラム陰性菌株を対象に投与すること、及び任意選択的に、
iii)異種タンパク質ががん細胞内の細菌エフェクタータンパク質からの送達シグナルから切断されるように融合タンパク質を切断すること
を含み、組換え弱毒化グラム陰性菌株は、対象を治療するのに十分である量で投与される。
i)本明細書に記載の組換え弱毒化グラム陰性菌株を培養すること、
ii)悪性固形腫瘍の細胞をi)の組換え弱毒化グラム陰性菌株と接触させること(ここで、細菌エフェクタータンパク質からの送達シグナルと異種タンパク質とを含む融合タンパク質が、組換え弱毒化グラム陰性菌株により発現され、悪性固形腫瘍の細胞内に移行される)により前記組換え弱毒化グラム陰性菌株を対象に投与すること、及び任意選択的に、
iii)異種タンパク質が悪性固形腫瘍細胞内の細菌エフェクタータンパク質からの送達シグナルから切断されるように融合タンパク質を切断すること
を含み、組換え弱毒化グラム陰性菌株は、前記対象を治療するために十分である量で投与される。
A)材料及び方法
菌株及び増殖条件。この研究で使用した株を図3A−Mに列挙する。プラスミド精製及びクローニングに使用した大腸菌Top10、及び接合伝達に使用した大腸菌Sm10λpir、並びにpKNG101を伝播するために使用した大腸菌BW1961031を、LB寒天プレート上及びLB培地中、37℃で常套的に増殖させた。アンピシリンを200μg/ml(エルシニア属)又は100μg/ml(大腸菌)で使用して、発現ベクターについて選択した。ストレプトマイシンを100μg/mlの濃度で使用して、自殺ベクターについて選択した。Y.エンテロコリティカMRS40(O:9、生物型2)20、非アンピシリン耐性E40派生物19及びそれに由来する株をブレインハートインフュージョン(BHI:Difco)上で、室温で常套的に増殖させた。Y.エンテロコリティカ株に、ナリジクス酸酸(35μg/ml)を添加し、全てのY.エンテロコリティカasd株に、さらに、100μg/mlのメソ−2,6−ジアミノピメリン酸(mDAP、Sigma Aldrich)を補充した。サルモネラ菌SL1344をLB寒天プレート上及びLB培地中、37℃で常套的に増殖させた。アンピシリンを100μg/mlの濃度で使用して、サルモネラ菌における発現ベクターについて選択した。
表I(プライマーNr.Si_:配列)
配列番号51:プライマーNo.:Si_285
CATACCATGGGAGTGAGCAAGGGCGAG
配列番号52:プライマーNo.:Si_286
GGAAGATCTttACTTGTACAGCTCGTCCAT
配列番号53:プライマーNo.:Si_287
CGGGGTACCTCAACTAAATGACCGTGGTG
配列番号54:プライマーNo.:Si_288
GTTAAAGCTTttcgaatctagactcgagCGTGGCGAACTGGTC
配列番号55:プライマーNo.:Si_387
CGTAtctagaATGGACTGTGAGGTCAACAA
配列番号56:プライマーNo.:Si_391
CGTAtctagaGGCAACCGCAGCA
配列番号57:プライマーNo.:Si_389
GTTAAAGCTTTCAGTCCATCCCATTTCTg
配列番号58:プライマーNo.:Si_436
CGTAtctagaATGCCCCGCCCC
配列番号59:プライマーNo.:Si_437
GTTAAAGCTTCTACCCACCGTACTCGTCAAT
配列番号60:プライマーNo.:Si_438
CGTAtctagaATGTCTGACACGTCCAGAGAG
配列番号61:プライマーNo.:Si_439
GTTAAAGCTTTCATCTTCTTCGCAGGAAAAAG
配列番号62:プライマーNo.:Si_463
CAGTctcgaggaaagcttgtttaaggggc
配列番号63:プライマーNo.:Si_464
cagtTTCGAAttagcgacggcgacg
配列番号64:プライマーNo.:Si_476
GTTAAAGCTTttACTTGTACAGCTCGTCCAT
配列番号65:プライマーNo.:Si_494
CGTAtctagaATGGCCGAGCCTTG
配列番号66:プライマーNo.:Si_495
GTTAAAGCTTttaTTGAAGATTTGTGGCTCC
配列番号67:プライマーNo.:Si_504
CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTATGCCCCGCCCC
配列番号68:プライマーNo.:Si_505
GTTAAAGCTTCCCACCGTACTCGTCAATtc
配列番号69:プライマーNo.:Si_508
CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTATGGCCGAGCCTTG
配列番号70:プライマーNo.:Si_509
GTTAAAGCTTTTGAAGATTTGTGGCTCCc
配列番号71:プライマーNo.:Si_511
CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTGTGAGCAAGGGCGAG
配列番号72:プライマーNo.:Si_512
CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTCCGCCGAAAAAAAAACGTAAAGTTGTGAGCAAGGGCGAG
配列番号73:プライマーNo.:Si_513
GTTAAAGCTTttAAACTTTACGTTTTTTTTTCGGCGGCTTGTACAGCTCGTCCAT
配列番号74:プライマーNo.:Si_515
CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTGATTATAAAGATGATGATGATAAAATGGCCGAGCCTTG
配列番号75:プライマーNo.:Si_677
TTACTATTCGAAGAAATTATTCATAATATTGCCCGCCATCTGGCCCAAATTGGTGATGAAATGGATCATTAAGCTTGGAGTA
配列番号76:プライマーNo.:Si_678
TACTCCAAGCTTAATGATCCATTTCATCACCAATTTGGGCCAGATGGCGGGCAATATTATGAATAATTTCTTCGAATAGTAA
配列番号77:プライマーNo.:Si_682
TTACTACTCGAGAAAAAACTGAGCGAATGTCTGCGCCGCATTGGTGATGAACTGGATAGCTAAGCTTGGAGTA
配列番号78:プライマーNo.:Si_683
TACTCCAAGCTTAGCTATCCAGTTCATCACCAATGCGGCGCAGACATTCGCTCAGTTTTTTCTCGAGTAGTAA
配列番号79:プライマーNo.:Si_580
catgccatggatttatggtcatagatatgacctc
配列番号80:プライマーNo.:Si_612
CGGGGTACCatgaggtagcttatttcctgataaag
配列番号81:プライマーNo.:Si_613
CGGGGTACCataattgtccaaatagttatggtagc
配列番号82:プライマーNo.:Si_614
catgccatggCGGCAAGGCTCCTC
配列番号83:プライマーNo.:Si_615
cggggtaccTTTATTTGTCAACACTGCCC
配列番号84:プライマーNo.:Si_616
cggggtaccTGCGGGGTCTTTACTCG
配列番号85:プライマーNo.:Si_585
CAGTctcgagATGCAGATCTTCGTCAAGAC
配列番号86:プライマーNo.:Si_586
GTTAAAGCTTgctagcttcgaaACCACCACGTAGACGTAAGAC
配列番号87:プライマーNo.:Si_588
cagtTTCGAAGATTATAAAGATGATGATGATAAAATGGCCGAGCCTTG
配列番号88:プライマーNo.733
TTACTACTCGAGGGTGCCATCGATGCCGAAGAAATTATTCATAATATTGCCCG
配列番号89:プライマーNo.735
TACTCCTTCGAATTAATGATCCATTTCATCACCAATTTG
配列番号90:プライマーNo.736
TTACTACTCGAGGGTGCCATCGATGCCAAAAAACTGAGCGAATGTCTGCG
配列番号91:プライマーNo.738
TACTCCTTCGAATTAGCTATCCAGTTCATCACCAATG
配列番号92:プライマーNo.734
TACTCCTTCGAAGGCACCATGATCCATTTCATCACCAATTTGG
配列番号93:プライマーNo.725:
TTACTATTCGAAGAAATTATTCATAATATTGCC
配列番号94:プライマーNo.726:
TACTCCAAGCTTACGGTTGAATATTATGATCCATTTCATCACCAATTTGG
配列番号95:プライマーNo.727:
TTACTATTCGAAGCCGGTGGTGCCGAAGAAATTATTCATAATATTGCCC
配列番号96:プライマーNo.728:
TACTCCAAGCTTAATGATCCATTTCATCA
配列番号97:プライマーNo.737:
TACTCCTTCGAAGGCACCGCTATCCAGTTCATCACCAATG
配列番号101: プライマーNo.869:
gatcgtcgacTTAAGTTCAATGGAGCGTTTAATATC
配列番号102:プライマーNo.870:
ctttgactggcgagaaacgcTCTTAACATGAGGCTGAGCTC
配列番号103:プライマーNo.871:
GAGCTCAGCCTCATGTTAAGAgcgtttctcgccagtcaaag
配列番号104:プライマーNo.872:
gatagcccccgagcctgtGCACTTTGTCATTAACCTCAGC
配列番号105:プライマーNo.873:
GCTGAGGTTAATGACAAAGTGCacaggctcgggggctatc
配列番号106:プライマーNo.874:
catgtctagaCCCTCAGCATAATAACGACTC
配列番号107:プライマーNo.600:
catgacatgtTGGCGTTTCTCGCC
配列番号108:プライマーNo.601:
catgacatgtATTAACCTCAGCCCTGACTATAAG
配列番号119:プライマーNo.1010:
cacatgtctagaCAACCGTTTCCGAAAGGTGATCTG
配列番号120:プライマーNo.1012:
atccCAagctTATTGGCGTTGGGTGGTAAAAATTTTG
配列番号121:プライマーNo.1021:
cacatgtctagaATGACCGCCGAACAACGC
配列番号122:プライマーNo.1022:
catgaagcttaCGGACCCGGATTTTGGCTC
>配列番号123:プライマーNo.1023:
catgaagcttaCGGTTCTTCTTGAATAAAAATTTGAATG
配列番号124:プライマーNo.1024:
catgaagcttaTTGCAGCACTTTCGGCCAATTT
配列番号125:プライマーNo.1025:
cacatgtctagaATGAGCATTGTGTGTAGCGC
配列番号126:プライマーNo.1026:
catgaagcttaGCTTTCATCCACGGCCGG
配列番号127:プライマーNo.1027:
catgaagcttaATTACCGGTTTGGCGCAGC
全ての動物実験は、認可されたものであり(license 1908;Kantonales Veterinaramt Basel−Stadt)、地域のガイドライン(Tierschutz−Verordnung;Basel−Stadt)及びスイス動物保護法(Tierschutz−Gesetz)に従って行った。6週齢C57Bl/6及びBALB/cマウスをJanvier Labsから取り寄せた。少なくとも1週間の馴化後、イソフルランを使用してマウスを麻酔し、10ul B16−F10又は4T1細胞(細胞1×105−1×106個)をC57Bl/6及びBALB/cマウスの側副部にそれぞれ皮下注射した。この実験を通して、マウスの行動及び身体的外見についてのスコアを付け、体表温度並びに体重を測定した。
YopE融合タンパク質の3型分泌に基づくタンパク質送達系
Y.エンテロコリティカT3SSエフェクターYopE(配列番号1)のまさにN末端は、異種タンパク質を移行させるのに十分な分泌シグナルを有する22が、そのシャペロン(SycE)についてはシャペロン結合部位(CBS)を含まない36。本発明者らは、YopEのN末端138アミノ酸(配列番号2)を選択して、送達すべきタンパク質と融合させた。このYopEのN末端138アミノ酸は、他の異種T3S基質の移行について最良の結果をもたらすことが証明されていた24からであった。YopEのこれらのN末端138アミノ酸はCBSを含有するので、本発明者らは、さらに、SycEを共発現させることを決めた。精製Y.エンテロコリティカpYV40毒性プラスミドからクローニングしたSycE−YopE1−138断片は、YopEの及びそのシャペロンSycEの内在性プロモーターを含有する(図2)。したがって、SycEと任意のYopE1−138の融合タンパク質は、室温での増殖から37℃への急速な温度シフトにより誘導される。37℃での培養時間は、細菌内に存在する融合タンパク質量に影響を与えることになる。多重クローニング部位(MCS)をYopE1−138の3’末端に付加させ(図2B)、その後、Myc及び6×Hisタグ及び終止コドンを付加させた。
YopE1−138断片は、細菌送達に大いに役立つものであるが、融合タンパク質の機能及び/又は局在化を妨げる可能性がある。したがって、タンパク質送達後にそれを除去することが最適であるだろう。この目的のために、本発明者らは、YopE1−138と融合パートナー(転写制御因子ET1−Myc(配列番号9及び11)44及びヒトINK4C(配列番号8及び配列番号10))との間に2つのTEV切断部位(ENLYFQS)41−43を導入した。提供する方法の優位性を保持するために、本発明者らは、別のY.エンテロコリティカ株においてTEVプロテアーゼ(S219Vバリアント;45)をYopE1−138(配列番号12)とさらに融合した。HeLa細胞を一度に両方の株に感染させた。タンパク質の移行画分のみの分析を可能にするために、感染HeLa細胞を、感染の2時間後に、細菌を溶解しないことが公知であるジギトニンを用いて溶解した(46;)。ウェスタンブロット分析は、細胞が対応する株に感染した場合にのみ、YopE1−138−2×TEV切断部位−ET1−Myc又はYopE1−138−2×TEV切断部位−Flag−INK4C−Mycの存在を明示した。この細胞溶解物を精製TEVプロテアーゼで一晩消化すると、シフトしたバンドを観察することができた。このバンドは、TEV切断部位のN末端に残存物がある、ET1−Myc又はFlag−INK4Cに対応し、この残存物は、1つのセリンのみの可能性が最も高い。TEVプロテアーゼを送達する株に細胞を同時感染させると、同じ切断ET1−Myc又はFlag−INK4C断片が目に見えるようになった。これは、T3SSを介して送達されたTEVプロテアーゼが機能性であること、及び単一細胞が両方の菌株に感染したことを示す。切断は完全なものではないが、移行したタンパク質の大多数が、感染の2時間後に既に切断され、精製TEVプロテアーゼで一晩消化しても、より高い切断率は得られなかった。報告されているように、TEVプロテアーゼ依存性切断は、融合タンパク質次第で最適化を必要とする可能性がある47、48。したがって、移行後のYopE1−138付属物のTEVプロテアーゼ依存性除去は、アミノ酸組成をN末端アミノ酸1つだけしか変化させずに、ほとんどの天然異種タンパク質のT3SSタンパク質送達をもたらす初めてのものである。
Y.エンテロコリティカの場合、毒性は、「エルシニア属外部タンパク質」(Yop)と呼ばれる6つの内在性エフェクタータンパク質、詳細には、YopH、O、P、E、M、T(MRS40 pIML421[yopHΔ1−352、yopOΔ65−558、yopP23、yopE21、yopM23、yopT135])の欠失により低減される37。これらのYopは、約70kbpサイズのプラスミドである、「エルシニア属毒性プラスミド」(pYV)上にコードされており、プラスミド上には、完全3型分泌系(T3SS)はもちろん他の毒性プレーヤーもコードされている(図4)。YopH、O、P、E、M及びTは、免疫系を調節し弱めるために細菌3型分泌系により宿主細胞に送達される6つのエフェクタータンパク質である。各Yopは、宿主細胞において特異的生化学的活性を有する。YopTは、Rho GTPaseのC末端システインを切り離し、かくて、GTPaseを膜に固定化するイソプレニル基を除去する。誤った局在化に起因するRhoのこの活性化により、マクロファージ及び好中球のような免疫細胞による食作用が回避される49。同じ経路で、YopEは、Rho GTPaseに対してGTPase活性化タンパク質(GAP)として作用して、Rho GTPaseを不活性化する。この結果、免疫細胞による食作用が減少し、IL−1ベータ放出が阻害されることになる49。さらに、YopOは、グアニジンヌクレオチド解離阻害因子(GDI)として作用して、Rho GTPaseを不活性化する。YopOは、アクチン細胞骨格に対してまだ定義されていない方法で作用する、セリン/スレオニンキナーゼドメインを有する49。YopHは、接着斑キナーゼ(Fak)のような接着斑タンパク質、パキシリン等に対して作用し、かくてマクロファージ及び好中球による食作用を強力に防止する、チロシンホスファターゼである49。YopPは、偽結核菌(Y.pseudotuberculosis)又はペスト菌(Y.pestis)ではYopJと称するものであり、免疫細胞におけるMAPK/NFkB経路を不活性化して、これらの細菌の存在による刺激を受けた免疫細胞からのTNFa及びIL−8放出を防止することが判明した。さらに、YopPは、免疫細胞においてアポトーシスを誘導することが判明しており、これは、MAPK経路における効果に関係がある可能性があり、これは活性化状態で細胞をアポトーシスから保護する49。YopMの役割は、まだ完全に明らかになってはいないが、YopMは、リボソームS6キナーゼ1(RSK1)及びプロテインキナーゼC様2(PRK2)に関連して見出された。YopMは、RSK1のリン酸化を刺激し、かくて、例えば細胞周期進行のような、下流の標的に作用するように思われる49。これらのYopのうちの1つ又はいくつかを欠失させることにより、免疫系に対する細菌の防御機構に劇的な影響を与えられる50。それぞれのyopの変異を、それぞれの領域に関してPCRにより確認し、またインビトロ分泌アッセイにより確認した。SDS−PAGE及びクマシーブルー染色によるインビトロでの分泌の分析により、完全長YopH、O、M及びYopEの非存在が確認された。
腫瘍特異的ビヒクルとしての、T3SSエフェクターのような重要な毒性決定因子の変異を有するグラム陰性菌を検証するために、十分に確証されているB16F10メラノーマモデル(ATCC番号CRL−6475)を使用するマウス同種移植腫瘍研究を行った。皮下腫瘍がある特定のサイズ(約100−200mm3)に達したら、マウスを2×105cfuのY.エンテロコリティカ亜種パレアークティカMRS40又はY.エンテロコリティカ亜種パレアークティカMRS40 ΔyopH、O、P、E、M、Tに静脈内感染させた。細菌増殖を可能にするために、感染の24時間前にマウスをデフェロキサミンで前治療した。wt Y.エンテロコリティカ亜種パレアークティカMRS40株に感染したマウスは、身体的外見及び行動について高いスコアが付き(図47−48)、感染の最初の48時間にわたって有意な体重減少を示し(図49)、これに促されて、本発明者らは、この群のマウス全てを既に感染後2日目に屠殺した。対照的に、Y.エンテロコリティカ亜種パレアークティカMRS40ΔyopH、O、P、E、M、T株に感染したマウスは、感染後4日目でも、有意な体重減少を示さず、身体的外見及び行動について正常なスコアが付与された(図47−49)。wt株(Y.エンテロコリティカ亜種パレアークティカMRS40)に感染したマウスでは、評価した全ての臓器において、及びさらには血液中で、生きている細菌が検出された(図51)。悪性固形腫瘍内に存在するwt細菌が認められたが、同程度に多い又はそれ以上に多い数が他の臓器において認められ、脾臓内が最も多かった(図51)。際だって対照的に、Y.エンテロコリティカ亜種パレアークティカMRS40ΔyopH、O、P、E、M、Tに感染したマウスでは、生きている細菌は、感染後1日目に主として悪性固形腫瘍内で認められ、少ない細菌数が脾臓、肝臓及び肺で観察された。特に、感染後4日目に、悪性固形腫瘍内の細菌数は、数桁増加した(腫瘍組織1g当り108cfu超に達した)が、評価した全ての他の臓器では、細菌数が検出限界未満に低下した(図50)。したがって、Y.エンテロコリティカ亜種パレアークティカMRS40ΔyopH、O、P、E、M、Tは、感染後4日目に、脾臓又は肝臓と比較して(検出限界に対して割当量を計算して)悪性固形腫瘍部位で約(最小限)100万倍の割当量で蓄積した。
遺伝子修飾Y.エンテロコリティカによる腫瘍定着実験を同系マウス同種移植モデル(4T1乳がんモデル)において繰り返し、細菌の定着を2週間にわたって追跡した。このとき、マウスを1×106コロニー形成単位(CFU)のY.エンテロコリティカΔyopH、O、P、E、M、Tに感染させた。感染後まだ間もない頃にB16F10モデルと同様の結果を得たが、本発明者らは、腫瘍定着が、感染後8日目に、そして14日目まで一貫して認められることをさらに明らかにすることができた(図7)。さらに、この定着は非常に特異的であり、評価した他の全ての臓器においてほんの少数の細菌しか検出されなかった(図8)。これらの研究結果は、Y.エンテロコリティカΔyopH、O、P、E、M、Tが、腫瘍の持続的定着を確立することによって、免疫系によるクリアランスを防止することができることを示す。
インビボで腫瘍細胞に送達されるYopE1−138−(tBID BH3)2の影響を評価するために、本発明者らは、4T1乳がん細胞を皮下に同種移植した野生型Balb/Cマウスにおいて研究を行った。本発明者らは、エルシニア属毒性プラスミドpYV上のYopEの天然部位に天然YopEプロモーターのもとでYopE1−138−(tBID BH3)2をコードしているY.エンテロコリティカΔHOPEMT株を評価し、それを、VirFの上流のhairpin I領域の欠失によりさらに最適化して送達されるタンパク質量を増加させることを目標とした。腫瘍が150−250mm3のサイズに達したら、PBS又は1×107のY.エンテロコリティカΔHOPEMT ΔHairpinI−virF pYV−YopE1−138−(tBID BH3)2をマウスに静脈内注射した。細菌の静脈内注射の日を0日目と定義した。その後数日(細菌の静脈内注射後0日目から9日目まで)にわたって腫瘍体積をノギスで測定した。腫瘍サイズの一切の初期均質性を補償するために、腫瘍体積を0日目の腫瘍体積に対して正規化した。Y.エンテロコリティカΔHOPEMT ΔHairpinI−VirF pYV−YopE1−138−(tBID BH3)2での治療は、腫瘍体積進行に対する影響を示し、細菌投与後8、9及び10日目に統計的に有意な腫瘍低減があった(図9)。重要なこととして、単独でのY.エンテロコリティカΔHOPEMTは、4T1マウスがんモデルにおいて腫瘍進行に影響を与えないことが判明した(図10)。これらの実験結果は、そのような細菌及びそれらのT3SSを腫瘍進行の妨害に用いることができることに光を当てるものである。
ほとんどの公知エルシニア属毒性遺伝子は、真核生物宿主外では発現されず、宿主環境への侵入後にしか誘導されない。これらの毒性遺伝子の発現は、宿主への侵入に関連する温度の急上昇により誘導される。特に、T3SS及びそのエフェクタータンパク質(Yop)のような、pYVにコードされた毒性因子は、このように制御される。室温(20−25℃)では、T3SSの制御、T3SS形成に必要な遺伝子それ自体、及び送達されたエフェクターは、発現されず、そのような温度が37℃に上昇された時のみ、発現が誘導される。pYVにコードされた毒性遺伝子(yadA及びT3SS関連遺伝子)の大多数の発現は、温度により誘導され、Y.エンテロコリティカではAraC型DNA結合タンパク質VirF(他のエルシニア属種ではLcrF)を必要とする。LcrFの発現の温度制御は、より高温でのmRNA内のRNAステム−ループの融解によって起こると考えられ、融解されない場合にはリボソーム結合部位を封鎖し、かくて翻訳を防止する51。対照的に、Y.エンテロコリティカの場合、VirFの転写が、主として、温度に依存することが証明されている52。つい最近の研究はより複雑な状況を示し、YmoAと呼ばれる熱不安定性制御因子の関与を示唆するが51、一方、LcrFの上流のRNA温度センサーが、LcrFの温度制御、したがって温度依存性毒性遺伝子の主な原因であることが判明した。
Y.エンテロコリティカΔyopH、O、P、E、M、Tは、本発明者らのマウス実験で固形腫瘍を標的とする非常に特異的な株であることが明らかになり、その一方で、細胞傷害性タンパク質のT3SS依存性送達はがん細胞に関する細胞培養で効果的であることが判明した。これら2つの形質を併用するために、数日又はさらには数週間にわたってインビボで細胞傷害性カーゴを安定的にコードするように固形腫瘍定着細菌を最適に操作する必要がある。制御要件のため、細菌内に外来プラスミドを維持するための古典的抗生物質耐性の使用は、不利である。それ故、マウス同種移植研究において、本発明者らは、抗生物質耐性のないプラスミド維持系を評価した。この系は、必須遺伝子(例えば、アスパラギン酸セミアルデヒドデヒドロゲナーゼ、asd)の染色体欠失、及び細菌増殖を維持するための異種プラスミド上の同遺伝子のコーディングに基づく。プラスミド維持のためのasd欠失の補完は以前に明らかにされている53が、再現性及び数日にわたっての持続性の相違が報告されている54。本発明者らは、この系をY.エンテロコリティカΔyopH、O、P、E、M、Tにおいての使用に適応させ、そこで、本発明者らは、染色体にコードされたasdをさらに欠失させ(その結果、Y.エンテロコリティカΔyopH、O、P、E、M、T Δasdを得)、次いで、中等度コピー数のプラスミドpBad−MycHisA上においてasdを戻した(pBad−MycHisA−asd)。asd遺伝子をY.エンテロコリティカ8081からクローニングし、pBad−MycHisAのPciI部位にその内在性プロモーター及び転写ターミネーターと共にそれぞれ順方向及び逆方向に挿入した。得られた株Y.エンテロコリティカΔyopH、O、P、E、M、T Δasd+pBad−MycHisA−asd(順又は逆asd方向)の増殖挙動を培養フラスコにおいてインビトロ(BHI培地)でwt及び親Y.エンテロコリティカΔyopH、O、P、E、M、T Δasd株と比較した(図12)。両方の方向で、pBad−MycHisA−asdは、asdの欠失時に観察された表現型を回復させた(図12)。対照的に、B16F10メラノーママウスモデル及び4T1モデルでは、本発明者らは、Y.エンテロコリティカΔyopH、O、P、E、M、T Δasd+pBad−MycHisA−asdが十分に固形腫瘍に定着しないことを見出し(図13及び14)、単離した少数のコロニーから本発明者らはpBad−MycHisA−asdプラスミドを回収することができなかった。さらに、B16F10メラノーママウスモデルから単離したコロニーを、アンピシリン耐性をコードするpBad−MycHisA−asdプラスミドの存在に有利になるアンピシリン含有プレート上で、増殖について検証した。静脈内注射後4日目に、単離したコロニーのわずかな割合しかアンピシリン耐性を示さなかった(図13)。これは、pBad−MycHisA−asdの欠損を示す可能性があったが、アンピシリン耐性遺伝子のみの欠損に、又はアンピシリンによる殺滅を回避するのに十分迅速なアンピシリン耐性遺伝子の発現の再始動の困難に関連する可能性もあった。いずれにせよ、B16F10メラノーマウスモデル及び4T1モデルにおいて観察されたエンテロコリティカΔyopH、O、P、E、M、T Δasd+pBad−MycHisA−asdでの腫瘍定着は、固形腫瘍内の細菌数の急減を示し、これは、Y.エンテロコリティカΔyopH,O,P,E,M,Tとは対照的である。このように、染色体asdの欠失及びpBad−MycHisAに対する補完は、インビボでの細菌の適応度の大幅な低下をもたらす。この観察は、バランスの取れていないasdレベルによる生存率低下に関する報告54と一致する。
4T1細胞(図9及び10)と同様の実験を、EMT6乳がんマウスモデルにおいて行い、この実験では、野生型Balb/CマウスにEMT6乳がん細胞を皮下に同種移植し、腫瘍が約80−250mm3のサイズに達したら細菌の単回静脈内投与で治療した。細菌の静脈内注射の日を0日目と定義し、全てのマウスにd0の前日にデスフェラールの腹腔内注射を施した。Y.エンテロコリティカΔHOPEMTでの治療は、生理食塩水と比較して腫瘍進行に影響を与えなかった。Y.エンテロコリティカΔHOPEMT pYV−YopE1−138−(tBID BH3)2は、腫瘍進行に対してわずかな影響を与え、これが、Y.エンテロコリティカΔHOPEMT ΔHairpinI−VirF pYV−YopE1−138−(tBID BH3)2の使用により強化された(図45)。これらの研究結果は、そのような細菌及びそれらのT3SSを腫瘍進行への干渉に利用することができること、並びにVirF活性の操作を、インビボでの投与時の細菌T3SS活性の調節に使用することができることに光を当てるものである。さらに、Y.エンテロコリティカΔHOPEMT ΔHairpinI−VirF Δasd pYV−asd−YopE1−138−(tBID BH3)2の使用は、腫瘍進行に対する影響をさらに強化した(図45)。このことにより、全身投与時の遺伝子安定性増大の利点が強調される。
サイトゾル核酸は、サイトゾル中の病原体由来RNAを検出するRIG−1様受容体(RLR)ファミリーメンバーのような受容体により感知される56。RIG−1及びMAD5は、2つのN末端CARDドメインと特定の核酸を感知する中心(DexD/H)ヘリカーゼドメインとからなる56。刺激性RNAへの結合は、オリゴマーを形成するための56(及びMDA5の場合はフィラメント形成への56)非固着K63連結ユビキチン鎖とのその後の会合のためにそのCARDを遊離するRIG−I(及びMAD5)の構造再構成を誘導する。RIG−I及びMDA5のオリゴマー化したCARDドメインは、MAVSのCARDドメインと相互作用する。この相互作用は、MAVSの単一のCARDドメインの重合を促進し、これが下流のシグナル伝達を誘導し、最終的にはI型IFN遺伝子の誘導に至る56。
cGAS/STING経路において、サイトゾル二本鎖DNAは、酵素サイクリックGMP−AMPシンターゼ(cGAS)と結合することにより検出される。dsDNAが結合すると、cGASは、活性化され、サイクリックCDN二次メッセンジャーであるサイクリックGMP−AMP(cGAMP)を産生する。次いで、cGAMPは、小胞体受容体タンパク質STING(IFN遺伝子の刺激因子)と直接結合する。cGAMPが結合すると、STINGは、活性化され、シグナル伝達経路を誘導して、I型IFN及び他の同時制御遺伝子の転写に至る57。ヒトcGASは、2’,3’cGAMP(2’−5’及び3’−5’ホスホジエステル結合を含有する)を産生するが、他のCDNは、マウス又はヒトSTINGを様々なレベルで誘導することが証明されている。これは、3’,3’cGAMP(例えば、コレラ菌(Vibrio cholera)DncV若しくは一部の真核生物cGASにより産生される)、サイクリックジAMP(例えば、様々なグラム陽性種のCdaA若しくはDisAにより産生される)又はサイクリックジGMP(例えば、緑膿菌(Pseudomonas aeruginosa)WspRにより産生される)を含む57、58。wtヒトSTING(及びマウスSTING)は、2’,3’cGAMP、3’,3’cGAMP、サイクリックジAMP及びサイクリックジGMPを認識するが、いくつかの天然ヒトSTING変異体は、これらのアゴニストに対して異なった応答をする59。
融合タンパク質YopE1−138−マウスRIG−1 CARD2、YopE1−138−コレラ菌DncV、YopE1−138−セレウス菌DisA様タンパク質及びYopE1−138−イソギンチャクcGASの送達を、免疫レポーター細胞株でI型IFN誘導について評価した。I型IFN刺激のためのマウスRAW264.7マクロファージレポーター細胞は、多量体ISREにより増強されたIFN誘導性ISG54プロモーターを含むI−ISG54プロモーターの調節下にある分泌型アルカリホスファターゼの活性に基づく。pBadMycHisA由来プラスミド(pBad_Si2)から発現し、YopE1−138−マウスRIG−1 CARD2、YopE1−138−コレラ菌DncV、YopE1−138−セレウス菌DisA様タンパク質及びYopE1−138−イソギンチャクcGASを移行させる菌株の様々な量(感染多重度)に、レポーター細胞を感染させた。YopE1−138−マウスRIG−1 CARD2、YopE1−138−コレラ菌DncV、及びYopE1−138−イソギンチャクcGAS全てが、この免疫レポーター細胞株においてI型IFN応答を用量依存的に誘導することを示した(図25)が、細菌バックグラウンド株(Y.エンテロコリティカΔHOPEMT)は、そのような応答を誘導することができなかった(図25)。YopE1−138−マウスRIG−1 CARD2が最も高い活性を示し、これに、3’,3’cGAMP産生イソギンチャク(ネマトステラ・ベクテンシス)cGAS及びコレラ菌DncV(3’,3’cGAMPを産生する)が続いた。セレウス菌DisA様タンパク質(サイクリックジAMPを産生する)は、I型IFN応答を弱くしか活性化しないことが判明した。
サイトゾル核酸は、サイトゾル中の病原体由来RNAを検出するRIG−1様受容体(RLR)ファミリーメンバーのような受容体により検知される56。RIG−1及びMAD5は、2つのN末端CARDドメインと特定の核酸を検知する中心(DexD/H)ヘリカーゼドメインとからなる56。刺激性RNAとの結合は、オリゴマーを形成するための56(及びMDA5の場合はフィラメント形成への56)非固着K63連結ユビキチン鎖とのその後の会合のためにそのCARDを遊離するRIG−I(及びMAD5)の構造再構成を誘導する。RIG−I及びMDA5のオリゴマー化したCARDドメインは、MAVSのCARDドメインと相互作用する。この相互作用は、MAVSの単一のCARDドメインの重合を促進し、これが下流のシグナル伝達を誘導し、最終的にI型IFN遺伝子の誘導に至る56。
サイクリックジヌクレオチドは、下流のI型IFNシグナル伝達誘導に至るSTING経路の周知アゴニストである。STINGアゴニストは、文献59に記載されており、主として免疫細胞に作用し、樹状細胞に対して最も高い活性を示すことが判明している59。対照的に、RLRシグナル伝達は、より遍在的に発現されることが判明した63。それ故、本発明者らは、免疫細胞(RAWマクロファージIFNレポーター細胞)及び非免疫細胞(B16F1メラノーマIFNレポーター細胞)上で、インターフェロン誘導能について、サイクリックジヌクレオチド生成酵素(YopE1−138−イソギンチャクcGAS及びYopE1−138−ヒトcGAS)を送達するY.エンテロコリティカΔHOPEMT細菌、又はYopE1−138−マウスRIG1 CARDドメイン1−218を送達する細菌を、小分子STINGアゴニスト2’3’−c−ジAM(PS)2(Rp、Rp)(Aduro BiotechからのADU−S100に類似)と比較した。免疫細胞で、同様の活性化能が、小分子STINGアゴニスト2’3’−c−ジAM(PS)2(Rp,Rp)、及びタンパク質(YopE1−138−イソギンチャクcGAS、YopE1−138−ヒトcGAS又はYopE1−138−マウスRIG1 CARDドメイン1−218)を送達する3つ全ての被験菌株について観察されたが、タンパク質を送達しないY.エンテロコリティカΔHOPEMT細菌は、非常に弱い活性化能を示した(図34−37)。非免疫細胞(がん細胞、メラノーマ)では、細菌により送達されるYopE1−138−マウスRIG1 CARDドメイン1−218、細菌により送達されるYopE1−138−イソギンチャクcGAS及びYopE1−138−ヒトcGASは、同様によく働き、ほとんどが小分子STINGアゴニストより優れており、これは、STINGと比較してRLRのより偏在的な存在を強調した(図34−37)。
厳密なT3SS依存性輸送を証明するために、真核細胞膜への移行孔を形成するT3SSタンパク質の1つを欠失させた(YopB)。そのようなyopB欠失細菌(Y.エンテロコリティカΔHOPEMT−yopBと呼ばれる)についてのYopE1−138−マウスRIG1 CARDドメイン1−246又はYopE1−138−ヒトMAVS CARD1−100発現能をRAWマクロファージIFNレポーター細胞株で評価し、同じくYopE1−138−マウスRIG1 CARDドメイン1−246又はYopE1−138−ヒトMAVS CARD1−100を発現する、yopB発現Y.エンテロコリティカΔHOPEMT細菌と比較した(図38)。YopE1−138−マウスRIG1 CARDドメイン1−246又はYopE1−138−ヒトMAVS CARD1−100を発現するyopB−野生型細菌は、I型IFN応答の用量依存性活性化を示したが、同じタンパク質を発現するyopB欠失株は、そのような応答を、送達されるタンパク質を発現しないバックグラウンド非細菌株により誘導されるバックグラウンドレベルより高く誘導することができなかった(図38)。これにより、YopE1−138−マウスRIG1 CARDドメイン1−246又はYopE1−138−ヒトMAVS CARD1−100は両方とも、T3SSニードルを通って標的真核細胞に輸送されることが検証される。
I型IFN応答を腫瘍微小環境内で開始させることができることを検証するために、本発明者らは、エクスビボで菌株に感染させた粗腫瘍単離物に関する分析を行い、続いてインターフェロンベータを用いてELISAを行った。EMT6乳がん細胞を皮下に同種移植した野生型Balb/Cマウスを、腫瘍が の体積に達したときに屠殺した。腫瘍をすりつぶし、消化し、24ウェルプレートに単一細胞懸濁液として播種した。2つの異なる腫瘍からのそのような細胞を、未感染で放置した(図39中の破線)か、又はY.エンテロコリティカΔHOPEMT、若しくはpBadMycHisA由来プラスミド上にYopE1−138−マウスRIG1 CARDドメイン1−246をコードしているY.エンテロコリティカΔHOPEMTに感染させた。インターフェロンベータを用いるELISAを使用してIFN刺激を評価し、これは、Y.エンテロコリティカΔHOPEMTが、インターフェロンベータ分泌を誘導することができないが、YopE1−138−マウスRIG1 CARDドメイン1−246をコードしているY.エンテロコリティカΔHOPEMTによる感染が、2つの異なる腫瘍の粗腫瘍単離物による用量依存性インターフェロンベータ分泌をもたらすことを示した(図39)。これにより、細菌により送達されるRIG1 CARDドメインは、がん細胞、免疫細胞及び腫瘍微小環境内の他の全ての細胞からなる混合細胞集団においてインターフェロン産生を誘導することができることが検証される。
インビボで腫瘍細胞に送達されるYopE1−138−マウスRIG1 CARDドメイン1−246及びYopE1−138−ヒトcGASの影響を評価するために、本発明者らは、EMT6乳がん細胞を皮下に同種移植した野生型Balb/Cマウスにおいて研究を行った。腫瘍が約60−130mm3のサイズに達したら、PBS(図40)又は7.5×107 Y.エンテロコリティカΔHOPEMT、Y.エンテロコリティカΔHOPEMT+YopE1−138マウスRIG1 CARDドメイン1−246若しくはY.エンテロコリティカΔHOPEMT+YopE1−138ヒトcGASをマウスに腫瘍内(it)注射した。細菌の初回腫瘍内注射の日を0日目と定義した。d0、d1、d5、d6、d10及びd11に、マウスに腫瘍内注射した。その後数日にわたって腫瘍体積をノギスで測定した。Y.エンテロコリティカ単独での治療は、腫瘍体積進行に対する影響を示し、4/14匹のマウスが完全腫瘍退縮を示した(図41)。I型IFN応答を誘導するタンパク質であって、RIG1 CARDS又はcGASであるタンパク質を送達するY.エンテロコリティカΔHOPEMTは、腫瘍進行に対してより顕著な影響をもたらし、8/14(RIG1 CARD)又は8/15(cGAS)匹のマウス各々が完全且つ永続的腫瘍退縮を示すことが判明した(図42−43)。これらの研究結果は、そのような細菌及びそれらのT3SSを、腫瘍進行への有意な干渉に利用することができること、及びI型IFN誘導タンパク質の送達が、原発性腫瘍の退縮を誘導するのによく適していることに光を当てるものである。
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Claims (14)
- 細菌エフェクタータンパク質からの送達シグナルをコードするヌクレオチド配列の3’末端とインフレームで融合している異種タンパク質をコードするヌクレオチド配列を含むヌクレオチド分子を含む組換え弱毒化グラム陰性菌株であって、
細菌エフェクタータンパク質からの送達シグナルをコードするヌクレオチド配列が、プロモーターに作用可能に連結され、異種タンパク質が、インターフェロン(IFN)応答の誘導又は制御に関与するタンパク質であり、IFN応答の誘導又は制御に関与するタンパク質が、I型IFN応答の誘導又は制御に関与するタンパク質であり、I型IFN応答の誘導又は制御に関与する異種タンパク質が、RIG−I様受容体(RLR)ファミリーと、抗ウイルスシグナル伝達及びI型IFN誘導に関与するタンパク質を含有する他のCARDドメインと、STINGの刺激をもたらすWspR、DncV、DisA及びDisA様、CdaA、CdaS並びにcGASからなる群から選択されるサイクリック−ジAMP、サイクリック−ジGMP及びサイクリック−ジGAMPシクラーゼ等のサイクリックジヌクレオチド生成酵素とからなる群から選択される、組換え弱毒化グラム陰性菌株。 - IFN応答の誘導又は制御に関与するタンパク質が、I型IFN応答の誘導又は制御に関与するタンパク質であり、I型IFN応答の誘導又は制御に関与するタンパク質が、RIG1、MDA5、MAVS/IPS−1、WspR、DncV、DisA及びDisA様、CdaA、並びにcGAS、又はその断片からなる群から選択される、請求項1に記載の組換え弱毒化グラム陰性菌株。
- 増殖に必須の内在性タンパク質をコードする染色体遺伝子の欠失、及びプロモーターに作用可能に連結されている増殖に必須の前記内在性タンパク質をコードする遺伝子を含むヌクレオチド配列を含む内在性毒性プラスミドをさらに含む、請求項1又は2に記載の組換え弱毒化グラム陰性菌株。
- 少なくとも1つの細菌エフェクタータンパク質の産生が不十分である、請求項3に記載の組換え弱毒化グラム陰性菌株。
- 増殖に必須の内在性タンパク質をコードする遺伝子が、アミノ酸産生に必須の酵素をコードする遺伝子、ペプチドグリカン生合成に関与する酵素をコードする遺伝子、LPS生合成に関与する酵素をコードする遺伝子、ヌクレオチド生合成に関与する酵素をコードする遺伝子、及び翻訳開始因子をコードする遺伝子から選択される、請求項3又は4に記載の組換え弱毒化グラム陰性菌株。
- 増殖に必須の内在性酵素をコードする遺伝子が、アミノ酸産生に必須の酵素をコードする遺伝子であり、アミノ酸産生に必須の酵素が、アスパラギン酸−ベータ−セミアルデヒドデヒドロゲナーゼ(asd)である、請求項3から5の何れか一項に記載の組換え弱毒化グラム陰性菌株。
- エルシニア属(Yersinia)株である、請求項1から6の何れか一項に記載の組換え弱毒化グラム陰性菌株。
- 内在性毒性プラスミド上に位置する増殖に必須の内在性酵素をコードする遺伝子が、その内在性プロモーター及びその内在性転写ターミネーターを含む、請求項3から7の何れか一項に記載の組換え弱毒化グラム陰性菌株。
- 増殖に必須の内在性酵素をコードする遺伝子、その内在性プロモーター、及びその内在性転写ターミネーターが、内在性毒性プラスミド上のorf155(SycO)の開始点の122bp上流に位置する、請求項8に記載の組換え弱毒化グラム陰性菌株。
- 内在性AraC型DNA結合タンパク質をコードする遺伝子の上流のRNA温度センサー領域内のモジュレーションをさらに含む、請求項1から9の何れか一項に記載の組換え弱毒化グラム陰性菌株。
- 内在性AraC型DNA結合タンパク質をコードする遺伝子の上流のRNA温度センサー領域内のモジュレーションが、内在性AraC型DNA結合タンパク質をコードする遺伝子の上流のRNAヘアピン構造又はその一部を除去する欠失を含む、請求項10に記載の組換え弱毒化グラム陰性菌株。
- AraC型DNA結合タンパク質が、VirFである、請求項10又は11に記載の組換え弱毒化グラム陰性菌株。
- エルシニア・エンテロコリティカ(Yersinia enterocolitica)である、請求項1から12の何れか一項に記載の組換え弱毒化グラム陰性菌株。
- 請求項1から13の何れか一項に記載の組換え弱毒化グラム陰性菌株を含む、対象のがんを治療するための医薬。
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