JP5891259B2 - ヒト化FcγRマウス - Google Patents
ヒト化FcγRマウス Download PDFInfo
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- JP5891259B2 JP5891259B2 JP2014087973A JP2014087973A JP5891259B2 JP 5891259 B2 JP5891259 B2 JP 5891259B2 JP 2014087973 A JP2014087973 A JP 2014087973A JP 2014087973 A JP2014087973 A JP 2014087973A JP 5891259 B2 JP5891259 B2 JP 5891259B2
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Description
本発明の分野は、内因性マウスFcγR遺伝子を欠く遺伝子改変された非ヒト動物であり、これには、内因性FcγR遺伝子の、ヒトFcγR遺伝子による置き換えを含む遺伝子改変された動物が含まれ、かつ少なくとも2、3、4、または5つの機能的ヒト低親和性FcγR遺伝子を発現することができるマウスが含まれ、かつ内因性低親和性FcγR遺伝子を発現しない免疫細胞を含む遺伝子改変されたマウスが含まれる。
Fc受容体(FcR)は、哺乳動物において免疫系の様々な機能を実行する、免疫系の細胞の表面上に見出されるタンパク質である。FcRは、様々な細胞上に、様々なタイプで存在し、たとえば、感染細胞または侵入している病原体に付いている抗体への結合、抗体媒介性食作用または抗体依存性細胞媒介性細胞傷害(ADCC)によって微生物または感染細胞を破壊するために、食作用性細胞または細胞傷害性細胞の刺激などのような、様々な免疫機能を媒介する。
特定の実施形態では、例えば以下が提供される:
(項目1)
遺伝子改変を含むマウスであって、該遺伝子改変が、内因性低親和性FcγR遺伝子の、該内因性マウス低親和性FcγR遺伝子の遺伝子座に配置される、少なくとも2つの低親和性ヒトFcγR遺伝子による置き換えを含み、該マウスが機能的FcR γ鎖を含む、マウス。
(項目2)
前記少なくとも2つの低親和性ヒトFcγR遺伝子が、FcγRIIA、FcγRIIB、FcγRIIC、FcγRIIIA、およびFcγRIIIBから選択される、項目1に記載のマウス。
(項目3)
前記少なくとも2つの低親和性ヒトFcγR遺伝子が、FcγRIIAおよびFcγRIIIAである、項目1に記載のマウス。
(項目4)
前記ヒトFcγRIIIAが、前記マウスのNK細胞上に発現される、項目3に記載のマウス。
(項目5)
前記少なくとも2つの低親和性ヒトFcγR遺伝子が、FcγRIIB、FcγRIIC、およびFcγRIIIBである、項目1に記載のマウス。
(項目6)
前記FcγRIIAおよびFcγRIIIAの遺伝子が、対立遺伝子変異体または多型を含む、項目3に記載のマウス。
(項目7)
ヒト低親和性FcγR対立遺伝子変異体を含み、前記対立遺伝子変異体が、(a)FcγRIIA対立遺伝子変異体131H、(b)FcγRIIIA対立遺伝子変異体158V、(c)FcγRIIB対立遺伝子変異体232I、(d)好中球抗原2(NA2)対立遺伝子によって特徴付けられるFcγRIIIB対立遺伝子変異体、および(e)それらの組合せから成る群から選択される、項目5に記載のマウス。
(項目8)
遺伝子改変されたマウスであって、該遺伝子改変が、FcγRIIB、FcγRIII、およびFcγRIVのαサブユニットをコードする内因性マウス遺伝子の、ヒトFcγRIIIA αサブユニットをコードする遺伝子による置き換えを含み、該マウスが、内因性マウスFcR γサブユニットを伴うヒトFcγRIIIAタンパク質を発現し、該ヒトFcγRIIIAタンパク質がマウスNK細胞の表面上に発現される、マウス。
(項目9)
マウスNK細胞が、前記マウスの血中で循環するNK細胞であり、該マウスNK細胞の表面上に発現される該FcγRIIIAタンパク質が、ヒトFcまたは改変ヒトFcを含むイムノアドヘシンまたは抗体に結合し、該イムノアドヘシンまたは抗体が、該マウスにおける標的細胞に特異的に結合し、該NK細胞の該FcγRIIIAへの、該イムノアドヘシンまたは該抗体の結合が、該標的細胞のNK細胞殺傷を媒介する、項目8に記載の遺伝子改変されたマウス。
(項目10)
前記標的細胞にヒト病原体が感染しており、前記イムノアドヘシンまたは抗体が該ヒト病原体のエピトープに特異的に結合する、項目9に記載の遺伝子改変されたマウス。
(項目11)
前記標的細胞が腫瘍細胞であり、前記イムノアドヘシンまたは抗体が該腫瘍細胞のエピトープに特異的に結合する、項目9に記載の遺伝子改変されたマウス。
(項目12)
治療薬依存性の細胞殺傷を測定するための方法であって、
(a)遺伝子改変されたマウスに、該マウスにおけるNK細胞でない標的細胞に特異的に結合する治療剤を投与するステップであって、該治療剤がヒトFcを含む、ステップと、(b)該マウスまたは該マウスの組織試料におけるNK媒介性の標的細胞殺傷のレベルを測定するステップと、
(c)前記治療剤によって媒介される標的細胞殺傷の量を決定し、それによって、治療薬依存性の細胞殺傷を測定するステップとを含み、該遺伝子改変が、マウスFcγRIIBのαサブユニットをコードする遺伝子の欠失、マウスFcγRIIIのαサブユニットをコードする遺伝子の欠失、およびマウスFcγRIVのαサブユニットをコードする遺伝子の欠失を含み、前記マウスが、ヒトFcγRIIIAのαサブユニットをコードする遺伝子を、内因性マウスαサブユニットの遺伝子座に含み、該マウスが機能的FcR γ鎖サブユニットを発現する、方法。
(項目13)
前記マウスが、前記内因性マウスαサブユニットの遺伝子座に、FcγRIIA αサブユニット、FcγRIIB αサブユニット、FcγRIIC αサブユニット、FcγRIIIB αサブユニット、およびそれらの組合せから選択されるヒトFcγR αサブユニットをコードする遺伝子をさらに含む、項目12に記載の方法。
(項目14)
前記標的細胞がヒト病原体であり、前記治療薬が、該ヒト病原体のエピトープに特異的に結合する、項目12に記載の方法。
(項目15)
前記標的細胞が、ヒト病原体が感染しているマウス細胞であり、前記治療薬が、該ヒト病原体のエピトープに特異的に結合する、項目12に記載の方法。
(項目16)
前記標的細胞が、ヒト病原体が感染しているヒト細胞であり、前記治療薬が、該ヒト病原体のエピトープに特異的に結合する、項目12に記載の方法。
(項目17)
前記標的細胞が、ヒト腫瘍細胞であり、前記治療薬が、該ヒト腫瘍細胞のエピトープに特異的に結合する、項目12に記載の方法。
(項目18)
前記組織試料が血液試料である、項目12に記載の方法。
(項目19)
前記治療薬が抗体である、項目12に記載の方法。
(項目20)
前記抗体がヒト抗体である、項目17に記載の方法。
本発明は、方法および条件が変更されてもよいので、記載される特定の方法および実験条件に限定されない。本明細書において使用される術語は、特定の実施形態を説明する目的のためのものであるにすぎず、本発明の範囲が請求項によってのみ限定されるので、限定を意図するものではない。
免疫グロブリンのFc(つまり定常)領域に対する受容体(FcR)は、免疫応答の調節において重要な役割を果たす。FcRは、抗体が結合した外来抗原を有効に処分するために、宿主の免疫系のアクセサリー細胞上に存在する。FcRはまた、免疫系のアクセサリー細胞の活性化および阻害性の応答の平衡を保つ際に重要な役割を果たす。FcRは、マクロファージによる食作用、肥満細胞の脱顆粒、抗体−抗原複合体の取り込み、および免疫応答の調節ならびに他の免疫系プロセスに関与する。
内因性低親和性マウスFcγR遺伝子を発現しないが、内因性マウスFcR γ鎖を発現する遺伝子改変された非ヒト動物が提供される。様々な実施形態では、FcR γ鎖は、野生型マウスと同じまたは実質的に同じである分布(つまり細胞型において)およびレベルでマウスにおいて発現される。内因性低親和性FcγR遺伝子は、免疫細胞の表面上にまたは動物の末梢において可溶性で発現することができる。内因性低親和性マウスFcγR遺伝子を発現しない非ヒト動物を作製するための遺伝子改変は、例証としてマウスを使用することによって好都合に説明される。本発明による遺伝子改変されたマウスは、様々な方法において作製することができ、これらの特定の実施形態は、本明細書において議論される。
低親和性ヒトFcγR遺伝子を発現する遺伝子改変された非ヒト動物が提供される。低親和性ヒトFcγR遺伝子は、動物の免疫系のアクセサリー細胞の表面上にまたは動物の末梢において可溶性で発現することができる。
内因性低親和性マウスFcγR遺伝子を発現しない遺伝子改変された非ヒト動物は、たとえば、免疫応答における個々の低親和性FcγR遺伝子の様々な機能を解明するのに、細胞性免疫(たとえばADCC)を介してのヒト治療用抗体の効能を測定するのに、免疫疾患または障害におけるFcγRの役割を決定するのに、免疫疾患または障害のモデルとして役立つのに、1つまたは複数のFcγRタンパク質に対する抗体を生成するのに、および対象の他の遺伝子改変されたマウスを生成するために交配仲間として役立つのに有用である。
低親和性FcγR遺伝子欠損マウスの生成
内因性低親和性マウスFcγR遺伝子座の欠失を導入するための標的構築物(下記に記載)を構築した(図1)。
低親和性FcγR遺伝子欠損マウスの特徴付け
脾臓を、FcγR欠損マウスおよび野生型マウスから採取し、無菌の使い捨てのバッグ中で10mLコラゲナーゼDを用いて灌流した。次いで、ただ1つの脾臓を含有するそれぞれのバッグを、Stomacher(登録商標)(Seward)の中に配置し、30秒間、中程度の設定でホモジナイズした。ホモジナイズした脾臓は、10cmペトリ皿に移し、37℃で25分間インキュベートした。細胞は、0.5M EDTAの1:50希釈液を使用して、ピペットを用いて分離し、37℃で5分間、さらにインキュベーションを続けた。次いで、細胞は、遠心分離機(10分間、1000rpm)を用いてペレットにし、赤血球は、3分間、4mL ACKバッファー(Invitrogen)中で溶解した。脾細胞は、RPMI−1640(Sigma)を用いて希釈し、再び遠心分離した。ペレットにした細胞は、10mL RPMI−1640中で再懸濁し、0.2μm細胞ストレーナーを用いてろ過した。
低親和性FcγR遺伝子欠損マウスにおけるB細胞のin vivo枯渇
ADCC経路を通してのB細胞枯渇に対するヒトまたはマウスFcアイソタイプの効果は、ヒト抗ヒトCD20抗体を使用して、ヒトCD20を発現するように遺伝子操作した低親和性FcγR遺伝子欠損マウスにおける様々なB細胞コンパートメントについて決定した。ヒトCD20を発現するマウスは、当技術分野において公知の技術を使用して、これとは別に遺伝子操作した。B細胞上にヒトCD20を発現し、低親和性FcγR遺伝子が欠損した(実施例1において記載)マウスは、2つの遺伝子操作系統の標準的な交配技術によって作製した。
FcγRIIIA/FcγRIIAヒト化マウスの生成
欠失させた内因性低親和性マウスFcγR遺伝子座(下記に記載)の中に2つの低親和性ヒトFcγR遺伝子を導入するための標的構築物を構築した(図4)。
FcγRIIIA/FcγRIIAヒト化マウスの特徴付け
脾臓は、ヒト化FcγRIIIA/FcγRIIA(伸長FcγRIIAプロモーター領域を欠くヘテロ接合体)および野生型マウスから採取し、FACのために調製した(上記に記載の通りに)。
低親和性FcγRヒト化マウスの生成
部分的にヒト化された内因性低親和性FcγR遺伝子座(下記に記載)の中に3つのさらなる低親和性ヒトFcγR遺伝子を導入するために標的構築物を構築した(図6)。
低親和性FcγRヒト化マウスの特徴付け
脾臓を、完全にヒト化したFcγR(ヘテロ接合体)および野生型マウスから採取し、FACSのために調製した(上記に記載の通りに)。
ヒト化FcγRマウスにおけるADCC
FcγR遺伝子欠損(つまりノックアウト)マウス、FcγRIIIA/FcγRIIA(ホモ接合体)マウス、FcγRIIIA/FcγRIIIB/FcγRIIA/FcγRIIB/FcγRIIC(ホモ接合体)マウス、および野生型マウスから単離した脾細胞を、細胞殺傷アッセイにおいて、ADCCを実行するそれらの能力について分析した(実施例2において上記に記載の通りに)。
Claims (11)
- 内因性FcγRIIB α鎖遺伝子におけるホモ接合性破壊、内因性FcγRIV α鎖遺伝子におけるホモ接合性破壊、および内因性FcγRIII α鎖遺伝子におけるホモ接合性破壊をそのゲノムに含む、トランスジェニックマウス。
- 前記マウスが、同じ抗原に関して、野生型マウスの能力と比較して、抗原に対する免疫応答を行う能力が低下している、請求項1に記載のマウス。
- 前記マウスが、抗体依存性細胞媒介性細胞傷害(ADCC)の少なくとも50%の減少を含む、請求項1に記載のマウス。
- 前記マウスが機能的FcR γ鎖を含む、請求項1に記載のマウス。
- 前記マウスがヒトCD20を発現する、請求項1〜4のいずれか一項に記載のマウス。
- 内因性FcγRIIB α鎖遺伝子のホモ接合性破壊、内因性FcγRIV α鎖遺伝子のホモ接合性破壊、および内因性FcγRIII α鎖遺伝子のホモ接合性破壊をそのゲノムに含む、遺伝子改変されたマウス細胞。
- 前記細胞が機能的FcR γ鎖を含む、請求項6に記載の細胞。
- 前記細胞が胚性幹(ES)細胞である、請求項6に記載の細胞。
- 前記細胞がナチュラルキラー(NK)細胞である、請求項6に記載の細胞。
- 前記細胞がヒトCD20遺伝子を発現する、請求項6〜9のいずれか一項に記載の細胞。
- 請求項8に記載の細胞の使用を包含する、内因性FcγRIIB α鎖、内因性FcγRIV α鎖、および内因性FcγRIII α鎖を発現しない遺伝子改変されたマウスの作製方法。
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