JP2016037488A - Chimeric protein with TGFβ inhibitory function - Google Patents
Chimeric protein with TGFβ inhibitory function Download PDFInfo
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- JP2016037488A JP2016037488A JP2014163803A JP2014163803A JP2016037488A JP 2016037488 A JP2016037488 A JP 2016037488A JP 2014163803 A JP2014163803 A JP 2014163803A JP 2014163803 A JP2014163803 A JP 2014163803A JP 2016037488 A JP2016037488 A JP 2016037488A
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Abstract
【課題】癌及び線維症の新規治療薬を提供する。【解決手段】ＴＧＦβ１、ＴＧＦβ２、ＴＧＦβ３のいずれにも結合することのできるキメラタンパク質、該キメラタンパク質をコードするＤＮＡ、該キメラタンパク質をコードするＤＮＡを有するベクター、該キメラタンパク質を発現する細胞、及び該キメラタンパク質を含む薬学的組成物に関する。【選択図】なしA novel therapeutic agent for cancer and fibrosis is provided. A chimeric protein capable of binding to any of TGFβ1, TGFβ2, and TGFβ3, a DNA encoding the chimeric protein, a vector having a DNA encoding the chimeric protein, a cell expressing the chimeric protein, and the It relates to a pharmaceutical composition comprising a chimeric protein. [Selection figure] None
Description
本発明は、ＴＧＦβ阻害機能を持つキメラタンパク質に関する。本発明はさらに、上記キメラタンパク質をコードするＤＮＡ、ベクター、ウイルス、細胞、上記キメラタンパク質の製造方法、並びに薬学的組成物に関する。 The present invention relates to a chimeric protein having a TGFβ inhibitory function. The present invention further relates to a DNA, vector, virus, cell encoding the chimeric protein, a method for producing the chimeric protein, and a pharmaceutical composition.
トランスフォーミング増殖因子（ＴＧＦ）βは、ヒトでは、３種のアイソフォーム、すなわちＴＧＦβ１、ＴＧＦβ２、およびＴＧＦβ３が存在する。これらは、生物学的に活性な状態において、二つの分子間のジスルフィド架橋によって結合された２つの１１２アミノ酸単量体を含む、２５ｋＤａのホモ二量体である。ＴＧＦβ１とＴＧＦβ２との間では２７個のアミノ酸が異なり、ＴＧＦβ１とＴＧＦβ３との間では２２個のアミノ酸が異なっている。ＴＧＦβは標的細胞の膜上に分布する特異的受容体に結合することで、細胞内にシグナルを伝達し、その機能を発揮する。ＴＧＦβの受容体にはＩ型受容体とＩＩ型受容体が存在し、ＴＧＦβとの結合によりヘテロ多量体からなる受容体複合体が形成される。 Transforming growth factor (TGF) β exists in humans in three isoforms, namely TGFβ1, TGFβ2, and TGFβ3. These are 25 kDa homodimers that, in biologically active state, contain two 112 amino acid monomers joined by a disulfide bridge between two molecules. Twenty-seven amino acids are different between TGFβ1 and TGFβ2, and 22 amino acids are different between TGFβ1 and TGFβ3. TGFβ binds to a specific receptor distributed on the membrane of a target cell, thereby transmitting a signal into the cell and exerting its function. TGFβ receptors include type I and type II receptors, and a receptor complex consisting of a heteromultimer is formed by binding to TGFβ.
ＴＧＦβは、細胞の増殖および分化、胚発生、細胞外基質形成、骨の発達、創傷治癒、造血、ならびに免疫反応および炎症反応に関与している多機能性サイトカインである（非特許文献１）。ＴＧＦβの調節異常は、ヒトにおいては、例えば、先天性欠損症、がん、慢性炎症性疾患、自己免疫性疾患、および線維症に関係があるとされている（非特許文献２及び非特許文献３）。 TGFβ is a multifunctional cytokine involved in cell proliferation and differentiation, embryogenesis, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses (Non-Patent Document 1). In humans, dysregulation of TGFβ is considered to be related to, for example, birth defects, cancer, chronic inflammatory diseases, autoimmune diseases, and fibrosis (Non-Patent Document 2 and Non-Patent Documents). 3).
ＴＧＦβは種々の線維症の成因に共通のサイトカインとして、その役割が強調されている。ＴＧＦβは血管内皮細胞や上皮細胞、リンパ球細胞には増殖抑制作用を示す一方で、線維芽細胞には増殖を促進し、またα平滑筋アクチン（αＳＭＡ）などを発現する筋線維芽細胞への形質転換を促す。さらにコラーゲンなどの細胞外基質の合成を強力に増加させ、同時にプラスミノーゲアクチベータ阻害因子（ＰＡＩ−１） などの線溶系阻害物質の産生を亢進するなど、組織線維化の原因となる分子である。それゆえ、線維症の治療にＴＧＦβ阻害剤が有用な可能性がある。 The role of TGFβ is emphasized as a cytokine common to the pathogenesis of various fibrosis. TGFβ has a growth inhibitory effect on vascular endothelial cells, epithelial cells, and lymphocyte cells, but promotes proliferation in fibroblasts, and also promotes myofibroblasts that express α-smooth muscle actin (αSMA) and the like. Encourage transformation. Furthermore, it is a molecule that causes tissue fibrosis, such as strongly increasing the synthesis of extracellular matrix such as collagen and at the same time enhancing the production of fibrinolytic inhibitors such as plasminogen activator inhibitor (PAI-1). . Therefore, TGFβ inhibitors may be useful for the treatment of fibrosis.
また、ＴＧＦβは細胞の運動性を増加させることに寄与し、上皮間葉転換（ＥＭＴ）を引き起こす作用があることが判明してきている（非特許文献４）。特にがん細胞の運動能の増加は転移の亢進などがんの悪性化に寄与するため、ＥＭＴを抑制することでがんの悪性化を抑制できると考えられている（非特許文献５）。 In addition, TGFβ has been found to contribute to increasing cell motility and to cause epithelial-mesenchymal transition (EMT) (Non-patent Document 4). In particular, an increase in the motility of cancer cells contributes to malignant transformation of cancer such as enhanced metastasis, and it is considered that malignant transformation of cancer can be suppressed by suppressing EMT (Non-patent Document 5).
ＴＧＦβシグナルに関連した疾患の治療を目的として、ＴＧＦβの中和抗体や低分子有機化合物である受容体拮抗物質がさかんに研究されてきたが、臨床応用されている化合物は存在していない。その一因としては、血中内薬物濃度が速やかに低下してしまうことが考えられている。 For the purpose of treating diseases related to TGFβ signal, neutralizing antibodies for TGFβ and receptor antagonists, which are low molecular weight organic compounds, have been studied extensively, but there are no compounds that have been clinically applied. One reason for this is thought to be a rapid decrease in the blood drug concentration.
ＴＧＦβ１及びＴＧＦβ３は、腫瘍部位や線維化部位、正常組織では骨髄などに発現が多く認められる。一方、ＴＧＦβ２は、特に脳腫瘍組織において高い発現が認められ、ＴＧＦβ２を標的としたアンチセンスオリゴが脳腫瘍分野で開発されているが、核酸であるため、細胞への送達の効率性、並びに安定性に関しては改善の余地があると考えられる（非特許文献６）。 TGFβ1 and TGFβ3 are often expressed in tumor sites, fibrosis sites, and normal tissues such as bone marrow. On the other hand, TGFβ2 is highly expressed especially in brain tumor tissues, and antisense oligos targeting TGFβ2 have been developed in the field of brain tumors. However, since it is a nucleic acid, it is related to the efficiency and stability of delivery to cells. Is considered to have room for improvement (Non-Patent Document 6).
ＴＧＦβ１及びＴＧＦβ３は、ＩＩ型受容体であるＴβＲＩＩとの親和性を持つ（非特許文献７）。そのため可溶性ＴβＲＩＩＦｃキメラタンパク質を用いて、ＴＧＦβシグナルを阻害することで、がん細胞の運動能を抑制するがん治療薬とする可能性について報告がある（非特許文献８）。Ｆｃキメラタンパク質はＦｃドメインを持つことによって抗体と同程度の血中滞留性を示すことが考えられ、しかも抗体よりも安価に生産しうる可能性を持っている。しかしながら、既存の可溶性ＴβＲＩＩＦｃキメラタンパク質は、ＴＧＦβ２との結合能を持たないために、ＴＧＦβ２より引き起こされると種々の疾患に関しては効果を持たないことが考えられる。 TGFβ1 and TGFβ3 have affinity for TβRII, which is a type II receptor (Non-patent Document 7). Therefore, there is a report on the possibility of using a soluble TβRIIFc chimeric protein as a cancer therapeutic agent that inhibits the motility of cancer cells by inhibiting the TGFβ signal (Non-patent Document 8). It is considered that the Fc chimeric protein has the same retention in blood as an antibody by having an Fc domain, and has the possibility of being produced at a lower cost than an antibody. However, since existing soluble TβRIIFc chimeric protein does not have the ability to bind to TGFβ2, it is considered that when it is caused by TGFβ2, it has no effect on various diseases.
上記の通り、ＴＧＦβシグナル関連疾患に対する治療を行う上で、薬物動態に課題点があり、十分にＴＧＦβシグナルをｉｎ ｖｉｖｏで抑制できない小分子化合物や、安価に供給することが難しい中和抗体にかわる新規な治療薬を開発することが望まれている。本発明は、優れた血中動態を示し、かつ安価に供給しうる、ＴＧＦβ阻害機能を持つキメラタンパク質を提供することを課題とする。本発明はさらに上記キメラタンパク質をコードするＤＮＡ、ベクター、ウイルス、細胞、上記キメラタンパク質の製造方法、並びに薬学的組成物を提供することを課題とする。 As described above, there are problems in pharmacokinetics in the treatment of TGFβ signal-related diseases, and there are small molecule compounds that cannot sufficiently suppress TGFβ signal in vivo, and neutralizing antibodies that are difficult to supply inexpensively. It is desirable to develop new therapeutic agents. An object of the present invention is to provide a chimeric protein having a TGFβ inhibitory function, which exhibits excellent blood dynamics and can be supplied at low cost. Another object of the present invention is to provide a DNA, vector, virus, cell, method for producing the chimeric protein, and pharmaceutical composition encoding the chimeric protein.
本発明者らは、上記課題を達成するために鋭意検討した結果、ＴＧＦβＩ型受容体細胞外ドメインとＴＧＦβＩＩ型受容体細胞外ドメインとを結合させたキメラタンパク質がＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３の全てに結合することを見出し、本発明を完成するに至った。 As a result of intensive studies to achieve the above-mentioned problems, the present inventors have found that a chimeric protein in which a TGFβ type I receptor extracellular domain and a TGFβ type II receptor extracellular domain are bound to all of TGFβ1, TGFβ2, and TGFβ3. As a result, the present invention has been completed.
すなわち本発明は、以下の（１）〜（２２）に関するものである。
（１） ＴＧＦβＩ型受容体細胞外ドメイン及びＴＧＦβＩＩ型受容体細胞外ドメインを有し、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質。
（２） ＴＧＦβＩ型受容体細胞外ドメインがＡＬＫ５細胞外ドメインである、（１）に記載のキメラタンパク質。
（３） ＴＧＦβＩＩ型受容体細胞外ドメインがＴβＲＩＩ細胞外ドメインである、（１）又は（２）に記載のキメラタンパク質。
（４） さらに二量体化ドメインを有する、（１）から（３）の何れかに記載のキメラタンパク質。
（５） 二量体化ドメインが免疫グロブリンドメインである、（４）に記載のキメラタンパク質。
（６） 免疫グロブリンドメインが、ＩｇＧのＦｃドメイン、ＩｇＧの重鎖、又はＩｇＧの軽鎖である、（５）に記載のキメラタンパク質。
（７） ＡＬＫ５細胞外ドメインのＣ末端にＴβＲＩＩ細胞外ドメインのアミノ末端（Ｎ末端）が結合し、ＴβＲＩＩ細胞外ドメインのカルボキシル末端（Ｃ末端）に免疫グロブリンドメインのＮ末端が結合している、（１）から（６）の何れかに記載のキメラタンパク質。
（８） ＴβＲＩＩ細胞外ドメインのＣ末端にＡＬＫ５細胞外ドメインのＮ末端が結合し、ＡＬＫ５細胞外ドメインのＣ末端に免疫グロブリンドメインのＮ末端が結合している、（１）から（６）の何れかに記載のキメラタンパク質。
（９） 以下の（ａ）〜（ｃ）の何れかである、（１）から（８）の何れかに記載のキメラタンパク質。
（ａ）配列番号４又は配列番号１５で表されるアミノ酸配列からなるキメラタンパク質；
（ｂ）配列番号４又は配列番号１５で表されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質；又は
（ｃ）配列番号４又は配列番号１５で表されるアミノ酸配列において１又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質；
（１０） アセチル修飾またはポリエチレングリコール修飾を受けている、（１）から（９）の何れかに記載のキメラタンパク質。
（１１） 二量体化している、（１）から（１０）の何れかに記載のキメラタンパク質。
（１２） 以下の（ａ）〜（ｃ）の何れかのキメラタンパク質をコードするＤＮＡ。
（ａ）配列番号４又は配列番号１５で表されるアミノ酸配列からなるキメラタンパク質；
（ｂ）配列番号４又は配列番号１５で表されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質；又は
（ｃ）配列番号４又は配列番号１５で表されるアミノ酸配列において１又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質。
（１３） 以下の（ｄ）又は（ｅ）の何れかのＤＮＡ。
（ｄ）配列番号８又は配列番号１６で表される塩基配列からなるＤＮＡ；又は
（ｅ）配列番号８又は配列番号１６で表される塩基配列において１又は数個の塩基が欠失、置換及び／又は付加している塩基配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質をコードする塩基配列からなるＤＮＡ。
（１４） （１２）又は（１３）に記載のＤＮＡを含むベクター。
（１５） （１４）に記載のベクターを有するウイルス。
（１６） （１４）に記載のベクター又は（１５）に記載のウイルスを有する細胞。
（１７） 細菌細胞、酵母細胞、昆虫細胞及び哺乳動物細胞から成る群から選択される細胞である、（１６）に記載の細胞。
（１８） （１６）又は（１７）に記載の細胞を培養する工程を含む、（１）から（１０）の何れかに記載のキメラタンパク質を製造する方法。
（１９） （１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞を含む、薬学的組成物。
（２０） ＴＧＦβ阻害剤である、（１９）に記載の薬学的組成物。
（２１） 抗線維症剤である、（１９）に記載の薬学的組成物。
（２２） 抗腫瘍剤である、（１９）に記載の薬学的組成物。
That is, the present invention relates to the following (1) to (22).
(1) A chimeric protein having a TGFβ type I receptor extracellular domain and a TGFβ type II receptor extracellular domain and binding to TGFβ1, TGFβ2, and TGFβ3.
(2) The chimeric protein according to (1), wherein the TGFβ type I receptor extracellular domain is an ALK5 extracellular domain.
(3) The chimeric protein according to (1) or (2), wherein the TGFβ type II receptor extracellular domain is a TβRII extracellular domain.
(4) The chimeric protein according to any one of (1) to (3), further having a dimerization domain.
(5) The chimeric protein according to (4), wherein the dimerization domain is an immunoglobulin domain.
(6) The chimeric protein according to (5), wherein the immunoglobulin domain is an IgG Fc domain, an IgG heavy chain, or an IgG light chain.
(7) The amino terminus (N terminus) of the TβRII extracellular domain is bound to the C terminus of the ALK5 extracellular domain, and the N terminus of the immunoglobulin domain is bound to the carboxyl terminus (C terminus) of the TβRII extracellular domain. The chimeric protein according to any one of (1) to (6).
(8) The N-terminus of the ALK5 extracellular domain is bound to the C-terminus of the TβRII extracellular domain, and the N-terminus of the immunoglobulin domain is bound to the C-terminus of the ALK5 extracellular domain. The chimeric protein according to any one of the above.
(9) The chimeric protein according to any one of (1) to (8), which is any of the following (a) to (c).
(A) a chimeric protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 15;
(B) a chimeric protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4 or 15 and binding to TGFβ1, TGFβ2, and TGFβ3; or (c) SEQ ID NO: 4 or A chimeric protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 15 and binding to TGFβ1, TGFβ2 and TGFβ3;
(10) The chimeric protein according to any one of (1) to (9), which is subjected to acetyl modification or polyethylene glycol modification.
(11) The chimeric protein according to any one of (1) to (10), which is dimerized.
(12) A DNA encoding any of the following chimeric proteins (a) to (c):
(A) a chimeric protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 15;
(B) a chimeric protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4 or 15 and binding to TGFβ1, TGFβ2, and TGFβ3; or (c) SEQ ID NO: 4 or A chimeric protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 15, and which binds to TGFβ1, TGFβ2 and TGFβ3.
(13) DNA of any of the following (d) or (e).
(D) DNA consisting of the base sequence represented by SEQ ID NO: 8 or SEQ ID NO: 16; or (e) one or several bases deleted or substituted in the base sequence represented by SEQ ID NO: 8 or SEQ ID NO: DNA consisting of a base sequence that encodes a chimeric protein that consists of a base sequence that is added and / or binds to TGFβ1, TGFβ2, and TGFβ3
(14) A vector comprising the DNA according to (12) or (13).
(15) A virus having the vector according to (14).
(16) A cell having the vector according to (14) or the virus according to (15).
(17) The cell according to (16), which is a cell selected from the group consisting of bacterial cells, yeast cells, insect cells and mammalian cells.
(18) A method for producing the chimeric protein according to any one of (1) to (10), comprising a step of culturing the cell according to (16) or (17).
(19) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), the vector according to (14), the virus according to (15), or (16 ) Or a pharmaceutical composition comprising the cell according to (17).
(20) The pharmaceutical composition according to (19), which is a TGFβ inhibitor.
(21) The pharmaceutical composition according to (19), which is an antifibrotic agent.
(22) The pharmaceutical composition according to (19), which is an antitumor agent.
さらに本発明によれば、以下の発明が提供される。
（２３） ＴＧＦβ阻害に使用するための、（１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞。
（２４） 抗線維症の予防及び／又は治療に使用するための、（１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞。
（２５） 腫瘍の予防及び／又は治療に使用するための、（１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞。
（２６） ＴＧＦβ阻害剤の製造のための、（１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞の使用。
（２７） 抗線維症剤の製造のための、（１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞の使用。
（２８） 抗腫瘍剤の製造のための、（１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞の使用。
（２９） （１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞を、ＴＧＦβ阻害を必要とする対象者に投与することを含む、ＴＧＦβの阻害方法。
（３０） （１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞を、対象者に投与することを含む、抗線維症の予防及び／又は治療方法。
（３１） （１）から（１１）の何れかに記載のキメラタンパク質、（１２）又は（１３）に記載のＤＮＡ、（１４）に記載のベクター、（１５）に記載のウイルス、あるいは（１６）又は（１７）に記載の細胞を、対象者に投与することを含む、腫瘍を抑制する方法。
Furthermore, according to the present invention, the following inventions are provided.
(23) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), the vector according to (14), (15) for use in TGFβ inhibition Or the cell according to (16) or (17).
(24) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13) for use in the prevention and / or treatment of antifibrosis, (14) The vector according to the above, the virus according to (15), or the cell according to (16) or (17).
(25) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), or the DNA according to (14) for use in prevention and / or treatment of a tumor A vector, the virus according to (15), or the cell according to (16) or (17).
(26) For the production of a TGFβ inhibitor, the chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), the vector according to (14), ) Or the cell according to (16) or (17).
(27) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), the vector according to (14), for the production of an antifibrotic agent, Use of the virus according to 15) or the cell according to (16) or (17).
(28) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), the vector according to (14), (15) for the production of an antitumor agent ) Or the cell according to (16) or (17).
(29) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), the vector according to (14), the virus according to (15), or (16 ) Or the cell according to (17), comprising administering to a subject in need of TGFβ inhibition.
(30) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), the vector according to (14), the virus according to (15), or (16 ) Or (17), a method for preventing and / or treating antifibrosis, comprising administering the subject to a subject.
(31) The chimeric protein according to any one of (1) to (11), the DNA according to (12) or (13), the vector according to (14), the virus according to (15), or (16 ) Or a method for suppressing tumor, comprising administering the cell according to (17) to a subject.
本発明のキメラタンパク質は、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３の全てに結合することができる新規なキメラタンパク質である。本発明のキメラタンパク質は、ＴＧＦβシグナル関連疾患において、より治療域が広くなるため、臓器の線維化や腫瘍のＥＭＴを抑制し、線維症や腫瘍の治療に有用であると考えられる。 The chimeric protein of the present invention is a novel chimeric protein that can bind to all of TGFβ1, TGFβ2, and TGFβ3. The chimeric protein of the present invention is considered to be useful for the treatment of fibrosis and tumors by suppressing organ fibrosis and tumor EMT because the therapeutic range is broader in TGFβ signal-related diseases.
（１）キメラタンパク質
本発明のキメラタンパク質は、ＴＧＦβＩ型受容体細胞外ドメイン及びＴＧＦβＩＩ型受容体細胞外ドメインを有し、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合することを特徴とする。
(1) Chimeric protein The chimeric protein of the present invention has a TGFβ type I receptor extracellular domain and a TGFβ type II receptor extracellular domain, and is characterized by binding to TGFβ1, TGFβ2, and TGFβ3.
ＴＧＦβＩ型受容体細胞外ドメインとしては、ＡＬＫ４細胞外ドメイン、ＡＬＫ５細胞外ドメイン、ＡＬＫ７細胞外ドメインなどが挙げられ、特にＡＬＫ５細胞外ドメインが好ましい。 Examples of the TGFβ type I receptor extracellular domain include ALK4 extracellular domain, ALK5 extracellular domain, ALK7 extracellular domain and the like, and ALK5 extracellular domain is particularly preferable.
ＡＬＫ５細胞外ドメインのアミノ酸配列としては、配列番号１で表されるアミノ酸配列、及び配列番号１で表されるアミノ酸配列において１又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列であって、リガンドであるＴＧＦβを介することで、ＴβＲＩＩおよびその他のＩＩ型受容体との複合受容体形成能を有するアミノ酸配列を挙げることができる。なお、ＡＬＫ５細胞外ドメインは、ヒト以外の生物由来のＡＬＫ５細胞外ドメインであってもＴβＲＩＩおよびその他のＩＩ型受容体との複合受容体形成能を有するものであれば本発明において利用可能である。 As the amino acid sequence of the ALK5 extracellular domain, the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence that has the ability to form a complex receptor with TβRII and other type II receptors through the ligand TGFβ. The ALK5 extracellular domain can be used in the present invention as long as it has an ability to form a complex receptor with TβRII and other type II receptors even if it is an ALK5 extracellular domain derived from a non-human organism. .
ＴＧＦβＩＩ型受容体細胞外ドメインとしては、ＴβＲＩＩ細胞外ドメイン、ＡｃｔＲＩＩ細胞外ドメイン、ＡｃｔＲＩＩＢ細胞外ドメイン、ＢＭＰＲＩＩ細胞外ドメイン、ＡＭＨＲＩＩ細胞外ドメインなどが挙げられ、特にＴβＲＩＩ細胞外ドメインが好ましい。 Examples of the TGFβ type II receptor extracellular domain include TβRII extracellular domain, ActRII extracellular domain, ActRIIB extracellular domain, BMPRII extracellular domain, AMHRII extracellular domain, and the like, and TβRII extracellular domain is particularly preferable.
ＴβＲＩＩ細胞外ドメインのアミノ酸配列としては、配列番号２で表されるアミノ酸配列、及び配列番号２で表されるアミノ酸配列において１又は数個のアミノ酸の欠失、置換及び／又は付加しているアミノ酸配列、及び、配列番号１３で表されるアミノ酸配列、及び配列番号１３で表されるアミノ酸配列において１３又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列であって、リガンドであるＴＧＦβを介することで、ＡＬＫ５およびその他のＩ型受容体との複合受容体形成能を有するアミノ酸配列を挙げることができる。なお、ＴβＲＩＩ細胞外ドメインは、ヒト以外の生物由来のＴβＲＩＩ細胞外ドメインであってもＡＬＫ５およびその他のＩ型受容体との複合受容体形成能を有するものであれば本発明において利用可能である。 The amino acid sequence of the TβRII extracellular domain includes the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids are deleted, substituted, and / or added. A sequence, an amino acid sequence represented by SEQ ID NO: 13, and an amino acid sequence represented by SEQ ID NO: 13, wherein 13 or several amino acids are deleted, substituted and / or added, An amino acid sequence having the ability to form a complex receptor with ALK5 and other type I receptors can be exemplified through TGFβ. The TβRII extracellular domain can be used in the present invention as long as it has the ability to form a complex receptor with ALK5 and other type I receptors even if it is a TβRII extracellular domain derived from a non-human organism. .
本発明においてはさらに二量体化ドメインを有するキメラタンパク質がより好ましい。二量体化ドメインにより本発明のキメラタンパク質は二量体化し、血中滞留性の向上が期待できる。 In the present invention, a chimeric protein further having a dimerization domain is more preferred. The chimeric protein of the present invention is dimerized by the dimerization domain, and improvement in blood retention can be expected.
本発明において二量体化ドメインは本発明のキメラタンパク質を二量体化させるものであれば特に限定されない。例えば、免疫グロブリンドメインを使用することができ、具体的には、ＩｇＧのＦｃドメイン（例えば、ヒトＩｇＧ１のＦｃドメイン、ヒトＩｇＧ２のＦｃドメイン、ヒトＩｇＧ３のＦｃドメイン、ヒトＩｇＧ４のＦｃドメインなど）が挙げられる。また、ＩｇＧの重鎖、又はＩｇＧの軽鎖でもよい。また、ヒト以外の生物由来の免疫グロブリンドメインでもよい。さらに、これらのアミノ酸配列において１又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列からなるものでもよい。本発明においてはキメラタンパク質の血中滞留性を向上させる免疫グロブリンドメインを使用することが好ましい。 In the present invention, the dimerization domain is not particularly limited as long as it dimerizes the chimeric protein of the present invention. For example, immunoglobulin domains can be used, specifically IgG Fc domains (eg, human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, etc.). Can be mentioned. Further, it may be an IgG heavy chain or an IgG light chain. Further, it may be an immunoglobulin domain derived from a non-human organism. Further, these amino acid sequences may be composed of amino acid sequences in which one or several amino acids are deleted, substituted and / or added. In the present invention, it is preferable to use an immunoglobulin domain that improves the retention of the chimeric protein in blood.
本発明において使用できる二量体化ドメインの一例としては、ヒトＩｇＧ１のＦｃドメインであって、配列番号３で表されるアミノ酸配列で示されるものである。 An example of a dimerization domain that can be used in the present invention is the Fc domain of human IgG1, which is represented by the amino acid sequence represented by SEQ ID NO: 3.
本発明のキメラタンパク質において、ＴＧＦβＩ型受容体細胞外ドメイン（例えば、ＡＬＫ５細胞外ドメイン）とＴＧＦβＩＩ型受容体細胞外ドメイン（例えば、ＴβＲＩＩ細胞外ドメイン）の配列順序は特に限定されない。即ち、ＴＧＦβＩ型受容体細胞外ドメインのＣ末端にＴＧＦβＩＩ型受容体細胞外ドメインのＮ末端が結合していてもよいし、ＴＧＦβＩＩ型受容体細胞外ドメインのＣ末端にＴＧＦβＩ型受容体細胞外ドメインのＮ末端が結合していてもよい。また、各ドメインがアミノ酸やペプチド、親水性ポリマーなど、何らかのリンカーを介して結合していてもよい。 In the chimeric protein of the present invention, the sequence order of the TGFβ type I receptor extracellular domain (eg, ALK5 extracellular domain) and the TGFβ type II receptor extracellular domain (eg, TβRII extracellular domain) is not particularly limited. That is, the N-terminus of the TGFβ type II receptor extracellular domain may be bound to the C terminus of the TGFβ type I receptor extracellular domain, or the TGFβ type I receptor extracellular domain may be bound to the C terminus of the TGFβ type II receptor extracellular domain. The N terminal of each may be bonded. In addition, each domain may be bound via some kind of linker such as an amino acid, a peptide, or a hydrophilic polymer.
ＴＧＦβＩ型受容体細胞外ドメインとしてＡＬＫ５細胞外ドメインを含み、ＴＧＦβＩＩ型受容体細胞外ドメインとしてＴβＲＩＩ細胞外ドメインを含み、さらに免疫グロブリンドメインを含むキメラタンパク質の場合の例としては、ＡＬＫ５細胞外ドメインのＣ末端にＴβＲＩＩ細胞外ドメインのＮ末端が結合し、ＴβＲＩＩ細胞外ドメインのＣ末端に免疫グロブリンドメインのＮ末端が結合しているキメラタンパク質、及びＴβＲＩＩ細胞外ドメインのＣ末端にＡＬＫ５細胞外ドメインのＮ末端が結合し、ＡＬＫ５細胞外ドメインのＣ末端に免疫グロブリンドメインのＮ末端が結合しているキメラタンパク質を挙げることができる。また、各ドメインがアミノ酸やペプチド、親水性ポリマーなど、何らかのリンカーを介して結合していてもよい。 As an example of a chimeric protein comprising an ALK5 extracellular domain as a TGFβ type I receptor extracellular domain, a TβRII extracellular domain as a TGFβ type II receptor extracellular domain, and further comprising an immunoglobulin domain, the ALK5 extracellular domain A chimeric protein in which the N-terminus of the TβRII extracellular domain is bound to the C-terminus, the N-terminus of the immunoglobulin domain is bound to the C-terminus of the TβRII extracellular domain, and the ALK5 extracellular domain is bound to the C-terminus of the TβRII extracellular domain A chimeric protein in which the N-terminus is bound and the N-terminus of the immunoglobulin domain is bound to the C-terminus of the ALK5 extracellular domain can be mentioned. In addition, each domain may be bound via some kind of linker such as an amino acid, a peptide, or a hydrophilic polymer.
リンカーがアミノ酸やペプチドの場合は、キメラタンパク質のベクターを構築する際に、各ドメインのＤＮＡ配列とリンカーのＤＮＡ配列を用いてベクターを構築すればよい。リンカーのペプチドとしては、例えば、ＣＧＧ−、ＣＡＡ−、ＣＬＬ−、ＣＶＶ−、ＣＬＡ−、ＣＧ−、ＣＧＧＧ−、ＣＧＧＧＧ−、ＣＧＧＧＧＧ−、（ＧＧＧＧＳ）3、（ＧＧＧＧＳ）6、ＶＣｉｔ、ＡＦ(（Ｇはグリシン、Ａはアラニン、Ｌはロイシン、Ｖはバリン、Ｓはセリン、Ｃｉｔはシトルリン、Ｆはフェニルアラニンをそれぞれ示す。）などを挙げることができる。また、リンカーの親水性ポリマーとしては、例えば、ヒドロキシ基、カルボキシル基、カルボキシレート基、ヒドロキシエチル基、ポリオキシエチル基、ヒドロキシプロピル基、ポリオキシプロピル基、アミノ基、アミノエチル基、アミノプロピル基、アンモニウム基、アミド基、カルボキシメチル基、スルホン酸基、リン酸基などの親水性基を有するものを挙げることができ、具体的には、アラビアゴム、カゼイン、ゼラチン、デンプン誘導体、カルボキシメチルセルロースおよびそのナトリウム塩、セルロースアセテート、アルギン酸ナトリウム、酢酸ビニル−マレイン酸コポリマー類、スチレン−マレイン酸コポリマー類、ポリアクリル酸類およびそれらの塩、ポリメタクリル酸類およびそれらの塩、ヒドロキシエチルメタクリレートのホモポリマーおよびコポリマー、ヒドロキシエチルアクリレートのホモポリマーおよびコポリマー、ヒドロキシピロピルメタクリレートのホモポリマーおよびコポリマー、ヒドロキシプロピルアクリレートのホモポリマーおよびコポリマー、ヒドロキシブチルメタクリレートのホモポリマーおよびコポリマー、ヒドロキシブチルアクリレートのホモポリマーおよびコポリマー、ポリエチレングリコール（ＰＥＧ）類、ヒドロキシプロピレンポリマー類、ポリビニルアルコール類、加水分解度が６０モル％以上、好ましくは８０モル％以上である加水分解ポリビニルアセテート、ポリビニルホルマール、ポリビニルブチラール、ポリビニルピロリドン、アクリルアミドのホモポリマーおよびコポリマー、メタクリルアミドのホモポリマーおよびポリマー、Ｎ−メチロールアクリルアミドのホモポリマーおよびコポリマー、アルコール可溶性ナイロン、２，２−ビス−（４−ヒドロキシフェニル）−プロパンとエピクロロヒドリンとのポリエーテルなどを挙げることができるが、ＰＥＧが好ましい。ＰＥＧの分子量は特に限定されず、例えば、ＰＥＧ２００、ＰＥＧ４００、ＰＥＧ５００、ＰＥＧ２０００、ＰＥＧ５０００、ＰＥＧ１００００、ＰＥＧ２００００、ＰＥＧ３００００、ＰＥＧ３５０００などを用いることができる。 When the linker is an amino acid or a peptide, the vector may be constructed using the DNA sequence of each domain and the DNA sequence of the linker when constructing a chimeric protein vector. Examples of the linker peptide include CGG-, CAA-, CLL-, CVV-, CLA-, CG-, CGGG-, CGGGGG-, CGGGGGG-, (GGGGGS) 3 , (GGGGS) 6 , VCit, AF (( G represents glycine, A represents alanine, L represents leucine, V represents valine, S represents serine, Cit represents citrulline, and F represents phenylalanine.) Examples of the hydrophilic polymer of the linker include: Hydroxy group, carboxyl group, carboxylate group, hydroxyethyl group, polyoxyethyl group, hydroxypropyl group, polyoxypropyl group, amino group, aminoethyl group, aminopropyl group, ammonium group, amide group, carboxymethyl group, List those having hydrophilic groups such as sulfonic acid groups and phosphoric acid groups Specifically, gum arabic, casein, gelatin, starch derivatives, carboxymethylcellulose and its sodium salt, cellulose acetate, sodium alginate, vinyl acetate-maleic acid copolymers, styrene-maleic acid copolymers, polyacrylic acids And salts thereof, polymethacrylic acids and salts thereof, homopolymers and copolymers of hydroxyethyl methacrylate, homopolymers and copolymers of hydroxyethyl acrylate, homopolymers and copolymers of hydroxypyrrole methacrylate, homopolymers and copolymers of hydroxypropyl acrylate, Homopolymers and copolymers of hydroxybutyl methacrylate, homopolymers of hydroxybutyl acrylate and Polymer, polyethylene glycol (PEG), hydroxypropylene polymers, polyvinyl alcohol, hydrolyzed polyvinyl acetate, polyvinyl formal, polyvinyl butyral, polyvinyl pyrrolidone, acrylamide having a hydrolysis degree of 60 mol% or more, preferably 80 mol% or more Homopolymers and copolymers of methacrylamide, homopolymers and polymers of methacrylamide, homopolymers and copolymers of N-methylolacrylamide, alcohol soluble nylon, poly of 2,2-bis- (4-hydroxyphenyl) -propane and epichlorohydrin Although ether can be exemplified, PEG is preferable, and the molecular weight of PEG is not particularly limited, and examples thereof include PEG200, PEG400, PEG500, PEG2000, EG5000, PEG10000, PEG20000, PEG30000, or the like can be used PEG35000.
本発明のキメラタンパク質の具体例としては、以下の（ａ）〜（ｃ）の何れかのキメラタンパク質を挙げることができる。
（ａ）配列番号４又は配列番号１５で表されるアミノ酸配列からなるキメラタンパク質；
（ｂ）配列番号４又は配列番号１５で表されるアミノ酸配列と９０％以上（好ましくは９５％以上、より好ましくは９７％以上、特に好ましくは９９％以上）の配列同一性を有するアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質；又は
（ｃ）配列番号４又は配列番号１５で表されるアミノ酸配列において１又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質；
Specific examples of the chimeric protein of the present invention include any of the following chimeric proteins (a) to (c).
(A) a chimeric protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 15;
(B) From an amino acid sequence having a sequence identity of 90% or more (preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more) with the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 15. A chimeric protein that binds to TGFβ1, TGFβ2, and TGFβ3; or (c) an amino acid in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 15 A chimeric protein consisting of a sequence and binding to TGFβ1, TGFβ2 and TGFβ3;
アミノ酸配列の同一性は、例えばＢＬＡＳＴ（Ｐｒｏ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９０，５８７３（１９９３））又はＦＡＳＴＡ（ＭｅｔｈｏｄｓＥｎｚｙｍｏｌ．，１８３，６３（１９９０））等を用いて決定することができる。このＢＬＡＳＴに基づいて、ＢＬＡＳＴＰ、ＢＬＡＳＴＮとよばれるプログラムが開発されているので（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ）、このプログラムをデフォルト設定で用いて配列同一性を計算することができる。 The identity of amino acid sequences can be determined using, for example, BLAST (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (Methods Enzymol., 183, 63 (1990)). Based on this BLAST, programs called BLASTP and BLASTN have been developed (http://www.ncbi.nlm.nih.gov), and this program is used with default settings to calculate sequence identity. Can do.
アミノ酸残基の置換は、あるアミノ酸残基が類似の側鎖を有するアミノ酸残基で置換される保存的置換でもよい。類似の側鎖を有するアミノ酸のファミリーとしては、塩基性側鎖を有するアミノ酸（リジン、アルギニン、ヒスチジン）、酸性側鎖を有するアミノ酸（アスパラギン酸、グルタミン酸）、非荷電性極性側鎖を有するアミノ酸（グリシン、アスパラギン、グルタミン、セリン、スレオニン、チロシン、システイン）、非極性側鎖を有するアミノ酸（アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン）、β位分岐側鎖を有するアミノ酸（スレオニン、バリン、イソロイシン）、芳香族側鎖を有するアミノ酸（チロシン、フェニルアラニン、トリプトファン、ヒスチジン）、ヒドロキシル基含有側鎖を有するアミノ酸（セリン、スレオニン、チロシン）、および硫黄含有側鎖を有するアミノ酸（システイン、メチオニン）を挙げることができる。 The amino acid residue substitution may be a conservative substitution in which an amino acid residue is substituted with an amino acid residue having a similar side chain. The family of amino acids with similar side chains includes amino acids with basic side chains (lysine, arginine, histidine), amino acids with acidic side chains (aspartic acid, glutamic acid), amino acids with uncharged polar side chains ( Glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (threonine) , Valine, isoleucine), amino acids with aromatic side chains (tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl group-containing side chains (serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (Cysteine, methionine) can be mentioned.
本明細書において「アミノ酸配列において１又は数個のアミノ酸の欠失、置換及び／又は付加している」における数個とは、好ましくは１〜１０個、より好ましくは１〜５個、さらに好ましくは１〜３個を意味する。 In the present specification, the term “several amino acids in which one or several amino acids are deleted, substituted and / or added” in the amino acid sequence is preferably 1 to 10, more preferably 1 to 5, even more preferably. Means 1-3.
本発明のキメラタンパク質は、アセチル修飾またはポリエチレングリコール修飾を受けているものでもよい。真核生物のタンパク質のＮ末端にあるαアミノ酸はアセチル化される場合があり、生細胞内で、タンパク質の翻訳後修飾としてアセチル化が行われる場合がある。また、ポリエチレングリコールをタンパク質に結合すると、タンパク質分解が抑制されることにより薬効の持続期間を延長したり、副作用を軽減することが可能になる。タンパク質のアセチル修飾またはポリエチレングリコール修飾は当業者に公知の手法により行うことができる。 The chimeric protein of the present invention may be subjected to acetyl modification or polyethylene glycol modification. An α-amino acid at the N-terminus of a eukaryotic protein may be acetylated and may be acetylated in a living cell as a post-translational modification of the protein. Further, when polyethylene glycol is bound to a protein, it is possible to prolong the duration of medicinal effect and reduce side effects by suppressing proteolysis. Protein acetyl modification or polyethylene glycol modification can be performed by techniques known to those skilled in the art.
本発明のキメラタンパク質は、二量体化したキメラタンパク質でもよい。本発明の二量体化キメラタンパク質としては、二量体化ドメインにより二量体化したもの、ペプチドリンカーによって二量体化された本発明のキメラタンパク質、化学結合により二量体化された本発明のキメラタンパク質を含む。 The chimeric protein of the present invention may be a dimerized chimeric protein. The dimerized chimeric protein of the present invention includes those dimerized by a dimerization domain, the chimeric protein of the present invention dimerized by a peptide linker, and a dimerized by chemical bonding. Including the chimeric protein of the invention.
（２）ＤＮＡ
本発明は、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３と結合することができるキメラタンパク質をコードするＤＮＡに関する。本発明のＤＮＡは、ｃＤＮＡであることが好ましい。具体的には、ＴＧＦβＩ型受容体細胞外ドメインをコードするＤＮＡ及びＴＧＦβＩＩ型受容体細胞外ドメインをコードするＤＮＡを有するＤＮＡが本発明に含まれる。さらにＡＬＫ５細胞外ドメインをコードするＤＮＡとＴβＲＩＩ細胞外ドメインをコードするＤＮＡ配列を有するＤＮＡ配列が好ましい。上記ＤＮＡにおけるＡＬＫ５細胞外ドメインをコードするＤＮＡとＴβＲＩＩ細胞外ドメインをコードするＤＮＡの配列順序は特に限定されない。本発明のＤＮＡは、免疫グロブリンドメインをコードするＤＮＡをさらに有することが好ましい。
(2) DNA
The present invention relates to a DNA encoding a chimeric protein capable of binding to TGFβ1, TGFβ2 and TGFβ3. The DNA of the present invention is preferably cDNA. Specifically, the present invention includes DNA having a DNA encoding a TGFβ type I receptor extracellular domain and a DNA having a TGFβ type II receptor extracellular domain. Furthermore, a DNA sequence having a DNA sequence encoding the ALK5 extracellular domain and a DNA sequence encoding the TβRII extracellular domain is preferred. The sequence order of the DNA encoding the ALK5 extracellular domain and the DNA encoding the TβRII extracellular domain in the DNA is not particularly limited. The DNA of the present invention preferably further has DNA encoding an immunoglobulin domain.
ＡＬＫ５細胞外ドメインをコードするＤＮＡ配列としては、配列番号５で表されるＤＮＡ配列、及び配列番号５で表されるＤＮＡ配列において１又は数個の塩基が欠失、置換及び／又は付加しているＤＮＡ配列であって、ＴβＲＩＩおよびその他のＩＩ型受容体との複合受容体形成能を有するタンパク質をコードするＤＮＡ配列を挙げることができる。なお、ＡＬＫ５細胞外ドメインのＤＮＡ配列はヒト以外の由来のものであってもＴβＲＩＩおよびその他のＩＩ型受容体との複合受容体形成能を有するポリペプチドを発現させるものであれば本発明において利用可能である。 As the DNA sequence encoding the ALK5 extracellular domain, one or several bases are deleted, substituted and / or added in the DNA sequence represented by SEQ ID NO: 5 and the DNA sequence represented by SEQ ID NO: 5. And a DNA sequence encoding a protein having the ability to form a complex receptor with TβRII and other type II receptors. The DNA sequence of the ALK5 extracellular domain can be used in the present invention as long as it expresses a polypeptide having the ability to form a complex receptor with TβRII and other type II receptors, even if it is derived from a source other than human. Is possible.
ＴβＲＩＩ細胞外ドメインをコードするＤＮＡ配列としては、配列番号６又は配列番号１４で表されるＤＮＡ配列、及び配列番号６又は配列番号１４で表されるＤＮＡ配列において１又は数個の塩基が欠失、置換及び／又は付加しているＤＮＡ配列であって、ＡＬＫ５およびその他のＩ型受容体との複合受容体形成能を有するタンパク質をコードするＤＮＡ配列を挙げることができる。なお、ＴβＲＩＩ細胞外ドメインのＤＮＡ配列はヒト以外の由来のものであってもＡＬＫ５およびその他のＩ型受容体との複合受容体形成能を有するポリペプチドを発現させるものであれば本発明において利用可能である。 As the DNA sequence encoding the TβRII extracellular domain, one or several bases are deleted in the DNA sequence represented by SEQ ID NO: 6 or SEQ ID NO: 14 and the DNA sequence represented by SEQ ID NO: 6 or SEQ ID NO: 14 And a DNA sequence that is substituted and / or added and encodes a protein having the ability to form a complex receptor with ALK5 and other type I receptors. The DNA sequence of the TβRII extracellular domain can be used in the present invention as long as it expresses a polypeptide having the ability to form a complex receptor with ALK5 and other type I receptors, even if it is derived from a source other than human. Is possible.
本明細書において「ＤＮＡ配列において１又は数個の塩基が欠失、置換及び／又は付加している」又は「塩基配列において１又は数個の塩基が欠失、置換及び／又は付加している」における数個とは、好ましくは１〜１０個、より好ましくは１〜５個、さらに好ましくは１〜３個を意味する。 In the present specification, “one or several bases are deleted, substituted and / or added in the DNA sequence” or “one or several bases are deleted, substituted and / or added in the base sequence” “Several” in the formula means preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3.
本発明において二量体化ドメインをコードするＤＮＡは、例えば配列番号７で表されるＤＮＡ配列からなるヒトＩｇＧ１のＦｃドメインである。 In the present invention, the DNA encoding the dimerization domain is, for example, the Fc domain of human IgG1 consisting of the DNA sequence represented by SEQ ID NO: 7.
本発明のＤＮＡの具体例としては、以下の（ａ）〜（ｃ）の何れかのキメラタンパク質をコードするＤＮＡを挙げることができる。
（ａ）配列番号４又は配列番号１５で表されるアミノ酸配列からなるキメラタンパク質；
（ｂ）配列番号４又は配列番号１５で表されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質；又は
（ｃ）配列番号４又は配列番号１５で表されるアミノ酸配列において１又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質。
Specific examples of the DNA of the present invention include DNA encoding any of the following chimeric proteins (a) to (c).
(A) a chimeric protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 15;
(B) a chimeric protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4 or 15 and binding to TGFβ1, TGFβ2, and TGFβ3; or (c) SEQ ID NO: 4 or A chimeric protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 15, and which binds to TGFβ1, TGFβ2 and TGFβ3.
本発明のＤＮＡの更なる具体例としては、以下の（ｄ）又は（ｅ）の何れかのＤＮＡを挙げることができる。
（ｄ）配列番号８又は配列番号１６で表される塩基配列からなるＤＮＡ；又は
（ｅ）配列番号８又は配列番号１６で表される塩基配列において１又は数個の塩基が欠失、置換及び／又は付加している塩基配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質をコードする塩基配列からなるＤＮＡ。
As a further specific example of the DNA of the present invention, any of the following DNAs (d) and (e) can be mentioned.
(D) DNA consisting of the base sequence represented by SEQ ID NO: 8 or SEQ ID NO: 16; or (e) one or several bases deleted or substituted in the base sequence represented by SEQ ID NO: 8 or SEQ ID NO: 16; DNA consisting of a base sequence that encodes a chimeric protein that consists of a base sequence that is added and / or that binds to TGFβ1, TGFβ2, and TGFβ3.
（３）ベクター、ウイルス、細胞、及びキメラタンパク質の製造方法
本発明はさらに、本発明のキメラタンパク質をコードするＤＮＡ（好ましくはｃＤＮＡ）を含むベクターに関する。本発明のベクターとしては、任意のプラスミドに、ＴＧＦβＩ型受容体細胞外ドメインをコードするＤＮＡ（好ましくはｃＤＮＡ）、及びＴＧＦβＩＩ型受容体細胞外ドメインをコードするＤＮＡ（好ましくはｃＤＮＡ）を組み込むことにより得られるベクターが挙げられる。さらに本発明のベクターとしては、任意のプラスミドに、ＴＧＦβＩ型受容体細胞外ドメインをコードするＤＮＡ（好ましくはｃＤＮＡ）、ＴＧＦβＩＩ型受容体細胞外ドメインをコードするＤＮＡ（好ましくはｃＤＮＡ）、及び二量体化ドメインをそれぞれコードするＤＮＡ（好ましくはｃＤＮＡ）を組み込んだものを挙げることができる。プラスミドは例えばｐＢｌｕｅＳｃｒｉｐｔ、ｐｃＲＩＩ−Ｔｏｐｏ、ｐＥＮＴＲ２０１などが挙げられる。
(3) Method for Producing Vector, Virus, Cell, and Chimeric Protein The present invention further relates to a vector comprising DNA (preferably cDNA) encoding the chimeric protein of the present invention. As the vector of the present invention, a DNA encoding TGFβ type I receptor extracellular domain (preferably cDNA) and a DNA encoding TGFβ type II receptor extracellular domain (preferably cDNA) are incorporated into any plasmid. Examples of the resulting vector include. Furthermore, as the vector of the present invention, DNA encoding TGFβ type I receptor extracellular domain (preferably cDNA), DNA encoding TGFβ type II receptor extracellular domain (preferably cDNA), and dimer Examples thereof include those incorporating DNAs (preferably cDNAs) each encoding an integration domain. Examples of the plasmid include pBlueScript, pcRII-Topo, and pENTR201.
本発明のベクターは、クローニングした、または、購入したＴＧＦβＩ型受容体細胞外ドメイン、ＴＧＦβＩＩ型受容体細胞外ドメイン及び二量体化ドメインそれぞれのＤＮＡ（好ましくはｃＤＮＡ）を用いて作製することができる。任意のプラスミドに各ドメインのｃＤＮＡを組み込む順序に制限はない。本発明のベクターの作成手順に制限はないが、例えば、二量体化ドメインを含む本発明のキメラタンパク質のベクターを作成する場合、クローニングしたＴＧＦβＩ型受容体細胞外ドメイン及びＴＧＦβＩＩ型受容体細胞外ドメインの各ＤＮＡをマルチクローニングサイトでタンデム発現できるようにサブクローニングし、該サブクローニングされたＴＧＦβＩ型受容体細胞外ドメイン及びＴＧＦβＩＩ型受容体細胞外ドメインの各ＤＮＡを二量体化ドメインのＤＮＡを有するベクターに組み込むことで得られる。二量体化ドメインのＤＮＡを有するベクターは市販のベクター、例えばＩｎｖｉｖｏｇｅｎ社から販売されているｐＦＵＳＥ−Ｆｃシリーズなどを利用してもよい。 The vector of the present invention can be prepared using cloned or purchased TGFβ type I receptor extracellular domain, TGFβ type II receptor extracellular domain, and dimerization domain DNA (preferably cDNA). . There is no restriction on the order of incorporating the cDNA of each domain into any plasmid. There is no limitation on the procedure for preparing the vector of the present invention. For example, when preparing a vector of the chimeric protein of the present invention containing a dimerization domain, the cloned TGFβ type I receptor extracellular domain and TGFβ type II receptor extracellular Subcloning each domain DNA so that it can be expressed in tandem at the multiple cloning site, and subcloned TGFβ type I receptor extracellular domain and TGFβ type II receptor extracellular domain vector having a dimerization domain DNA It is obtained by incorporating it into A vector having a dimerization domain DNA may be a commercially available vector such as the pFUSE-Fc series sold by Invivogen.
本発明のベクターは、本発明のキメラタンパク質をコードするＤＮＡを有する発現ベクターでもよい。本発明の発現ベクターは、上記した本発明のベクターから、本発明のキメラタンパク質をコードするＤＮＡを切り出し、任意のプロモーター領域を有するベクターに組み込むことで得ることができる。プロモーター領域を有するベクターとしては、例えばｐｃＤＮＡ、ｐＧＬ、ｐＣＳＩＩ−ＥＦ−ＲｆＡが挙げられる。 The vector of the present invention may be an expression vector having a DNA encoding the chimeric protein of the present invention. The expression vector of the present invention can be obtained by cutting out the DNA encoding the chimeric protein of the present invention from the above-described vector of the present invention and incorporating it into a vector having any promoter region. Examples of the vector having a promoter region include pcDNA, pGL, and pCSII-EF-RfA.
本発明は、本発明のベクターを有するウイルスに関する。本発明のウイルスとしては、本発明のベクター（好ましくは発現ベクター）により形質転換されたウイルスを挙げることができる。具体的には、本発明の発現ベクターを、後記する本発明のキメラタンパク質産生細胞の宿主細胞の形質転換に適したウイルスに組み込むことによって、本発明のウイルスを得ることができる。用いられるウイルスは特に限定されないが、例えばアデノウイルス、レトロウイルス、レンチウイルス属のウイルスが挙げられる。 The present invention relates to a virus having the vector of the present invention. Examples of the virus of the present invention include a virus transformed with the vector of the present invention (preferably an expression vector). Specifically, the virus of the present invention can be obtained by incorporating the expression vector of the present invention into a virus suitable for transformation of the host cell of the chimeric protein-producing cell of the present invention described later. The virus to be used is not particularly limited, and examples thereof include adenovirus, retrovirus, and lentivirus viruses.
本発明はさらに、本発明のベクター又は本発明のウイルスを有する細胞に関する。本発明の細胞は、宿主細胞を、本発明のベクター又は本発明のウイルスにより形質転換することにより得ることができる。好ましくは、本発明の細胞は、本発明のキメラタンパク質を産生する細胞である。本発明の細胞の宿主細胞は、本発明のキメラタンパク質の発現に適した細胞であれば制限はなく、例えば、大腸菌、酵母、昆虫細胞、又は哺乳動物細胞が挙げられる。哺乳動物細胞としてはＭＤＡ−ＭＢ−２３１Ｄ、ＣＯＳ若しくはＣＨＯなどが挙げられる。本発明の細胞は、各宿主細胞に適した公知の培養方法により継代される。 The present invention further relates to a cell having the vector of the present invention or the virus of the present invention. The cell of the present invention can be obtained by transforming a host cell with the vector of the present invention or the virus of the present invention. Preferably, the cell of the present invention is a cell that produces the chimeric protein of the present invention. The host cell of the cell of the present invention is not limited as long as it is a cell suitable for expression of the chimeric protein of the present invention, and examples thereof include Escherichia coli, yeast, insect cells, and mammalian cells. Mammalian cells include MDA-MB-231D, COS, and CHO. The cells of the present invention are passaged by a known culture method suitable for each host cell.
本発明の細胞は、電子穿孔、リポフェクション、又はリン酸カルシウム媒介トランスフェクション等により細胞へ本発明のベクターを導入することで得ることができる。また、本発明の細胞は、本発明のウイルスを感染させることにより得ることができる。 The cell of the present invention can be obtained by introducing the vector of the present invention into the cell by electroporation, lipofection, calcium phosphate-mediated transfection, or the like. The cell of the present invention can be obtained by infecting the virus of the present invention.
本発明は、上記した本発明の細胞を培養することによって、本発明のキメラタンパク質を製造する方法にも関する。本発明のキメラタンパク質の製造方法は、上記した本発明のキメラタンパク質産生細胞を用いて行われる。 The present invention also relates to a method for producing the chimeric protein of the present invention by culturing the above-described cells of the present invention. The method for producing a chimeric protein of the present invention is performed using the above-described chimeric protein-producing cell of the present invention.
任意の本発明のキメラタンパク質産生細胞により産生された本発明のキメラタンパク質は、活性を保持することのできる任意の方法によって精製される。例えば、これらに限定されないが、可溶性タンパク質又は封入体としてのいずれかで細胞からこれらの融合タンパク質を回収してもよく、細胞から８Ｍのグアニジン塩酸塩及び透析によって定量的に抽出してもよい。これらのキメラタンパク質をさらに精製するために、これらに限定されないが、従来のイオン交換クロマトグラフィ、アフィニティクロマトグラフィ、様々な糖クロマトグラフィ、疎水性相互作用クロマトグラフィ、逆相クロマトグラフィ又はゲル濾過を含む任意数の精製法を使用してもよい。 The chimeric protein of the present invention produced by any chimeric protein producing cell of the present invention is purified by any method capable of retaining activity. For example, without limitation, these fusion proteins may be recovered from the cells, either as soluble proteins or inclusion bodies, and may be quantitatively extracted from the cells by 8M guanidine hydrochloride and dialysis. Any number of purification methods to further purify these chimeric proteins, including but not limited to conventional ion exchange chromatography, affinity chromatography, various sugar chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration May be used.
（４）薬学的組成物
本発明はさらに、上記した本発明のキメラタンパク質、ＤＮＡ、ベクター、ウイルス又は細胞を含む、薬学的組成物に関する。本発明の薬学的組成物は、ＴＧＦβ阻害剤、抗線維症剤、又は抗腫瘍剤として使用することができる。
(4) Pharmaceutical Composition The present invention further relates to a pharmaceutical composition comprising the chimeric protein, DNA, vector, virus or cell of the present invention described above. The pharmaceutical composition of the present invention can be used as a TGFβ inhibitor, antifibrotic agent, or antitumor agent.
本発明の薬学的組成物は、少なくとも本発明のキメラタンパク質、ＤＮＡ、ベクター、ウイルス又は細胞を含み、必要に応じて担体、その他成分を含む。本発明のキメラタンパク質は標的化分子（例えば抗体、ホルモン、成長因子等）と連結させたり、又はリポソーム、マイクロカプセルに組み込んでもよい。担体としては、滅菌水、生理食塩水、リン酸バッファー又はデキストロース溶液等が挙げられるがこれらに制限されない。担体は固体（ 例えばワックス） 又は半固体（例えばゲル状）でもよい。その他成分は薬学的に許容された任意の成分であって、例えば、賦形剤、結合剤、崩壊剤、潤沢剤、被覆剤、矯味剤、可溶化剤等である。 The pharmaceutical composition of the present invention contains at least the chimeric protein, DNA, vector, virus or cell of the present invention, and optionally contains a carrier and other components. The chimeric protein of the present invention may be linked to a targeting molecule (eg, antibody, hormone, growth factor, etc.), or incorporated into a liposome or microcapsule. Examples of the carrier include, but are not limited to, sterilized water, physiological saline, phosphate buffer, or dextrose solution. The carrier may be solid (eg, wax) or semi-solid (eg, gel). The other components are any pharmaceutically acceptable components such as excipients, binders, disintegrants, lubricants, coating agents, flavoring agents, solubilizers, and the like.
本発明の薬学的組成物の投与経路には限定がなく、全身又は局所から適宜選択される。本発明の薬学的組成物の投与経路は静脈内、髄腔内、動脈内、鼻内、経口、皮下、腹腔内、又は局所の注射若しくは外科的移植を含む。本発明の薬学的組成物の剤型は特に制限がなく、錠剤、顆粒剤、散剤、液剤、注射剤、座剤等、任意の剤型から選ばれる。 There is no limitation on the route of administration of the pharmaceutical composition of the present invention, and it is appropriately selected from systemic or local. The route of administration of the pharmaceutical composition of the present invention includes intravenous, intrathecal, intraarterial, intranasal, oral, subcutaneous, intraperitoneal, or local injection or surgical implantation. The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and is selected from arbitrary dosage forms such as tablets, granules, powders, solutions, injections, suppositories and the like.
本発明の薬学的組成物の投与量は、性別や体重、年齢、人種、症状等に応じて当業者により適宜決定される。有効成分であるキメラタンパク質、ＤＮＡ、ベクター、ウイルス又は細胞の投与量は、例えば、０.００１μｇ／ｋｇ〜１０００ｍｇ／ｋｇから適宜調整される。 The dosage of the pharmaceutical composition of the present invention is appropriately determined by those skilled in the art according to sex, body weight, age, race, symptoms and the like. The dose of the chimera protein, DNA, vector, virus or cell as the active ingredient is appropriately adjusted, for example, from 0.001 μg / kg to 1000 mg / kg.
本発明は、遺伝子療法を含む。遺伝子療法としては、例えば、本発明のＤＮＡ、発現ベクター、ウイルス又はキメラタンパク質産生細胞を生体に移植することによって生体内で本発明のキメラタンパク質を産生させるようにすることを含む。 The present invention includes gene therapy. Gene therapy includes, for example, producing the chimeric protein of the present invention in vivo by transplanting the DNA, expression vector, virus or chimeric protein-producing cell of the present invention into the living body.
遺伝子療法において本発明のＤＮＡ配列を用いる場合、例えば、ｎａｋｅｄ ＤＮＡの直接注射、脂質若しくは細胞表面受容体と親和性のあるタンパク質などでコーティングされた、リポソーム、微粒子、若しくはマイクロカプセル、又は核移行性ペプチド等に連結して投与することが挙げられる。 When the DNA sequence of the present invention is used in gene therapy, for example, direct injection of naked DNA, liposomes, microparticles or microcapsules coated with a protein having affinity for lipid or cell surface receptor, or nuclear transferability Administration by linking to a peptide or the like can be mentioned.
遺伝子療法において本発明のＤＮＡを含む発現ベクターを用いる場合、ベクターとしては欠損、弱毒化レトロウイルスベクター、レトロウイルスベクター、アデノウイルスベクター及びアデノ関連ウイルスなどが挙げられるがこれに限られない。 When an expression vector containing the DNA of the present invention is used in gene therapy, examples of the vector include, but are not limited to, deletion, attenuated retrovirus vector, retrovirus vector, adenovirus vector, and adeno-associated virus.
遺伝子療法のために本発明のＤＮＡを導入することができる細胞は、上皮細胞、内皮細胞、角化細胞、線維芽細胞、筋肉細胞、肝細胞； Ｔ―リンパ球、Ｂ−リンパ球、単球、マクロファージ、好中球、好酸球、巨核球、顆粒球等の血液細胞； 様々な幹細胞又は前駆細胞、特に例えば骨髄、臍帯血、末梢血、胎児肝臓から得られる造血幹細胞又は前駆細胞等が挙げられるが、これらに限定されない。 Cells to which the DNA of the present invention can be introduced for gene therapy are epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; T-lymphocytes, B-lymphocytes, monocytes Blood cells such as macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem cells or progenitor cells, in particular hematopoietic stem cells or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. For example, but not limited to.
以下、本発明を実施例により具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
実施例１：本発明のｃＤＮＡの調製及び本発明のベクターの調製
ＡＬＫ５細胞外ドメインをコードするｃＤＮＡ配列およびＴβＲＩＩ細胞外ドメインをコードするｃＤＮＡは、ヒト乳がん細胞（ＭＤＡ−ＭＢ−２３１）のｃＤＮＡをもとに、ＡＬＫ５細胞外ドメインは配列番号９及び１０に示すプライマーにて、ＴβＲＩＩ細胞外ドメインは配列番号１１及び１２に示すプライマーにてＰＣＲ法により増幅し、クローニングした。クローニングした各ｃＤＮＡ（配列番号５で表されるｃＤＮＡおよび配列番号６又は配列番号１４で表されるｃＤＮＡ）をｐＣＲＩＩ−Ｔｏｐｏベクター（ｉｎｖｉｔｒｏｇｅｎ社）に組み込んだ。次いでｐＥＮＴＲ２０１ベクター（Ｉｎｖｉｔｒｏｇｅｎ社）のマルチクローニングサイト（ｍｃｓ）に配列番号５で表されるｃＤＮＡおよび配列番号６又は配列番号１４で表されるｃＤＮＡを組み込んだ（図１参照）。次いで本発明のｃＤＮＡをｍｃｓでタンデム発現できるようにサブクローニングするため、ｐＥＮＴＲ２０１ベクターから切り出した配列番号５で表されるｃＤＮＡおよび配列番号６又は配列番号１４で表されるｃＤＮＡをｐＦＵＳＥ−ｈＩｇＧ１−Ｆｃ（Ｉｎｖｉｖｏｇｅｎ社）のｈＩｇＧ１−ＦｃのｃＤＮＡ配列（配列番号３）の５’末端側（コードされるアミノ酸配列のＮ末端側）に組み込み、本発明のキメラタンパク質をコードするｃＤＮＡ（配列番号６及び配列番号１６）及び本発明のベクター（ｐＥＮＴＲ２０１ ＡＬＫ５ＴβＲＩＩ−Ｆｃ）を得た（図１参照）。
Example 1: Preparation of cDNA of the present invention and preparation of vector of the present invention The cDNA sequence encoding the ALK5 extracellular domain and the cDNA encoding the TβRII extracellular domain are the cDNAs of human breast cancer cells (MDA-MB-231). Originally, the ALK5 extracellular domain was amplified by the PCR method with primers shown in SEQ ID NOs: 9 and 10, and the TβRII extracellular domain was amplified with the primers shown in SEQ ID NOs: 11 and 12, and cloned. Each cloned cDNA (cDNA represented by SEQ ID NO: 5 and cDNA represented by SEQ ID NO: 6 or SEQ ID NO: 14) was incorporated into a pCRII-Topo vector (Invitrogen). Subsequently, the cDNA represented by SEQ ID NO: 5 and the cDNA represented by SEQ ID NO: 6 or 14 were incorporated into the multicloning site (mcs) of the pENTR201 vector (Invitrogen) (see FIG. 1). Subsequently, in order to subclone the cDNA of the present invention so that it can be expressed in tandem with mcs, the cDNA represented by SEQ ID NO: 5 and the cDNA represented by SEQ ID NO: 6 or 14 excised from the pENTR201 vector were transformed into pFUSE-hIgG1-Fc ( CDNA encoding the chimeric protein of the present invention (SEQ ID NO: 6 and SEQ ID NO: 6) incorporated into the 5 ′ terminal side (N-terminal side of the encoded amino acid sequence) of hIgG1-Fc cDNA sequence (SEQ ID NO: 3) of Invivogen) 16) and the vector of the present invention (pENTR201 ALK5TβRII-Fc) was obtained (see FIG. 1).
実施例２：本発明の発現ベクター調製
実施例１で得られた本発明のベクターからクローニングした本発明のｃＤＮＡをｐＣＳＩＩ−ＥＦ−Ｒｆａ（理化学研究所より分与）へＬＲ反応を用いて組み込み、本発明の発現ベクターを得た（図１参照）。
Example 2: Preparation of expression vector of the present invention The cDNA of the present invention cloned from the vector of the present invention obtained in Example 1 was incorporated into pCSII-EF-Rfa (distributed from RIKEN) using LR reaction, The expression vector of the present invention was obtained (see FIG. 1).
実施例３：本発明のキメラタンパク質の作製
１０％ＦＢＳ含有ＤＭＥＭ培地を用いて、ＨＥＫ２９３Ｔ細胞（Ｉｎｖｉｔｒｏｇｅｎ社）３×１０6個を直径１０ｃｍ ｄｉｓｈに撒きこみ、一晩培養したのちにＦｕｇｅｎｅＨＤ（登録商標）（Ｒｏｃｈｅ社）を用いて実施例２で得られた本発明の発現ベクターのうち配列番号６が組み込まれた発現ベクターを２０μｇトランスフェクトし、本発明のキメラタンパク質産生細胞を得た。得られた本発明のキメラタンパク質産生細胞を４時間、３７℃で培養した後に培地を除去し、ＦＢＳを含まないＯＰＴＩ−ＭＥＭ培地に置き換えて、２４時間インキュベーター内で培養し、培養上清を回収した。回収した上清は０．２２μｍのポアサイズのフィルターを用いてろ過し、本発明のキメラタンパク質を含む培養上清液を得た。本発明のキメラタンパク質の濃度をヒトＩｇＧＦｃＥＬＩＳＡキット（Ｂｅｔｈｙｌ社）を用いて測定した結果、ＡＬＫ５ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃは１８００ｎｇ/ｍＬの濃度であった。
実施例２で得られた本発明の発現ベクターのうち配列番号１６が組み込まれた発現ベクターはＡ５４９細胞（ＡＴＣＣ）にトランスフェクトし、本発明のキメラタンパク質産生細胞を得た他は配列番号６が組み込まれた発現ベクターと同様に処理し、本発明のキメラタンパク質を含む培養上清液を得た。ＡＬＫ５ＴβＲＩＩ（ｌｏｎｇ）Ｆｃは１２００ｎｇ/ｍＬの濃度であった。
Example 3 Production of Chimeric Protein of the Present Invention Using 10% FBS-containing DMEM medium, 3 × 10 6 HEK293T cells (Invitrogen) were seeded in a 10 cm diameter dish and cultured overnight, then FugeneHD (registered trademark). (Roche) was used to transfect 20 μg of the expression vector in which SEQ ID NO: 6 was incorporated among the expression vectors of the present invention obtained in Example 2 to obtain chimeric protein-producing cells of the present invention. The obtained chimeric protein-producing cells of the present invention were cultured for 4 hours at 37 ° C., then the medium was removed, replaced with OPTI-MEM medium containing no FBS, and cultured in an incubator for 24 hours, and the culture supernatant was recovered. did. The collected supernatant was filtered using a 0.22 μm pore size filter to obtain a culture supernatant containing the chimeric protein of the present invention. The concentration of the chimeric protein of the present invention was measured using a human IgG Fc ELISA kit (Bethyl). As a result, ALK5TβRII (short) Fc was 1800 ng / mL.
Of the expression vector of the present invention obtained in Example 2, the expression vector in which SEQ ID NO: 16 was incorporated was transfected into A549 cells (ATCC), and the chimeric protein producing cell of the present invention was obtained. The culture was carried out in the same manner as the incorporated expression vector to obtain a culture supernatant containing the chimeric protein of the present invention. ALK5TβRII (long) Fc was at a concentration of 1200 ng / mL.
比較例：比較用キメラタンパク質の作製
比較のため、配列番号５で表されるｃＤＮＡ、配列番号６で表されるｃＤＮＡおよび配列番号１４で表されるｃＤＮＡを組み込んだｐＦＵＳＥ−ｈＩｇＧ１−ＦｃのｈＩｇＧ１−ＦｃのｃＤＮＡの５’末端側に組み込んだｃＤＮＡをそれぞれ作成し、実施例２と同様にｐＣＳＩＩ−ＥＦ−ＲｆａへＬＲ反応を用いて組み込み、ＡＬＫ５Ｆｃ発現ベクター及びＴβＲＩＩＦｃ発現ベクターを得た。ＡＬＫ５Ｆｃ発現ベクター及びＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃ発現ベクターを実施例３と同様にＨＥＫ２９３Ｔ細胞に処理し、ＴβＲＩＩ（ｌｏｎｇ）Ｆｃ発現ベクターを実施例３と同様にＡ５４９細胞に処理し、培養して、ＡＬＫ５Ｆｃを含む培養上清及びＴβＲＩＩＦｃを含む培養上清を得た。得られた培養上清は実施例３と同様にろ過し、比較用キメラタンパク質の濃度を、ヒトＩｇＧＦｃＥＬＩＳＡキットを用いて測定した。その結果、ＡＬＫ５Ｆｃが２３００ｎｇ/ｍＬ、ＴβＲＩＩ（ｌｏｎｇ）Ｆｃが２０００ｎｇ/ｍＬ、ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃが２１００ｎｇ/ｍＬの濃度であった。
Comparative Example: Production of a chimeric protein for comparison For comparison, hIgG1- of pFUSE-hIgG1-Fc incorporating cDNA represented by SEQ ID NO: 5, cDNA represented by SEQ ID NO: 6, and cDNA represented by SEQ ID NO: 14 CDNAs incorporated into the 5 ′ end of the Fc cDNA were prepared, respectively, and incorporated into pCSII-EF-Rfa using the LR reaction in the same manner as in Example 2 to obtain an ALK5Fc expression vector and a TβRIIFc expression vector. The ALK5Fc expression vector and the TβRII (short) Fc expression vector were treated with HEK293T cells in the same manner as in Example 3. The TβRII (long) Fc expression vector was treated with A549 cells in the same manner as in Example 3, cultured, and ALK5Fc was cultured. A culture supernatant containing and a culture supernatant containing TβRIIFc were obtained. The obtained culture supernatant was filtered in the same manner as in Example 3, and the concentration of the comparative chimeric protein was measured using a human IgG Fc ELISA kit. As a result, ALK5Fc was 2300 ng / mL, TβRII (long) Fc was 2000 ng / mL, and TβRII (short) Fc was 2100 ng / mL.
実施例４−１：本発明のキメラタンパク質のＴＧＦβシグナル阻害活性確認
ＨＥＫ２９３Ｔ細胞を、２×１０4個を２４ ｗｅｌｌ ｐｌａｔｅ直径１０ｃｍ ｄｉｓｈに撒きこんで一晩培養した。そののちに、ＴＧＦβに応答してホタルルシフェラーゼを発現するプラスミド（ＣＡＧＡ）１２−ＭＬＰ−Ｌｕｃ（ＥＭＢＯ Ｊ., １７ ３０９１（１９９８）に基づき樹立）と補正用のウミシイタケルシフェラーゼ発現プラスミドｐＧＬ４−ＴＫ−Ｒｅｎｉｌｌａ−Ｌｕｃ（Ｉｎｖｉｔｒｏｇｅｎ社）を、共にＦｕｇｅｎｅＨＤ（登録商標）を用いてＨＥＫ２９３Ｔに導入した。ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３（いずれも１ｎｇ／ｍＬ）を、実施例３で得られた配列番号４で表される本発明のキメラタンパク質溶液又はＡＬＫ５Ｆｃ溶液又はＴβＲＩＩＦｃ溶液と共に、図２に記載の濃度となるようにＨＥＫ２９３Ｔ細胞の培養液に添加し、２４時間培養した。２４時間後にＤｕａｌ−Ｌｕｃｉｆｅｒａｓｅ（登録商標） Ｒｅｐｏｒｔｅｒ Ａｓｓａｙ Ｓｙｓｔｅｍ（プロメガ社）を用いて細胞を溶解して、Ｍｉｔｈｒａｓ ＬＢ ９４０ Ｍｕｌｔｉｍｏｄｅ Ｍｉｃｒｏｐｌａｔｅ Ｒｅａｄｅｒ（ＢＥＲＴＨＯＬＤ ＴＥＣＨＮＯＬＯＧＩＥＳ社)で発光強度を測定した。得られたホタルルシフェラーゼ発光強度の値は、ウミシイタケルシフェラーゼ発光強度により補正を行った。ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３を処理した場合のそれぞれの発光強度を図２に示す。発光強度と、キメラタンパク質とＴＧＦβとの結合の強さは反比例し、発光強度が低い場合はキメラタンパク質とＴＧＦβとが結合し、高い場合はキメラタンパク質とＴＧＦβとが結合していないことを示す。
実施例３で得られた配列番号１５で表される本発明のキメラタンパク質に関しては、Ａ５４９細胞を用いたこと、及び、配列番号４で表される本発明のキメラタンパク質溶液、配列番号１５で表される本発明のキメラタンパク質溶液、ＡＬＫ５Ｆｃ溶液又はＴβＲＩＩＦｃ溶液は終濃度５００ｎｇ／ｍＬとした他は、配列番号４で表される本発明のキメラタンパク質と同様にして実験を行った。ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３を処理した場合のそれぞれの発光強度を図３に示す。
Example 4-1 Confirmation of TGFβ Signal Inhibitory Activity of Chimeric Protein of the Present Invention HEK293T cells were cultured overnight by inoculating 2 × 10 4 cells in a 24 well plate diameter 10 cm dish. Thereafter, a plasmid (CAGA) 12-MLP-Luc (established based on EMBO J., 17 3091 (1998)) that expresses firefly luciferase in response to TGFβ, and a Renilla luciferase expression plasmid pGL4-TK- for correction Both Renilla-Luc (Invitrogen) were introduced into HEK293T using FugeneHD (registered trademark). TGFβ1, TGFβ2, and TGFβ3 (all at 1 ng / mL) have the concentrations shown in FIG. 2 together with the chimeric protein solution of the present invention represented by SEQ ID NO: 4 obtained in Example 3, the ALK5Fc solution, or the TβRIIFc solution. Thus, it was added to the culture solution of HEK293T cells and cultured for 24 hours. After 24 hours, cells were lysed using a Dual-Luciferase (registered trademark) Reporter Assay System (Promega), and luminescence intensity was measured with a Mitras LB 940 Multimode Microplate Reader (BERTHOLD TECHNOLOGIES). The value of the obtained firefly luciferase luminescence intensity was corrected by the Renilla luciferase luminescence intensity. FIG. 2 shows the respective luminescence intensities when TGFβ1, TGFβ2 and TGFβ3 were treated. The intensity of the luminescence and the strength of the binding between the chimeric protein and TGFβ are inversely proportional. When the luminescence intensity is low, the chimeric protein and TGFβ are bound, and when the intensity is high, the chimeric protein and TGFβ are not bound.
Regarding the chimeric protein of the present invention represented by SEQ ID NO: 15 obtained in Example 3, A549 cells were used and the chimeric protein solution of the present invention represented by SEQ ID NO: 4, represented by SEQ ID NO: 15 The chimeric protein solution of the present invention, ALK5Fc solution or TβRIIFc solution was subjected to an experiment in the same manner as the chimeric protein of the present invention represented by SEQ ID NO: 4 except that the final concentration was 500 ng / mL. FIG. 3 shows the respective luminescence intensities when TGFβ1, TGFβ2 and TGFβ3 were treated.
ＴβＲＩＩ細胞外ドメインのバリアント違いに関わらず、ＡＬＫ５ＴβＲＩＩＦｃは、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３全てのタイプに結合能が認められた。一方、ＡＬＫ５ＦｃはＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３にいずれに対しても結合能は認められず、ＴβＲＩＩＦｃはＴβＲＩＩ細胞外ドメインのバリアント違いに関わらず、ＴＧＦβ１及びＴＧＦβ３にのみ結合が認められた。 Regardless of the variant of the TβRII extracellular domain, ALK5TβRIIFc was able to bind to all types of TGFβ1, TGFβ2, and TGFβ3. On the other hand, ALK5Fc did not bind to TGFβ1, TGFβ2 and TGFβ3, and TβRIIFc was found to bind only to TGFβ1 and TGFβ3 regardless of the variant of the TβRII extracellular domain.
実施例５：本発明のウイルスの調製
実験例２で得られた配列番号５で表される本発明のキメラタンパク質のｃＤＮＡを組み込んだ本発明の発現ベクター又は比較例で得られた比較用ベクター２種と、パッケージング用プラスミドのｐＣＡＧ−ＨＩＶ（理化学研究所より分与）及びｐＣＭＶ−ＶＳＶ−Ｇ−ＲＳＶ−Ｒｅｖ（理化学研究所より分与）をＨＥＫ２９３ＦＴ（Ｉｎｖｉｔｒｏｇｅｎ社）にトランスフェクトし、実施例３と同様に培養して、レンチウイルスを含む培養上清を回収し、本発明の発現ベクター又は比較例で得られた比較用ベクター２種をそれぞれ含むレンチウイルス液を得た。
Example 5: Preparation of the virus of the present invention The expression vector of the present invention incorporating the cDNA of the chimeric protein of the present invention represented by SEQ ID NO: 5 obtained in Experimental Example 2 or the comparative vector 2 obtained in the Comparative Example Seed and packaging plasmids pCAG-HIV (distributed from RIKEN) and pCMV-VSV-G-RSV-Rev (distributed from RIKEN) into HEK293FT (Invitrogen), Examples After culturing in the same manner as in No. 3, the culture supernatant containing the lentivirus was recovered to obtain a lentivirus solution containing each of the expression vector of the present invention or the two comparative vectors obtained in the comparative examples.
実施例６：キメラタンパク質のＴＧＦβシグナル阻害活性
実施例５で得られたレンチウイルス液をＭＤＡ−ＭＢ−２３１ＤＬｕｃ（Ａｎｔｉｃａｎｃｅｒ Ｒｅｓ．，２５，３８１７（２００５）に基づき樹立したＭＤＡ−ＭＢ−２３１の高骨転移性株にレンチウイルスによりルシフェラーゼを導入した株）に処理し、本発明のキメラタンパク質（ＡＬＫ５ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃ）及び比較用キメラタンパク質２種（ＡＬＫ５Ｆｃ、ＴβＲＩＩ（ｌｏｎｇ）Ｆｃ）をそれぞれ発現するＭＤＡ−ＭＢ−２３１ＤＬｕｃを作成した。これらの細胞を６ｗｅｌｌ ｐｌａｔｅに２×１０6個の細胞を１０％ＦＢＳ含有Ｄ−ＭＥＭで２ｍＬの容量で撒き、コンフルエントな条件下で２日間培養した。ＴＧＦβ１、ＴＧＦβ２、及びＴＧＦβ３（いずれも０．２ｎｇ／ｍＬ）を培養液中に添加して３０分後に細胞を回収した。回収した細胞からタンパク質を抽出し、Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇによってリン酸化Ｓｍａｄ２（Ｐ−Ｓｍａｄ２）の発現量及びＳｍａｄ２及びＳｍａｄ３の総発現量（Ｔｏｔａｌ Ｓｍａｄ２／３）をそれぞれ、抗Ｐ−Ｓｍａｄ２抗体（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ社）、抗Ｔｏｔａｌ Ｓｍａｄ２／３（ＢＤバイオサイエンス社）を用いて確認した。リン酸化Ｓｍａｄ２（Ｐ−Ｓｍａｄ２）の発現量及びＳｍａｄ２及びＳｍａｄ３の総発現量（Ｔｏｔａｌ Ｓｍａｄ２／３）を図４に示す。Ｓｍａｄ２はＴＧＦβによりリン酸化される細胞内情報伝達分子である。ＮＴは無処理条件の略称である。
Example 6: TGFβ signal inhibitory activity of chimeric protein The lentiviral solution obtained in Example 5 was used as the high bone of MDA-MB-231 established based on MDA-MB-231DLuc (Anticancer Res., 25, 3817 (2005)) MDA expressing a chimeric protein (ALK5TβRII (short) Fc) of the present invention and two comparative chimeric proteins (ALK5Fc, TβRII (long) Fc) by treating the metastatic strain with a luciferase introduced by lentivirus -MB-231DLuc was created. These cells were seeded in a 6- well plate at 2 × 10 6 cells in a volume of 2 mL with D-MEM containing 10% FBS, and cultured for 2 days under confluent conditions. TGFβ1, TGFβ2, and TGFβ3 (all 0.2 ng / mL) were added to the culture medium, and the cells were collected 30 minutes later. Proteins were extracted from the collected cells, and the expression level of phosphorylated Smad2 (P-Smad2) and the total expression level of Smad2 and Smad3 (Total Smad2 / 3) were respectively determined by Western blotting, anti-P-Smad2 antibody (Cell Signaling) This was confirmed using anti-Total Smad2 / 3 (BD Bioscience). The expression level of phosphorylated Smad2 (P-Smad2) and the total expression level of Smad2 and Smad3 (Total Smad2 / 3) are shown in FIG. Smad2 is an intracellular signaling molecule that is phosphorylated by TGFβ. NT is an abbreviation for no-treatment condition.
本発明のキメラタンパク質（ＡＬＫ５ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃ）を発現するＭＤＡ−ＭＢ−２３１ＤＬｕｃでは、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３のいずれによってもＰ−Ｓｍａｄ２の発現がみられなかった。ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３によるＳｍａｄ２のリン酸化を本発明のキメラタンパク質が抑制することがわかる。一方、ＡＬＫ５Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃではＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３のいずれに対してもＰ−Ｓｍａｄ２の発現が認められた。ＡＬＫ５ＦｃはＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３の刺激によるＳｍａｄ２のリン酸化を抑制しないことがわかる。ＴβＲＩＩ（ｌｏｎｇ）Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃではＴＧＦβ１及びＴＧＦβ３によるＰ−Ｓｍａｄ２の発現がみられなかったが、ＴＧＦβ２によるＰ−Ｓｍａｄ２の発現がみられた。ＴβＲＩＩＦｃはＴＧＦβ１及びＴＧＦβ３によるＳｍａｄ２のリン酸化を抑制することがわかる。 In MDA-MB-231DLuc expressing the chimeric protein (ALK5TβRII (short) Fc) of the present invention, no expression of P-Smad2 was observed by any of TGFβ1, TGFβ2, and TGFβ3. It can be seen that the chimeric protein of the present invention inhibits Smad2 phosphorylation by TGFβ1, TGFβ2 and TGFβ3. On the other hand, in ALK5Fc expression MDA-MB-231DLuc, expression of P-Smad2 was observed for all of TGFβ1, TGFβ2, and TGFβ3. It can be seen that ALK5Fc does not suppress phosphorylation of Smad2 by stimulation with TGFβ1, TGFβ2, and TGFβ3. In TβRII (long) Fc expression MDA-MB-231DLuc, expression of P-Smad2 by TGFβ1 and TGFβ3 was not observed, but expression of P-Smad2 by TGFβ2 was observed. It can be seen that TβRIIFc suppresses phosphorylation of Smad2 by TGFβ1 and TGFβ3.
実施例７：キメラタンパク質のＴＧＦβによる細胞遊走抑制作用
実施例６で得られた本発明のキメラタンパク質（ＡＬＫ５ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃ）及び比較用キメラタンパク質２種をそれぞれ発現するＭＤＡ−ＭＢ−２３１ＤＬｕｃ（ＡＬＫ５ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃ、ＡＬＫ５Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃ、ＴβＲＩＩ（ｌｏｎｇ）Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃ）を用いて、ＴＧＦβにより促進される細胞の遊走能を評価した。８．０μｍポアサイズの２４ウェルセルカルチャーインサート（ＢＤファルコン社）内に１ｍＭ塩酸で３ｍｇ／ｍＬに希釈したＩ型コラーゲン（新田ゼラチン社）をコートした後にピペットで余分な水分を除き、３×１０5個の上記ＭＤＡ−ＭＢ−２３１ＤＬｕｃをインサート内に加え、ＴＧＦβ２及びＴＧＦβ３を最終濃度が０．３ｎｇ／ｍＬとなるように、リザーバー側のウェルとインサート内に加えて、無血清のＤ−ＭＥＭで４８時間培養した。培養後にインサートを回収し、１０％ホルマリンに１０分間浸漬して細胞を固定した。クリスタルバイオレッド液で細胞を染色し、インサートの裏側に遊走した細胞を顕微鏡観察下でカウントした。測定結果を図５に示す。
Example 7: Cell migration inhibitory action of chimeric protein by TGFβ MDA-MB-231DLuc (ALK5TβRII) expressing the chimeric protein of the present invention (ALK5TβRII (short) Fc) obtained in Example 6 and two kinds of comparative chimeric proteins, respectively (Short) Fc-expressing MDA-MB-231DLuc, ALK5Fc-expressing MDA-MB-231DLuc, TβRII (long) Fc-expressing MDA-MB-231DLuc) were used to evaluate the TGFβ-stimulated cell migration ability. After coating type I collagen (Nitta Gelatin) diluted to 3 mg / mL with 1 mM hydrochloric acid in a 24-well cell culture insert (BD Falcon) with a pore size of 8.0 μm, excess water was removed with a pipette and 3 × 10 Add 5 of the above MDA-MB-231DLuc into the insert, and add TGFβ2 and TGFβ3 to the well and insert on the reservoir side so that the final concentration is 0.3 ng / mL, and use serum-free D-MEM. Cultured for 48 hours. After culturing, the insert was recovered and immersed in 10% formalin for 10 minutes to fix the cells. Cells were stained with crystal violet solution, and the cells that migrated to the back of the insert were counted under a microscope. The measurement results are shown in FIG.
ＡＬＫ５ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃはＴＧＦβ２及びＴＧＦβ３処理のいずれによっても遊走した細胞数はコントロールと同程度であった。ＴＧＦβ２及びＴＧＦβ３の遊走促進作用を本発明のキメラタンパク質が抑制することがわかる。ＡＬＫ５Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃはＴＧＦβ２及びＴＧＦβ３処理によって遊走した細胞数がコントロールに比較して増加した。ＡＬＫ５ＦｃはＴＧＦβ２及びＴＧＦβ３の遊走促進作用に少なくとも影響を及ぼさないことがわかる。ＴβＲＩＩ（ｌｏｎｇ）Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃはＴＧＦβ３処理では遊走した細胞数はコントロールと同程度であったが、ＴＧＦβ２処理による遊走した細胞数がコントロールに比較して増加した。ＴβＲＩＩ（ｌｏｎｇ）ＦｃはＴＧＦβ２の遊走促進作用に少なくとも影響を及ぼさないものの、ＴＧＦβ３の遊走促進作用は抑制することがわかる。 ALK5TβRII (short) Fc-expressing MDA-MB-231DLuc migrated by both TGFβ2 and TGFβ3 treatments in the same number as the control. It can be seen that the chimeric protein of the present invention suppresses the migration promoting action of TGFβ2 and TGFβ3. In ALK5Fc-expressing MDA-MB-231DLuc, the number of cells that migrated by TGFβ2 and TGFβ3 treatment increased compared to the control. It can be seen that ALK5Fc has at least no effect on the migration promoting action of TGFβ2 and TGFβ3. TβRII (long) Fc-expressing MDA-MB-231DLuc migrated with TGFβ3 treatment at the same level as the control, but the number of migrated cells with TGFβ2 treatment increased compared to the control. It can be seen that TβRII (long) Fc does not affect at least the TGFβ2 migration promoting action, but suppresses the TGFβ3 migration promoting action.
実施例８：キメラタンパク質のＴＧＦβ誘導性遺伝子の発現抑制作用
実施例６で得られた本発明のキメラタンパク質及び比較用キメラタンパク質２種をそれぞれ発現するＭＤＡ−ＭＢ−２３１ＤＬｕｃを２．５×１０5個／ウェルを１２ ｗｅｌｌ ｐｌａｔｅに１０％ＦＢＳ含有Ｄ−ＭＥＭで撒き、一晩培養した後にＴＧＦβ２及びＴＧＦβ３（いずれも１ｎｇ／ｍＬ）を添加して２４時間後にＲＮｅａｓｙ（キアゲン社）を用いてｔｏｔａｌ ＲＮＡの回収を行った。回収したＲＮＡは濃度を測定後、各処理群で同量のＲＮＡを使用してＰｒｉｍｅＳｃｒｉｐｔTM ＲＴ ｒｅａｇｅｎｔ Ｋｉｔ（タカラバイオ社）を用いてｃＤＮＡの合成を行った。得られたｃＤＮＡを水で２５倍に希釈し、ＳＹＢＲ（登録商標） Ｇｒｅｅｎ ＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（アプライドバイオシステム社）を用いて、定量的ＰＣＲを行い、ＳｔｅｐＯｎｅＰｌｕｓリアルタイムＰＣＲシステム（アプライドバイオシステム社）によってＴＧＦβ誘導性の遺伝子であるＰＡＩ−１、Ｓｍａｄ７、ＩＬ−１０、ＰＴＨｒＰの発現量を測定した。これら遺伝子の発現量は同時に測定したＧＡＰＤＨの発現量を用いて補正した。図６にこれら遺伝子の補正済み発現量を示す。
Example 8: TGFβ-inducible gene expression inhibitory action of chimeric protein MDA-MB-231DLuc expressing each of the chimeric protein of the present invention and two chimeric proteins for comparison obtained in Example 6 was 2.5 × 10 5. Cells / well were seeded in 12-well plate with D-MEM containing 10% FBS, cultured overnight, TGFβ2 and TGFβ3 (both 1 ng / mL) were added, and 24 hours later, total RNA was used with RNeasy (Qiagen). Was collected. After measuring the concentration of the collected RNA, cDNA was synthesized using PrimeScript ™ RT reagent Kit (Takara Bio Inc.) using the same amount of RNA in each treatment group. The obtained cDNA was diluted 25-fold with water, quantitative PCR was performed using SYBR (registered trademark) Green PCR Master Mix (Applied Biosystems), and TGFβ was performed by StepOnePlus real-time PCR system (Applied Biosystems). The expression levels of inducible genes PAI-1, Smad7, IL-10, and PTHrP were measured. The expression levels of these genes were corrected using the simultaneously measured GAPDH expression levels. FIG. 6 shows the corrected expression levels of these genes.
ＡＬＫ５ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃはＴＧＦβ２及びＴＧＦβ３処理によって、いずれの遺伝子の誘導も認められなかった。本発明のキメラタンパク質がＴＧＦβ２及びＴＧＦβ３のシグナルを阻害していることがわかる。ＡＬＫ５Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃはＴＧＦβ２及びＴＧＦβ３処理によってそれぞれＰＡＩ−１、Ｓｍａｄ７、ＩＬ−１１、ＰＴＨｒＰの発現誘導が認められた。ＡＬＫ５ＦｃにはＴＧＦβ２及びＴＧＦβ３のシグナルの阻害作用がないことがわかる。また、ＴβＲＩＩ（ｌｏｎｇ）Ｆｃ発現ＭＤＡ−ＭＢ−２３１ＤＬｕｃはＴＧＦβ３によるこれら遺伝子の誘導は認められなかったものの、ＴＧＦβ２により誘導が認められた。ＴβＲＩＩＦｃはＴＧＦβ３のシグナルの阻害作用はあるものの、ＴＧＦβ２のシグナルの阻害作用はないことがわかる。 ALK5TβRII (short) Fc-expressing MDA-MB-231DLuc was not induced by any gene by treatment with TGFβ2 and TGFβ3. It can be seen that the chimeric protein of the present invention inhibits TGFβ2 and TGFβ3 signals. Induction of PAI-1, Smad7, IL-11, and PTHrP was observed in ALK5Fc-expressing MDA-MB-231DLuc by treatment with TGFβ2 and TGFβ3, respectively. It can be seen that ALK5Fc has no inhibitory action on TGFβ2 and TGFβ3 signals. In addition, although TβRII (long) Fc-expressing MDA-MB-231DLuc did not induce these genes by TGFβ3, it was induced by TGFβ2. It can be seen that TβRIIFc has an inhibitory effect on the TGFβ3 signal, but has no inhibitory effect on the TGFβ2 signal.
以上の結果から、本発明のキメラタンパク質はＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３のいずれにも結合し、ＴＧＦβからＴＧＦβ受容体を経たＳｍａｄ２のリン酸化を阻害することが明らかとなった。本発明のキメラタンパク質はＴＧＦβ２及びＴＧＦβ３による癌細胞の遊走とＴＧＦβ誘導性の遺伝子であるＰＡＩ−１、Ｓｍａｄ７、ＩＬ−１１、ＰＴＨｒＰの発現も抑制することが確認できた。よって、本発明はＴＧＦβシグナルに関連する疾患の治療、予防に有用である。 From the above results, it was revealed that the chimeric protein of the present invention binds to any of TGFβ1, TGFβ2 and TGFβ3 and inhibits Smad2 phosphorylation from TGFβ via the TGFβ receptor. It was confirmed that the chimeric protein of the present invention suppresses the migration of cancer cells by TGFβ2 and TGFβ3 and the expression of TGFβ-inducible genes PAI-1, Smad7, IL-11 and PTHrP. Therefore, the present invention is useful for the treatment and prevention of diseases associated with TGFβ signal.
本発明のキメラタンパク質は、ＴＧＦβ阻害機能を有することから、がん及び転移臓器の線維化を伴う疾患、例えば肺線維症、肝硬変、動脈硬化症、強皮症、経皮経管冠動脈血管拡張術後の冠動脈再狭窄、間質性肺炎、間質性心筋炎、間質性膀胱炎、糸球体腎炎、血管炎、糖尿病性腎症、高血圧性腎硬化症、ＨＩＶ腎症、ＩｇＡ腎症、ループス腎症、間質性腎炎、尿管閉塞による閉塞腎、熱傷後の皮膚瘢痕化などやそれらの合併症の治療に有効である。本発明のキメラタンパク質は、医薬分野において有用である。 Since the chimeric protein of the present invention has a TGFβ inhibitory function, it is a disease accompanied by fibrosis of cancer and metastatic organs such as pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, percutaneous transluminal coronary vasodilation Later coronary restenosis, interstitial pneumonia, interstitial myocarditis, interstitial cystitis, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus It is effective in the treatment of nephropathy, interstitial nephritis, obstructive kidney due to ureteral obstruction, skin scarring after burns, and other complications. The chimeric protein of the present invention is useful in the pharmaceutical field.
配列番号１：ＡＬＫ１細胞外ドメインのアミノ酸配列
配列番号２：ＴβＲＩＩ細胞外ドメイン（ｓｈｏｒｔ）のアミノ酸配列
配列番号３：ｈＩｇＧ１Ｆｃのアミノ酸配列
配列番号４：ＡＬＫ１ＴβＲＩＩ（ｓｈｏｒｔ）Ｆｃのアミノ酸配列
配列番号５：ＡＬＫ１細胞外ドメインのｃＤＮＡ配列
配列番号６：ＴβＲＩＩ細胞外ドメイン（ｓｈｏｒｔ）のｃＤＮＡ配列
配列番号７：ｈＩｇＧ１ＦｃのｃＤＮＡ配列
配列番号８：ＡＬＫ１ＴβＲＩＩ（ｓｈｏｒｔ）ＦｃのｃＤＮＡ配列
配列番号９：ＡＬＫ１細胞外ドメインのフォワードプライマー
配列番号１０：ＡＬＫ１細胞外ドメインのリバースプライマー
配列番号１１：ＴβＲＩＩ細胞外ドメイン（ｓｈｏｒｔ）のフォワードプライマー
配列番号１２：ＴβＲＩＩ細胞外ドメイン（ｓｈｏｒｔ）のリバースプライマー
配列番号１３：ＴβＲＩＩ細胞外ドメイン（ｌｏｎｇ）のアミノ酸配列
配列番号１４：ＴβＲＩＩ細胞外ドメイン（ｌｏｎｇ）のｃＤＮＡ配列
配列番号１５：ＡＬＫ１ＴβＲＩＩ（ｌｏｎｇ）Ｆｃのアミノ酸配列
配列番号１６：ＡＬＫ１ＴβＲＩＩ（ｌｏｎｇ）ＦｃのｃＤＮＡ配列
SEQ ID NO: 1: amino acid sequence of ALK1 extracellular domain SEQ ID NO: 2: amino acid sequence of TβRII extracellular domain (short) SEQ ID NO: 3: amino acid sequence of hIgG1Fc SEQ ID NO: 4: amino acid sequence of ALK1TβRII (short) Fc SEQ ID NO: 5: ALK1 CDNA sequence of extracellular domain SEQ ID NO: 6: cDNA sequence of TβRII extracellular domain (short) SEQ ID NO: 7: cDNA sequence of hIgG1Fc SEQ ID NO: 8: cDNA sequence of ALK1TβRII (short) Fc SEQ ID NO: 9: Forward of ALK1 extracellular domain Primer SEQ ID NO: 10: Reverse primer of ALK1 extracellular domain SEQ ID NO: 11: Forward primer of TβRII extracellular domain (short) SEQ ID NO: 12: Redirection of TβRII extracellular domain (short) SEQ ID NO: 13: amino acid sequence of TβRII extracellular domain (long) SEQ ID NO: 14: cDNA sequence of TβRII extracellular domain (long) SEQ ID NO: 15: amino acid sequence of ALK1TβRII (long) Fc SEQ ID NO: 16: ALK1TβRII (long) Fc CDNA sequence of
Claims (22)
（ａ）配列番号４又は配列番号１５で表されるアミノ酸配列からなるキメラタンパク質；
（ｂ）配列番号４又は配列番号１５で表されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質；又は
（ｃ）配列番号４又は配列番号１５で表されるアミノ酸配列において１又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質； The chimeric protein according to any one of claims 1 to 8, which is any of the following (a) to (c).
(A) a chimeric protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 15;
(B) a chimeric protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4 or 15 and binding to TGFβ1, TGFβ2, and TGFβ3; or (c) SEQ ID NO: 4 or A chimeric protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 15 and binding to TGFβ1, TGFβ2 and TGFβ3;
（ａ）配列番号４又は配列番号１５で表されるアミノ酸配列からなるキメラタンパク質；
（ｂ）配列番号４又は配列番号１５で表されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質；又は
（ｃ）配列番号４又は配列番号１５で表されるアミノ酸配列において１又は数個のアミノ酸が欠失、置換及び／又は付加しているアミノ酸配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質。 DNA encoding any of the following chimeric proteins (a) to (c):
(A) a chimeric protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 15;
(B) a chimeric protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4 or 15 and binding to TGFβ1, TGFβ2, and TGFβ3; or (c) SEQ ID NO: 4 or A chimeric protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 15, and which binds to TGFβ1, TGFβ2 and TGFβ3.
（ｄ）配列番号８又は配列番号１６で表される塩基配列からなるＤＮＡ；又は
（ｅ）配列番号８又は配列番号１６で表される塩基配列において１又は数個の塩基が欠失、置換及び／又は付加している塩基配列からなり、ＴＧＦβ１、ＴＧＦβ２及びＴＧＦβ３に結合するキメラタンパク質をコードする塩基配列からなるＤＮＡ。 DNA of either of the following (d) or (e).
(D) DNA consisting of the base sequence represented by SEQ ID NO: 8 or SEQ ID NO: 16; or (e) one or several bases deleted or substituted in the base sequence represented by SEQ ID NO: 8 or SEQ ID NO: 16; DNA consisting of a base sequence that encodes a chimeric protein that consists of a base sequence that is added and / or that binds to TGFβ1, TGFβ2, and TGFβ3.
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