JP2017101012A - Immunotherapeutic formulation - Google Patents
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Abstract
Description
本発明は、遺伝子導入した細胞から得られるエクソソームからなる癌などの免疫治療薬および微生物感染予防・治療薬に関する。 The present invention relates to an immunotherapeutic agent such as cancer composed of exosomes obtained from a gene-transferred cell and a preventive / therapeutic agent for microbial infection.
癌などの疾患を対象とした免疫治療としては、様々な腫瘍関連抗原（以下ＴＡＡｓ）をコードしたＤＮＡワクチンが試みられ、限定的ではあるがその効果が報告されている（非特許文献１）。しかしＴＡＡｓは、もともと遺伝子的に同一の正常細胞がガン化した細胞の抗原であり、正常細胞にも量の差はあれ多少は発現しているのが普通である。そのためＴＡＡｓは一般に免疫原性が低く、ＴＡＡｓタンパクそのもの、あるいはそれをコードしたＤＮＡを投与しても、十分な免疫応答が誘導できないことが常に問題となり、抗イディオタイプ・ワクチンによるＢ細胞腫の治療など一部の例を除いては、十分な効果は認められていない（非特許文献１）。 As immunotherapy for diseases such as cancer, DNA vaccines encoding various tumor-associated antigens (hereinafter TAAs) have been tried, and their effects have been reported, although limited (Non-patent Document 1). However, TAAs are antigens of cells in which normal cells that are genetically identical are originally cancerous, and are usually also expressed to some extent in normal cells, although their amounts differ. For this reason, TAAs are generally low in immunogenicity, and it has always been a problem that even when TAAs protein itself or DNA encoding it is administered, sufficient immune response cannot be induced. Treatment of B cell tumor with anti-idiotype vaccine Except for some examples, sufficient effects are not recognized (Non-Patent Document 1).
一方近年、エクソソームを用いた新しいガンの診断、治療法が注目を集めている。特に、２００７年にエクソソームが分泌細胞由来のｍＲＮＡやｍｉｃｒｏＲＮＡを内包することが報告されて以来、エクソソームをガンの診断や核酸医薬のキャリアーとして癌治療に用いる研究が急速に発展した。腫瘍細胞から放出されたエクソソームは腫瘍関連抗原（ＴＡＡｓ）を持つことが知られており、エクソソームを用いた抗腫瘍免疫応答の導入が試行された。しかし、ＴＡＡｓは一般に免疫原性が低いため、サイトカインである顆粒球単球コロニー刺激因子（ＧＭ−ＣＳＦ）などの併用がないと、エクソソームそのものを投与しても免疫応答を誘導する効果は認められなかった（非特許文献２）。そこで、サイトカインであるインターロイキン−１２（ＩＬ−１２）で修飾した腫瘍細胞由来エクソソームが作られ、これらには免疫抑制を抑える効果があることがイン・ビトロの実験で示された（非特許文献３）が、生体中での治癒効果までは確認されていない。また、腫瘍細胞由来エクソソームに代わって、ＴＡＡｓでパルスした樹状細胞（ＤＣ）に由来するエクソソームを用いた免疫誘導が試みられた。しかし、これらのＤＣ由来エクソソームは、成熟ＤＣに対しては免疫導入効果を示すが、未成熟ＤＣには効果は認められなかった（非特許文献４）。 On the other hand, in recent years, new cancer diagnosis and treatment methods using exosomes have attracted attention. In particular, since it was reported in 2007 that exosomes contain mRNA and microRNA derived from secretory cells, research on the use of exosomes for cancer diagnosis as cancer carriers and nucleic acid drug carriers has rapidly developed. Exosomes released from tumor cells are known to have tumor-associated antigens (TAAs), and attempts have been made to introduce an anti-tumor immune response using exosomes. However, since TAAs are generally low in immunogenicity, the effect of inducing an immune response is observed even if exosomes themselves are administered without the use of cytokines such as granulocyte monocyte colony stimulating factor (GM-CSF). There was not (nonpatent literature 2). Therefore, tumor cell-derived exosomes modified with cytokine, interleukin-12 (IL-12), were made, and in vitro experiments showed that these have the effect of suppressing immunosuppression (non-patent literature). 3), but the healing effect in the living body has not been confirmed. In addition, immune induction was attempted using exosomes derived from dendritic cells (DC) pulsed with TAAs instead of tumor cell-derived exosomes. However, these DC-derived exosomes show an immunization effect on mature DCs, but no effect was observed on immature DCs (Non-patent Document 4).
他方、ガンばかりでなく、感染症も毎年多くの人の命を奪い続けている重要な疾患である。三大感染症と呼ばれるエイズ、結核、マラリアだけで毎年３００万人以上が亡くなっており、いまだに多くの中・低所得国で主要な死因の一つである。エクソソームを用いた感染予防の研究も近年盛んになった。結核菌に感染したマクロファージから分泌されたエクソソームを用いる研究（非特許文献５）や、トキソプラズマの抗原で刺激したＤＣからのエクソソームを用いた感染予防の研究（非特許文献６）が行われている。 On the other hand, not only cancer but also infectious diseases are important diseases that continue to kill many people every year. More than 3 million people die each year from AIDS, tuberculosis and malaria alone, which are called the three major infectious diseases. Research on infection prevention using exosomes has also become active in recent years. Studies using exosomes secreted from macrophages infected with Mycobacterium tuberculosis (Non-Patent Document 5) and studies on prevention of infection using exosomes from DC stimulated with Toxoplasma antigen (Non-Patent Document 6) have been conducted. .
原発性の腫瘍細胞の腫瘍関連抗原（ＴＡＡｓ）は、上述のように抗原性が弱く、また正常細胞にもしばしば発現しているため、腫瘍細胞から放出されたエクソソームや、ＴＡＡｓでパルスした樹状細胞（ＤＣ）に由来するエクソソームだけでは抗腫瘍免疫を惹起するのに不十分であり、十分な治癒効果は得られない。免疫システムを稼動させるための外来「デンジャー・シグナル」の存在が無いと、抗原性の弱いＴＡＡｓに対する免疫を惹起させるのは難しい。 Tumor-associated antigens (TAAs) of primary tumor cells are weakly antigenic as described above, and are often expressed in normal cells, so exosomes released from tumor cells and dendrites pulsed with TAAs are used. Only exosomes derived from cells (DC) are insufficient to elicit anti-tumor immunity, and a sufficient healing effect cannot be obtained. Without the presence of an exogenous “danger signal” to activate the immune system, it is difficult to elicit immunity against TAAs with weak antigenicity.
また、上で述べたように、エクソソームを用いた感染予防の研究が行われている。しかし、細菌感染や、単離したトキソプラズマ抗原による抗原提示細胞の刺激は、煩雑な手法や、特殊な設備、技術を必要とするため、広く一般的に応用できる、容易で汎用的な手法の開拓が望まれている。 In addition, as described above, research on infection prevention using exosomes has been conducted. However, bacterial infection and stimulation of antigen-presenting cells with isolated toxoplasma antigen require complicated methods, special equipment, and technology, and therefore cultivate easy and versatile methods that can be widely applied in general. Is desired.
すなわち、本発明は、抗原性の高いタンパク遺伝子を培養腫瘍細胞に導入し、そこから得られたエクソソームを抗腫瘍免疫製剤として利用する方法に関する。 That is, the present invention relates to a method for introducing a highly antigenic protein gene into cultured tumor cells and utilizing the exosomes obtained therefrom as an antitumor immunity preparation.
原発性の腫瘍細胞の腫瘍関連抗原（ＴＡＡｓ）は、上述のように抗原性が弱く、また正常細胞にもしばしば発現しているため、サイトカインの併用だけでは抗腫瘍免疫を惹起するのに不十分であると考えた。そこで、本発明者らは、樹状細胞を成熟させるためには「デンジャー・シグナル」の存在が必要であると考え、上記欠点を解決すべく鋭意研究を行った結果、「抗原遺伝子導入細胞由来のデンジャー・シグナル担持エクソソーム製剤」を考案した。すなわち、抗原性の高いタンパクの遺伝子を導入した培養細胞から得たエクソソームを用いて効果的に免疫を惹起、誘導する製剤である。 Since tumor-associated antigens (TAAs) of primary tumor cells are weakly antigenic as described above and are often expressed in normal cells, the combined use of cytokines alone is insufficient to elicit anti-tumor immunity. I thought. Therefore, the present inventors considered that the presence of a “danger signal” is necessary for maturation of dendritic cells, and as a result of earnest research to solve the above-mentioned drawbacks, "Danger signal carrying exosome preparation" was devised. That is, it is a preparation that effectively induces and induces immunity using exosomes obtained from cultured cells into which a highly antigenic protein gene has been introduced.
親細胞として腫瘍細胞を用いた場合には、分泌されるエクソソームは「デンジャー・シグナル」と、ＴＡＡｓの両者を兼ね備えた強力なガンワクチンとなる。実際に、デンジャーシグナルとして結核菌抗原タンパクの遺伝子をコードしたＤＮＡの複合体を用いて、腫瘍細胞に「デンジャー・シグナル」遺伝子を導入後、培養を続けてエクソソームを分泌させ、回収、単離したエクソソームを同じ腫瘍細胞を移植したマウスの腫瘍内に投与してみた。すると驚くべきことに、遺伝子導入していない（デンジャーシグナルを持たない）腫瘍細胞由来のエクソソームと比べて著しく高い治療効果が見られ、「抗原遺伝子導入細胞由来のデンジャー・シグナル担持エクソソーム製剤」の優れた抗腫瘍効果が確認された。と、同時に、エクソソームを用いた抗腫瘍免疫治療における「デンジャー・シグナル」の重要性が確認された。 When tumor cells are used as parental cells, the secreted exosome is a powerful cancer vaccine that combines both “danger signals” and TAAs. In fact, using a DNA complex that encodes the gene of Mycobacterium tuberculosis antigen protein as a danger signal, after introducing the "anger signal" gene into tumor cells, the culture was continued and the exosomes were secreted, recovered and isolated Exosomes were administered into the tumors of mice transplanted with the same tumor cells. Surprisingly, the therapeutic effect is significantly higher than that of exosomes derived from tumor cells that do not have gene transfer (no denger signal). Anti-tumor effect was confirmed. At the same time, the importance of “danger signals” in anti-tumor immunotherapy using exosomes was confirmed.
本発明のエクソソームは、血中安定性が高く、腫瘍組織内への蓄積性が高く、また、毒性が低く副作用が少ないため、局所投与、腹腔内投与、動脈内投与、静脈内投与、頭蓋内投与などの各種投与が可能である。同様の実験を、デンジャー・シグナル担持エクソソームの静脈内投与で行ってみたところ、同等の高い抗腫瘍効果が確認された。エクソソームは腫瘍細胞や抗原提示細胞への高い取り込み効率を持つことが期待される。そのために、全身投与に於いても高い治療効果を発揮したと考えられ、また、転移癌の治療にも有効であることが示された。 The exosome of the present invention has high blood stability, high accumulation in a tumor tissue, and low toxicity and few side effects. Therefore, local administration, intraperitoneal administration, intraarterial administration, intravenous administration, intracranial administration Various administrations such as administration are possible. A similar experiment was conducted by intravenous administration of a angersome bearing a danger signal, and the same high antitumor effect was confirmed. Exosomes are expected to have high uptake efficiency into tumor cells and antigen-presenting cells. Therefore, it was considered that a high therapeutic effect was exhibited even in systemic administration, and it was shown to be effective in the treatment of metastatic cancer.
さらに驚くべきことに、抗原遺伝子導入した腫瘍細胞由来のエクソソームは、同種の腫瘍を担持したマウスに対してだけでなく、他種の腫瘍を持つマウスに対しても高い腫瘍増殖抑制効果を示した。また、その効果は、親細胞と異系列のマウスに対しても示された。腫瘍細胞や樹状細胞は貪食作用が盛んであり、エクソソームを取り込みやすいこと、さらに取り込んだエクソソームの持つ抗原、あるいはエピトープは自己の主要組織適合遺伝子複合体への再結合が可能であるため、と思われる。すなわち、抗原遺伝子導入した腫瘍細胞由来のエクソソームは、他家であっても、また、他種の腫瘍に対しても効果的に用いることができることが示された。 Surprisingly, exosomes derived from antigen-transfected tumor cells showed a high tumor growth inhibitory effect not only on mice bearing the same type of tumor but also on mice with other types of tumors. . Moreover, the effect was shown also with respect to the parent cell and the mouse | mouth of a different lineage. Tumor cells and dendritic cells are active in phagocytosis, are easy to take up exosomes, and the antigens or epitopes of the taken up exosomes can be rebound to their major histocompatibility complex, Seem. That is, it was shown that the exosome derived from a tumor cell into which an antigen gene has been introduced can be used effectively against other types of tumors even in other families.
また、病原体の抗原遺伝子を導入した細胞から分泌されたエクソソームは、「デンジャー・シグナル」としてこれらの病原体の抗原またはそのエピトープを含む。そのため、これらの抗原に対する強い細胞性免疫を誘導することが見出された。これは、本発明のエクソソームが微生物の感染予防、治癒にも非常に有効であることを示すものである。
本発明のエクソソームは、腫瘍組織内への蓄積性が高く、毒性が低いため、局所投与、腹腔内投与、動脈内投与、静脈内投与、頭蓋内投与などの各種投与が可能である。また、調製・取り扱いが簡便で、保存性にも優れている。さらに、本発明の一態様においては、他の抗腫瘍免疫治療製剤と併用することで治癒効果を向上させることができる。In addition, exosomes secreted from cells into which pathogen antigen genes have been introduced contain these pathogen antigens or epitopes thereof as “danger signals”. Therefore, it was found to induce strong cellular immunity against these antigens. This indicates that the exosome of the present invention is very effective for prevention and cure of microorganism infection.
Since the exosome of the present invention has high accumulation in tumor tissue and low toxicity, various administrations such as local administration, intraperitoneal administration, intraarterial administration, intravenous administration and intracranial administration are possible. In addition, preparation and handling are simple and storage is excellent. Furthermore, in one embodiment of the present invention, the curative effect can be improved by using in combination with other antitumor immunotherapy preparations.
本発明で行う細胞に導入するデンジャー・シグナル遺伝子としては、抗腫瘍効果を期待するのであれば、対象とする患者、罹患動物において抗原性を有するものであればあらゆる種類のタンパク、ペプチド、またはこれらのエピトープを含むタンパク、ペプチドの遺伝子を使用することができる。デンジャー・シグナルとしての効果は、その抗原性の高さに由来するため、高い効果を期待するのであれば、抗原性の高いタンパク、ペプチド、またはこれらのエピトープを含むタンパク、ペプチドの遺伝子を用いるのがよい。例えば、ウィルス、バクテリア、原虫、異種生物の抗原性タンパク遺伝子などが使用できる。
感染予防効果を期待するのであれば、対象となる疾患の病原体の抗原タンパク、ペプチド、またはそのエピトープを含むタンパク、ペプチドの遺伝子が良い。As a danger signal gene to be introduced into a cell according to the present invention, any anti-tumor effect can be expected. It is possible to use a gene of a protein or peptide containing the above-mentioned epitope. Since the effect as a danger signal is derived from its high antigenicity, if a high effect is expected, a highly antigenic protein or peptide, or a protein or peptide gene containing these epitopes should be used. Is good. For example, antigenic protein genes of viruses, bacteria, protozoa, and heterologous organisms can be used.
As long as an anti-infection effect is expected, an antigen protein, peptide, or protein or peptide gene containing the epitope of the pathogen of the target disease is preferable.
本発明でデンジャー・シグナル遺伝子を導入し、エクソソームを分泌させる細胞にとしては、培養可能な細胞であれば、自家、他家を問わず、腫瘍細胞、繊維芽細胞、ＤＣ、マクロファージなど、あらゆる動物細胞を使用することができる。 In the present invention, the cells that introduce the danger signal gene and secrete exosomes can be any kind of animal, including tumor cells, fibroblasts, DCs, macrophages, etc. Cells can be used.
本発明で行う細胞への遺伝子導入法には、ウィルスを用いる方法、リン酸カルシウム、ポリカチオン、カチオン性脂質またはその集合体を用いる方法、エレクトロポレーション法、マイクロインジェクション法などを使用することができる。実施例では、特許文献７記載と同じＤＮＡ複合体を用いた。その複合体の特徴を以下に述べる。ただし、この特徴の記載は、本発明を説明するためのものであって、本発明を何ら限定するものではない。 As a method for introducing a gene into a cell according to the present invention, a method using a virus, a method using calcium phosphate, a polycation, a cationic lipid or an aggregate thereof, an electroporation method, a microinjection method and the like can be used. In the examples, the same DNA complex as described in Patent Document 7 was used. The characteristics of the composite are described below. However, the description of this feature is for explaining the present invention and does not limit the present invention.
培養細胞に人工のベクター（ポリカチオン、カチオン性脂質など）を用いて遺伝子導入しても、その遺伝子発現期間は短く、通常３〜４日後には発現が消えてしまう。また、発現を高めようと、多くのＤＮＡ複合体を細胞に加えると、その毒性のために細胞は弱り、一両日中に死んでしまう。効率よくデンジャー・シグナルを担持したエクソソームを調製するためには、細胞が導入した遺伝子を発現しながら長期間エクソソームを分泌する条件が必要である。我々は、以前生体内で高発現する遺伝子導入システムを得るために、通常のＤＮＡ／ポリカチオン（またはカチオン性脂質）複合体ではなく、これにさらにポリアニオンを加えたＤＮＡ三元複合体システムを開発し、様々な効果を上げてきた（特許文献１）。このＤＮＡ三元複合体システムは遺伝子発現が比較的長期間持続すること（非特許文献７）、また、細胞毒性が低いこと（非特許文献８）から、エクソソームの効率良い調製に適している。 Even if a gene is introduced into a cultured cell using an artificial vector (polycation, cationic lipid, etc.), the gene expression period is short, and the expression usually disappears after 3 to 4 days. Also, when many DNA complexes are added to cells to increase expression, the cells become weak due to their toxicity and die within a day or two. In order to efficiently prepare exosomes carrying a danger signal, conditions for secreting exosomes for a long period of time while expressing genes introduced by cells are necessary. We have developed a DNA ternary complex system in which a polyanion is added to the conventional DNA / polycation (or cationic lipid) complex to obtain a gene transfer system that is highly expressed in vivo. However, various effects have been achieved (Patent Document 1). This DNA ternary complex system is suitable for efficient preparation of exosomes because gene expression lasts for a relatively long time (Non-patent Document 7) and low cytotoxicity (Non-patent Document 8).
様々な手法で遺伝子導入した細胞を数日間培養し、その上澄みから超遠心分離法、エクソソーム単離試薬など、通常の方法によって、目的のデンジャー・シグナルを担持したエクソソームを容易に得ることができる Cells transfected with various methods can be cultured for several days, and exosomes carrying the desired danger signal can be easily obtained from the supernatant by ordinary methods such as ultracentrifugation and exosome isolation reagents.
得られたデンジャー・シグナルを担持したエクソソームは、注射用シリンジ、点滴用具などを用いて、生体に腫瘍内投与、皮下投与、静脈内投与、動脈内投与などにより容易に投与することができる。 The obtained exosome carrying a danger signal can be easily administered to a living body by intratumoral administration, subcutaneous administration, intravenous administration, intraarterial administration or the like using an injection syringe, an infusion device, or the like.
本発明を、実施例により更に具体的に説明する。なお、これらの実施例は、本発明を説明するためのものであって、本発明を何ら限定するものではない。 The present invention will be described more specifically with reference to examples. In addition, these Examples are for demonstrating this invention, Comprising: This invention is not limited at all.
結核菌の抗原タンパクであるＥａｒｌｙ Ｓｅｃｒｅｔｏｒｙ Ａｎｔｉｇｅｎｉｃ Ｔａｒｇｅｔ−６（ＥＳＡＴ−６）の遺伝子を導入したＢ１６メラノーマ細胞からのエクソソーム（ＥＳ−Ｂ１６−Ｅｘ）および遺伝子導入していないＢ１６メラノーマ細胞からのコントロールの空エクソソーム（Ｅｍｐｔｙ−Ｂ１６−Ｅｘ）の単離
ＥＳＡＴ−６プラスミド、およびＰＥＩ″Ｍａｘ″は、非特許文献８に記載と同じものを用いた。ＨＡは、生化学工業株式会社の「トリトサカ由来」のヒアルロン酸を用いた。ＰＢＳはＲｏｍａｎ Ｉｎｄｕｓｔｒｉｅｓ社製のＰｈｏｓｐｈａｔｅ Ｂｕｆｆｅｒｅｄ Ｓａｌｔｓ（Ｔａｂｌｅｔ）を蒸留したイオン交換水に溶解したものを用いた。以降の実施例でも同様である。 Control sky from exosomes (ES-B16-Ex) from B16 melanoma cells into which the gene for Early Secret Antigen Target-6 (ESAT-6), an antigenic protein of Mycobacterium tuberculosis, was introduced, and B16 melanoma cells from which no gene was introduced Isolation of Exosomes (Empty-B16-Ex) The same ESAT-6 plasmid and PEI “Max” as described in Non-Patent Document 8 were used. As the HA, hyaluronic acid “derived from Tritosaka” manufactured by Seikagaku Corporation was used. The PBS used was a solution of Phosphate Buffered Salts (Tablet) manufactured by Roman Industries in distilled ion-exchanged water. The same applies to the following embodiments.
操作手順
▲１▼ 遺伝子を導入する３日前に、２４穴マルチプレートに１ウェルあたり１．０×１０４個のＢ１６メラノーマ細胞をまき、１０％ ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍ（ＦＢＳ）、ペニシリン（１００ｕｎｉｔ／ｍＬ）、およびストレプトマイシン（０．１ｍｇ／ｍＬ）を含むＥａｇｌｅ’ｓ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（ＥＭＥＭ）培地を用いて３晩インキュベートした。
▲２▼ 培養した培地を取り除き、１０％エクソソーム・フリーＦＢＳ、ペニシリン（１００ｕｎｉｔ／ｍＬ）、およびストレプトマイシン（０．１ｍｇ／ｍＬ）を含むＥＭＥＭ培地５００μｌをウェルに入れた。
▲３▼ ＥＳＡＴ−６プラスミド１．５μｇを含む水溶液４．８４μｌを１．７８μｌのＣＳ溶液（５ｍｇ／ｍｌ）とＤＮＡ／ＣＳ比（電荷モル比）が８となるように混合した後、０．８８μｌのＰＥＩ″Ｍａｘ″水溶液（５ｍｇ／ｍｌ）とＤＮＡ／ＰＥＩ″Ｍａｘ″比（電荷モル比）が１２となるように混合し、２０分後に２倍濃縮ＰＢＳを７．５μｌ加えた。。
▲４▼ ▲３▼で調製したＤＮＡ複合体懸濁液１５μＬをウェルに加えた（ＥＳＡＴ−６プラスミド１．５μｇ／ウェル）。
▲５▼ ３７℃、５％ＣＯ２−９５％ ａｉｒ下で５時間インキュベートした。
▲６▼ 培地を新しい１０％エクソソーム・フリーＦＢＳと２５Ｕのペニシリンと２５μｇのストレプトマイシンを含むＥＭＥＭ １ｍｌと取り換え、３７℃で７２時間インキュベートした。
▲７▼ ７２時間のインキュベート後、ウェル中の上澄みを回収した。
▲８▼ 遠心分離（３０００ｘ ｇ、１５分）した後、上清を採取し、体積にして半分量のＴｏｔａｌ Ｅｘｏｓｏｍｅ Ｉｓｏｌａｔｉｏｎ（ｆｒｏｍ ｃｅｌｌ ｃｕｌｔｕｒｅ ｍｅｄｉａ）（Ｔｈｅ Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ，Ｉｎｃ．Ｃｏｒｐｏｒａｔｅ（Ｗａｌｔｈａｍ，ＭＡ，ＵＳＡ）．）を加え、一晩４℃で静置した。
▲９▼ 遠心分離（１００００ｘ ｇ、１時間）した後、沈殿したエクソソームを０．１６ｍｌのＰＢＳに懸濁した。エクソソーム懸濁液は、４℃で保管、一週間以内に使用した。
▲１０▼ 同様の実験を遺伝子導入していないＢ１６細胞に対しても行い、コントロールの空エクソソーム（Ｅｍｐｔｙ−Ｂ１６−Ｅｘ）懸濁液を作成した。Operating procedure {circle around (1)} Three days before gene introduction, 1.0 × 10 4 B16 melanoma cells are seeded per well in a 24-well multiplate, 10% fetal bovine serum (FBS), penicillin (100 units / mL) And Eagle's Minimum Essential Medium (EMEM) medium containing streptomycin (0.1 mg / mL).
(2) The cultured medium was removed, and 500 μl of EMEM medium containing 10% exosome-free FBS, penicillin (100 units / mL), and streptomycin (0.1 mg / mL) was added to the wells.
(3) 4.84 μl of an aqueous solution containing 1.5 μg of ESAT-6 plasmid was mixed with 1.78 μl of CS solution (5 mg / ml) so that the DNA / CS ratio (charge molar ratio) was 8, 88 μl of PEI “Max” aqueous solution (5 mg / ml) was mixed with DNA / PEI “Max” ratio (charge molar ratio) to 12, and after 20 minutes, 7.5 μl of 2-fold concentrated PBS was added. .
(4) 15 μL of the DNA complex suspension prepared in (3) was added to the well (1.5 μg / well of ESAT-6 plasmid).
(5) The plate was incubated at 37 ° C. and 5% CO 2 -95% air for 5 hours.
(6) The medium was replaced with 1 ml of EMEM containing fresh 10% exosome-free FBS, 25 U penicillin and 25 μg streptomycin, and incubated at 37 ° C. for 72 hours.
(7) After 72 hours of incubation, the supernatant in the well was collected.
(8) After centrifugation (3000 × g, 15 minutes), the supernatant was collected, and half the volume of Total Exosome Isolation (from cell cell culture media) (The Thermo Fisher Scientific, Inc., Corporate, Mass.). USA).) Was added and left at 4 ° C. overnight.
(9) After centrifugation (10000 × g, 1 hour), the precipitated exosome was suspended in 0.16 ml of PBS. The exosome suspension was stored at 4 ° C. and used within a week.
(10) A similar experiment was performed on B16 cells into which no gene had been introduced, and a control empty exosome (Empty-B16-Ex) suspension was prepared.
結果
遺伝子導入したもの、していないものともに、Ｅｘｏｓｏｍｅ Ｉｓｏｌａｔｉｏｎ （ｆｒｏｍ ｃｅｌｌ ｃｕｌｔｕｒｅ ｍｅｄｉａ）と混合し、一晩後遠心分離することによって、白色のエクソソームの沈殿が得られた。再懸濁した液中のエクソソームをＥｘｏ−ｇｒｅｅｎ または Ｅｘｏ−ｒｅｄ （ともにＳｙｓｔｅｍ Ｂｉｏｓｃｉｅｎｃｅｓ （Ｓａｎ Ｆｒａｎｃｉｓｃｏ，Ｉｎｃ．，ＣＡ，ＵＳＡ））で染色し、ＥｘｏＱｕｉｃｋ−ＴＣ （ＳｙｓｔｅｍＢｉｏｓｃｉｅｎｃｅｓ（Ｓａｎ Ｆｒａｎｃｉｓｃｏ，Ｉｎｃ．，ＣＡ，ＵＳＡ））で単離し、蛍光顕微鏡で観察したところ、直径数十ｎｍの輝点が浮遊しているのが同じ位置に観察され、確かにエクソソーム懸濁液が得られたことが確認された。Results Both exogenous and non-transfected genes were mixed with Exosome Isolation (from cell cell media) and centrifuged overnight to obtain a white exosome precipitate. The exosomes in the resuspended solution are stained with Exo-green or Exo-red (both System Biosciences (San Francisco, Inc., CA, USA)), and ExoQuick-TC (System Biosciences, San Francisco, Inc., San Francisco, Inc.). USA)) and observed with a fluorescence microscope, a bright spot with a diameter of several tens of nanometers was observed at the same position, and it was confirmed that an exosome suspension was obtained.
結核菌の抗原タンパクであるＭｙｃｏｂａｃｔｅｒｉｕｍ ｔｕｂｅｒｃｕｌｏｓｉｓ Ｍａｊｏｒ Ｓｅｃｒｅｔｅｄ ＰｒｏｔｅｉｎＡｎｔｉｇｅｎ８５Ｂ（Ａｇ８５Ｅ）の遺伝子を導入したＢ１６メラノーマ細胞からのエクソソーム（Ａｇ−Ｂ１６−Ｅｘ）の単離
実施例１と同様の手法で、Ｂ１６メラノーマ細胞にＡｇ８５Ｂの遺伝子を導入した後、その培養上澄からＡｇ８５Ｂ遺伝子を導入したＢ１６メラノーマ細胞由来のエクソソーム（Ａｇ−Ｂ１６−Ｅｘ）を単離した。 Isolation of exosomes (Ag-B16-Ex) from B16 melanoma cells into which the gene of Mycobacterium tuberculosis Major Major Secreted Protein Antigen 85B (Ag85E), which is an antigenic protein of Mycobacterium tuberculosis, was introduced in the same manner as in Example 1, and B16A 85 cells From the culture supernatant, B16 melanoma cell-derived exosome (Ag-B16-Ex) into which the Ag85B gene was introduced was isolated from the culture supernatant.
アデノウィルスの抗原タンパクであるＡｄｅｎｏｖｉｒｕｓ ｄｅａｔｈ ｐｒｏｔｅｉｎ（ＡＤＰ）の遺伝子を導入したＢ１６メラノーマ細胞からのエクソソーム（ＡＤ−Ｂ１６−Ｅｘ）の単離
実施例１と同様の手法で、Ｂ１６メラノーマ細胞にＡＤＰの遺伝子を導入した後、その培養上澄みからＡＤＰ遺伝子を導入したＢ１６メラノーマ細胞由来のエクソソーム（ＡＤ−Ｂ１６−Ｅｘ）を単離した。 Isolation of exosomes (AD-B16-Ex) from B16 melanoma cells into which an adenovirus antigen protein (ADP) gene, which is an adenovirus antigen protein, was introduced In the same manner as in Example 1, the ADP gene was transferred to B16 melanoma cells. After the introduction, B16 melanoma cell-derived exosomes (AD-B16-Ex) into which the ADP gene was introduced were isolated from the culture supernatant.
結核菌の抗原タンパクであるｅａｒｌｙ ｓｅｃｒｅｔｏｒｙ ａｎｔｉｇｅｎｉｃ ６ ｋＤａ（ＥＳＡＴ−６）の遺伝子を導入したＣＨＯ（Ｃｈｉｎｅｓｅ Ｈａｍｓｔｅｒ Ｏｖａｒｙ）細胞からのエクソソーム（ＥＳ−ＣＨＯ−Ｅｘ）の単離
実施例１と同様の手法で、ＣＨＯ細胞にＥＳＡＴ−６の遺伝子を導入した後、その培養上澄みからＥＳＡＴ−６遺伝子を導入したＣＨＯ細胞由来のエクソソーム（ＥＳ−ＣＨＯ−Ｅｘ）、および遺伝子導入していないＣＨＯ細胞からのエクソソーム（Ｅｍｐｔｙ−ＣＨＯ−Ｅｘ）を単離した。 Isolation of exosome (ES-CHO-Ex) from CHO (Chinese Hamster Ovary) cell into which early secretory antigenic 6 kDa (ESAT-6) gene, which is an antigenic protein of Mycobacterium tuberculosis, was introduced In the same manner as in Example 1. , CHO cells derived from CHO cells into which ESAT-6 gene has been introduced from the culture supernatant after introduction of ESAT-6 gene into CHO cells (ES-CHO-Ex), and exosomes from CHO cells into which no gene has been introduced (ES-CHO-Ex) (Empty-CHO-Ex) was isolated.
デンジャーシグナル遺伝子を導入した細胞から分泌したエクソソームの抗腫瘍効果（１）
▲１▼ Ｃ５７ＢＬ／６マウス（オス、５週齢）の皮下にＢ１６細胞を移植し、坦癌モデルマウスを作成した。
▲２▼ 腫瘍の直径が５ｍｍを越えたところで、実施例１〜３で得られた各エクソソーム懸濁液（２００μＬ）を腫瘍内に一回投与し、その後の腫瘍のサイズの変化を調べた。 Antitumor effects of exosomes secreted from cells introduced with a danger signal gene (1)
(1) B16 cells were transplanted subcutaneously into C57BL / 6 mice (male, 5 weeks old) to prepare a cancer model mouse.
{Circle around (2)} When the diameter of the tumor exceeded 5 mm, each exosome suspension (200 μL) obtained in Examples 1 to 3 was administered once into the tumor, and the subsequent change in the size of the tumor was examined.
結果
結果を図１に示す。ここで、図中の値は３例以上の平均値を表している。微生物遺伝子を導入していないＢ１６細胞から得られたエクソソーム、Ｅｍｐｔｙ−Ｂ１６−Ｅｘはほとんど抗腫瘍効果を示さなかったのに対し、微生物遺伝子を導入したＢ１６細胞から得られたエクソソーム、ＡＤ−Ｂ１６−Ｅｘ、Ａｇ−Ｂ１６−Ｅｘ、ＥＳ−Ｂ１６−Ｅｘは明らかな抗腫瘍効果を示した。Results The results are shown in FIG. Here, the value in the figure represents an average value of three or more cases. The exosome obtained from B16 cells not transfected with the microbial gene, Empty-B16-Ex, showed little antitumor effect, whereas the exosome obtained from B16 cells introduced with the microbial gene, AD-B16- Ex, Ag-B16-Ex, ES-B16-Ex showed a clear antitumor effect.
デンジャーシグナル遺伝子を導入した細胞から分泌したエクソソームの抗腫瘍効果（２）
▲１▼ Ｃ５７ＢＬ／６マウス（オス、５週齢）の皮下にＢ１６細胞を移植し、坦癌モデルマウスを作成した。
▲２▼ 腫瘍の直径が５ｍｍを越えたところで、実施例４で得られた各エクソソーム懸濁液（２００μＬ）を腫瘍内に一回投与し、その後の腫瘍のサイズの変化を調べた。 Anti-tumor effect of exosome secreted from cells introduced with a danger signal gene (2)
(1) B16 cells were transplanted subcutaneously into C57BL / 6 mice (male, 5 weeks old) to prepare a cancer model mouse.
(2) When the diameter of the tumor exceeded 5 mm, each exosome suspension (200 μL) obtained in Example 4 was administered once into the tumor, and the subsequent change in the size of the tumor was examined.
［結果］
結果を図２に示す。ここで、図中の値は３例以上の平均値を表している。微生物遺伝子を導入していないＣＨＯ細胞から得られたエクソソーム、Ｅｍｐｔｙ−ＣＨＯ−Ｅｘはほとんど抗腫瘍効果を示さなかったのに対し、ＥＳＡＴ−６遺伝子を導入したＣＨＯ細胞から得られたエクソソーム、ＥＳ−ＣＨＯ−Ｅｘは明らかな抗腫瘍効果を示し、異種の腫瘍に対しても効果があること、また他家のエクソソームも抗腫瘍効果を導くことが確認された。[result]
The results are shown in FIG. Here, the value in the figure represents an average value of three or more cases. Exosome obtained from CHO cells into which microbial genes have not been introduced, Empty-CHO-Ex, showed little antitumor effect, whereas exosome obtained from CHO cells into which ESAT-6 gene was introduced, ES- It was confirmed that CHO-Ex has a clear antitumor effect, is effective against different types of tumors, and that exosomes from other families also induce an antitumor effect.
ＥＳＡＴ−６の遺伝子を導入した細胞から分泌したエクソソームの細胞性免疫導入効果（１）
▲１▼ 実施例１で得られたエクソソーム懸濁液をＣ５７ＢＬ／６マウス（オス、５週齢）の両後足の足底に７５μＬずつ一回投与した。
▲２▼ ９日目に両後足の膝裏のリンパ節を採取し、つぶしてメッシュを通した後、ＲＰＭＩ１６４０培地に懸濁させた。
▲３▼ 得られたリンパ球懸濁液をマイトマイシン処理したＢ１６メラノーマ細胞とともに９６穴培養プレートに加え、３７℃で２日間インキュベート後、分泌されたＩＦＮ−ＹをＭｏｕｓｅ ＩＦＮ−ｇａｍｍａ Ｑｕａｎｔｉｋｉｎｅ ＥＬＩＳＡ Ｋｉｔ（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ，Ｉｎｃ．（Ｍｉｎｎｅａｐｏｌｉｓ，ＭＮ，ＵＳＡ））で定量した。 Cellular immunity transduction effect of exosome secreted from cells introduced with ESAT-6 gene (1)
(1) The exosome suspension obtained in Example 1 was administered once by 75 μL to the soles of both hind paws of C57BL / 6 mice (male, 5 weeks old).
(2) On the ninth day, lymph nodes on the back of knees of both hind paws were collected, crushed and passed through a mesh, and suspended in RPMI 1640 medium.
▲ 3 ▼ resulting lymphocyte suspension was added to 96-well culture plate with B16 melanoma cells mitomycin treated, after incubation for 2 days at 37 ° C., the secreted IFN- Y Mouse IFN-gamma Quantikine ELISA Kit (R & D Quantified by Systems, Inc. (Minneapolis, MN, USA).
［結果］
結果を図３に示す。ここで、図中の値は３例以上の平均値を表している。ＥＳＡＴ−６遺伝子を導入したＢ１６細胞から得られたエクソソーム、ＥＳ−Ｂ１６−Ｅｘを投与したマウスのリンパ球は、ＥＳＡＴ−６遺伝子を導入していないＢ１６細胞から得られたエクソソーム、Ｅｍｐｔｙ−Ｂ１６−Ｅｘを投与したマウスのリンパ球に比べて、Ｂ１６細胞と２日間インキュベートすることで明らかに多くのＩＦＮ−Ｙを分泌した。このことから、微生物遺伝子を導入した腫瘍細胞から得られたエクソソームは腫瘍細胞に対する細胞性免疫を惹起する効果を持つことが確認された。[result]
The results are shown in FIG. Here, the value in the figure represents an average value of three or more cases. Exosome obtained from B16 cell introduced with ESAT-6 gene, lymphocyte of mouse administered with ES-B16-Ex, exosome obtained from B16 cell not introduced with ESAT-6 gene, Empty-B16- Compared to the lymphocytes of mice administered with Ex, incubation with B16 cells for 2 days clearly secreted more IFN- Y . From this, it was confirmed that exosomes obtained from tumor cells into which a microbial gene was introduced had an effect of inducing cellular immunity against tumor cells.
ＥＳＡＴ−６の遺伝子を導入した細胞から分泌したエクソソームの細胞性免疫導入効果（２）
▲１▼ 実施例１で得られたエクソソーム懸濁液をＣ５７ＢＬ／６マウス（オス、５週齢）の両後足の足底に７５μＬずつ一回投与した。
▲２▼ ９日目に両後足の膝裏のリンパ節を採取し、つぶしてメッシュを通した後、ＲＰＭＩ１６４０培地に懸濁させた。
▲３▼ 得られたリンパ球懸濁液を１μｇのＥＳＡＴ−６を含むＱｕａｎｔｉＦＥＲＯＮ−ＴＢ Ｇｏｌｄ ｂｌｏｏｄの検査管に加え、３７℃で一晩インキュベート後、分泌されたＩＦＮ−ＹをＭｏｕｓｅ ＩＦＮ−ｇａｍｍａ Ｑｕａｎｔｉｋｉｎｅ ＥＬＩＳＡ Ｋｉｔ（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ，Ｉｎｃ．（Ｍｉｎｎｅａｐｏｌｉｓ，ＭＮ，ＵＳＡ））で定量した。 Cellular immunity transduction effect of exosome secreted from cells introduced with ESAT-6 gene (2)
(1) The exosome suspension obtained in Example 1 was administered once by 75 μL to the soles of both hind paws of C57BL / 6 mice (male, 5 weeks old).
(2) On the ninth day, lymph nodes on the back of knees of both hind paws were collected, crushed and passed through a mesh, and suspended in RPMI 1640 medium.
(3) The obtained lymphocyte suspension was added to a Quantferon-TB Gold blood test tube containing 1 μg of ESAT-6, incubated at 37 ° C. overnight, and then secreted IFN- Y was added to Mouse IFN-gamma Quantikine. Quantification was performed by ELISA Kit (R & D Systems, Inc. (Minneapolis, MN, USA)).
［結果］
結果を図４に示す。ここで、図中の値は３例以上の平均値を表している。ＥＳＡＴ−６遺伝子を導入したＢ１６細胞から得られたエクソソーム、ＥＳ−Ｂ１６−Ｅｘを投与したマウスのリンパ球は、ＥＳＡＴ−６遺伝子を導入していないＢ１６細胞から得られたエクソソーム、Ｅｍｐｔｙ−Ｂ１６−Ｅｘを投与したマウスのリンパ球に比べて、ＥＳＡＴ−６タンパクと一晩インキュベートすることで明らかに多くのＩＦＮ−Ｙを分泌した。このことから、微生物遺伝子を導入した細胞から得られたエクソソームは、当該微生物に対する細胞性免疫を惹起する効果を持つことが確認された。[result]
The results are shown in FIG. Here, the value in the figure represents an average value of three or more cases. Exosome obtained from B16 cell introduced with ESAT-6 gene, lymphocyte of mouse administered with ES-B16-Ex, exosome obtained from B16 cell not introduced with ESAT-6 gene, Empty-B16- Compared to the lymphocytes of mice administered with Ex, incubation with ESAT-6 protein overnight secreted more IFN- Y . From this, it was confirmed that the exosome obtained from the cell into which the microbial gene was introduced has an effect of inducing cellular immunity against the microorganism.
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