JP7430914B2 - Pharmaceutical composition for the treatment of cancer - Google Patents
Pharmaceutical composition for the treatment of cancer Download PDFInfo
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- JP7430914B2 JP7430914B2 JP2020539476A JP2020539476A JP7430914B2 JP 7430914 B2 JP7430914 B2 JP 7430914B2 JP 2020539476 A JP2020539476 A JP 2020539476A JP 2020539476 A JP2020539476 A JP 2020539476A JP 7430914 B2 JP7430914 B2 JP 7430914B2
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Description
本発明は、がんの治療のための医薬組成物等に関する。 TECHNICAL FIELD The present invention relates to pharmaceutical compositions and the like for the treatment of cancer.
がんの主な治療には外科治療、薬物療法および放射線治療がある。薬物治療のなかでもがん細胞に特異的な分子を標的とする分子標的治療が注目されている。最近になって、がん細胞に特異的に発現するデスレセプター（death receptor）を標的としたがん治療の研究が進んでいる。デスレセプターを刺激することにより、がん細胞のアポトーシスを誘導することができる。デスレセプターのリガンドとして腫瘍壊死因子刺激因子１０（ＴＮＦＳＦ１０）（ＴＲＡＩＬともいう）が知られている（非特許文献１等参照）。このサイトカインを癌治療に用いるための研究が行われている。しかしながら、ＴＲＡＩＬは血中での分解が早く、がん細胞を効果的に死滅させることができないという問題がある。
The main treatments for cancer include surgery, drug therapy, and radiation therapy. Among drug treatments, molecular target therapy that targets molecules specific to cancer cells is attracting attention. Recently, research has been progressing on cancer treatments that target death receptors, which are specifically expressed in cancer cells. Apoptosis of cancer cells can be induced by stimulating death receptors. Tumor necrosis factor stimulating factor 10 (TNFSF10) (also referred to as TRAIL) is known as a death receptor ligand (see
ＴＲＡＩＬが血中で早く分解されるという問題を解決し、ＴＲＡＩＬを用いてがん細胞を効果的に死滅させることが必要であった。 It was necessary to solve the problem that TRAIL is rapidly degraded in the blood and to effectively kill cancer cells using TRAIL.
本発明者らは、上記課題を解決するために鋭意研究を重ね、生理活性ポリペプチド、リポ多糖（ＬＰＳ）、二本鎖ＲＮＡ（ｄｓＲＮＡ）またはポリイノシン酸－ポリシチジル酸（ｐｏｌｙ（Ｉ：Ｃ））で処理された間葉系組織由来の接着性細胞がＴＲＡＩＬを産生することを見出し、本発明を完成させるに至った。 The present inventors have conducted extensive research to solve the above problems, and have developed bioactive polypeptides, lipopolysaccharide (LPS), double-stranded RNA (dsRNA), or polyinosinic acid-polycytidylic acid (poly(I:C)). It was discovered that adherent cells derived from mesenchymal tissue treated with TRAIL produced TRAIL, and the present invention was completed.
すなわち本発明は以下のものを提供する。
（１）生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された、腫瘍壊死因子刺激因子１０（ＴＲＡＩＬ）を産生する間葉系組織由来の接着性細胞および医薬上許容される担体および／または賦形剤を含む、がんの治療のための医薬組成物。
（２）生理活性ポリペプチドが、インターフェロンアルファ（ＩＦＮα）、インターフェロンベータ（ＩＦＮβ）、インターフェロンガンマ（ＩＦＮγ）、腫瘍壊死因子アルファ（ＴＮＦα）、腫瘍壊死因子ベータ（ＴＮＦβ）、インターロイキン１α（ＩＬ－１α）、インターロイキン１ベータ（ＩＬ－１β）およびオンコスタチンＭからなる群より選択される１種またはそれ以上のポリペプチドである、（１）記載の医薬組成物。
（３）生理活性ポリペプチドが、インターフェロンベータ（ＩＦＮβ）、インターフェロンガンマ（ＩＦＮγ）および腫瘍壊死因子アルファ（ＴＮＦα）からなる群より選択される１種またはそれ以上のポリペプチドである、（１）記載の医薬組成物。
（４）間葉系組織由来の接着性細胞が、間葉系組織由来幹細胞（ＭＳＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、Ｍｕｓｅ細胞、骨髄組織、胞衣組織、軟骨組織、骨膜組織、滑膜組織、骨格筋組織に由来する細胞、幹細胞および間質細胞、ならびに経血細胞からなる群より選択される１種またはそれ以上の細胞である、（１）～（３）のいずれか記載の医薬組成物。
（５）間葉系組織由来の接着性細胞が、ＡＤＳＣ、ＡＤＭＰＣ、胞衣組織に由来する細胞、骨髄組織に由来する細胞、および滑膜組織に由来する細胞からなる群より選択される１種またはそれ以上の細胞である、（１）～（３）のいずれか記載の医薬組成物。
（６）がんの治療のための医薬組成物の製造方法であって、下記工程：
（ａ）間葉系組織由来の接着性細胞を生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理し、次いで、
（ｂ）工程（ａ）にて処理された、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞を医薬上許容される担体および／または賦形剤と混合する
を含む方法。
（７）生理活性ポリペプチドが、インターフェロンアルファ（ＩＦＮα）、インターフェロンベータ（ＩＦＮβ）、インターフェロンガンマ（ＩＦＮγ）、腫瘍壊死因子アルファ（ＴＮＦα）、腫瘍壊死因子ベータ（ＴＮＦβ）、インターロイキン１α（ＩＬ－１α）、インターロイキン１ベータ（ＩＬ－１β）およびオンコスタチンＭからなる群より選択される１種またはそれ以上のポリペプチドである、（６）記載の方法。
（８）生理活性ポリペプチドが、インターフェロンベータ（ＩＦＮβ）、インターフェロンガンマ（ＩＦＮγ）および腫瘍壊死因子アルファ（ＴＮＦα）からなる群より選択される１種またはそれ以上のポリペプチドである、（６）記載の方法。
（９）間葉系組織由来の接着性細胞が、間葉系組織由来幹細胞（ＭＳＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、Ｍｕｓｅ細胞、骨髄組織、胞衣組織、軟骨組織、骨膜組織、滑膜組織、骨格筋組織に由来する細胞、幹細胞および間質細胞、ならびに経血細胞からなる群より選択される１種またはそれ以上の細胞である、（６）～（８）のいずれか記載の方法。
（１０）間葉系組織由来の接着性細胞が、ＡＤＳＣ、ＡＤＭＰＣ、胞衣組織に由来する細胞、骨髄組織に由来する細胞、および滑膜組織に由来する細胞からなる群より選択される１種またはそれ以上の細胞である、（６）～（８）のいずれか記載の方法。
（１１）（１）～（５）のいずれか記載の医薬組成物の有効性を調べるための方法であって、がん細胞に（１）～（５）のいずれか記載の医薬組成物を添加し、がん細胞の生存率または死滅率を調べることを含む方法。
（１２）（１）～（５）のいずれか記載の医薬組成物を用いるがんの治療の有効性を予測する方法であって、がん細胞がデスレセプターを有しているかどうかを調べること、および／またはデスレセプターの量を調べることを含む方法。
（１３）生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）および医薬上許容される担体または賦形剤を含むがんの治療のための医薬組成物であって、がん関連線維芽細胞（ＣＡＦ）に投与される医薬組成物。
（１４）生理活性ポリペプチドが、インターフェロンアルファ（ＩＦＮα）、インターフェロンベータ（ＩＦＮβ）、インターフェロンガンマ（ＩＦＮγ）、腫瘍壊死因子アルファ（ＴＮＦα）、腫瘍壊死因子ベータ（ＴＮＦβ）、インターロイキン１α（ＩＬ－１α）、インターロイキン１ベータ（ＩＬ－１β）およびオンコスタチンＭからなる群より選択される１種またはそれ以上のポリペプチドである、（１３）記載の医薬組成物。
（１５）生理活性ポリペプチドが、インターフェロンベータ（ＩＦＮβ）、インターフェロンガンマ（ＩＦＮγ）および腫瘍壊死因子アルファ（ＴＮＦα）からなる群より選択される１種またはそれ以上のポリペプチドである、（１３）記載の医薬組成物。
（１６）間葉系組織由来の接着性細胞を生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理することを含む、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞の製造方法。
（１７）生理活性ポリペプチドが、インターフェロンアルファ（ＩＦＮα）、インターフェロンベータ（ＩＦＮβ）、インターフェロンガンマ（ＩＦＮγ）、腫瘍壊死因子アルファ（ＴＮＦα）、腫瘍壊死因子ベータ（ＴＮＦβ）、インターロイキン１α（ＩＬ－１α）、インターロイキン１ベータ（ＩＬ－１β）およびオンコスタチンＭからなる群より選択される１種またはそれ以上のポリペプチドである、（１６）記載の方法。
（１８）生理活性ポリペプチドが、ＩＦＮβ、ＩＦＮγおよびＴＮＦαからなる群より選択される１種またはそれ以上のポリペプチドである、（１６）記載の方法。
（１９）間葉系組織由来の接着性細胞が、間葉系組織由来幹細胞（ＭＳＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、Ｍｕｓｅ細胞、骨髄組織、胞衣組織、軟骨組織、骨膜組織、滑膜組織、骨格筋組織に由来する細胞、幹細胞および間質細胞、ならびに経血細胞からなる群より選択される１種またはそれ以上の細胞である、（１６）～（１８）のいずれか記載の方法。
（２０）間葉系組織由来の接着性細胞が、ＡＤＳＣ、ＡＤＭＰＣ、胞衣組織に由来する細胞、骨髄組織に由来する細胞、および滑膜組織に由来する細胞からなる群より選択される１種またはそれ以上の細胞である、（１６）～（１８）のいずれか記載の方法。
That is, the present invention provides the following.
(1) Adhesive cells derived from mesenchymal tissue producing tumor necrosis factor-stimulating factor 10 (TRAIL) treated with bioactive polypeptide, LPS, dsRNA or poly(I:C) and pharmaceutically acceptable A pharmaceutical composition for the treatment of cancer, comprising a carrier and/or excipient.
(2) The physiologically active polypeptide is interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), interleukin 1α (IL-1α). ),
(3) The description in (1), wherein the physiologically active polypeptide is one or more polypeptides selected from the group consisting of interferon beta (IFNβ), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα). Pharmaceutical composition.
(4) Adhesive cells derived from mesenchymal tissues include mesenchymal tissue-derived stem cells (MSCs), adipose tissue-derived stem cells (ADSCs), adipose tissue-derived multilineage progenitor cells (ADMPCs), Muse cells, bone marrow tissues, and cysts. One or more cells selected from the group consisting of cells derived from tissue, cartilage tissue, periosteal tissue, synovial tissue, skeletal muscle tissue, stem cells and stromal cells, and menstrual blood cells, (1) The pharmaceutical composition according to any one of (3).
(5) The adhesive cell derived from mesenchymal tissue is one type selected from the group consisting of ADSC, ADMPC, cells derived from alveolar tissue, cells derived from bone marrow tissue, and cells derived from synovial tissue, or The pharmaceutical composition according to any one of (1) to (3), which is a cell larger than that.
(6) A method for producing a pharmaceutical composition for the treatment of cancer, comprising the following steps:
(a) Adherent cells derived from mesenchymal tissue are treated with bioactive polypeptide, LPS, dsRNA or poly(I:C), and then
(b) A method comprising mixing the TRAIL-producing mesenchymal tissue-derived adherent cells treated in step (a) with a pharmaceutically acceptable carrier and/or excipient.
(7) The physiologically active polypeptide is interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), interleukin 1α (IL-1α). ),
(8) The description in (6), wherein the physiologically active polypeptide is one or more polypeptides selected from the group consisting of interferon beta (IFNβ), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα). the method of.
(9) Adhesive cells derived from mesenchymal tissue include mesenchymal tissue-derived stem cells (MSCs), adipose tissue-derived stem cells (ADSCs), adipose tissue-derived multilineage progenitor cells (ADMPCs), Muse cells, bone marrow tissues, and cysts. One or more cells selected from the group consisting of cells derived from tissue, cartilage tissue, periosteal tissue, synovial tissue, skeletal muscle tissue, stem cells and stromal cells, and menstrual blood cells, (6) ~ (8) The method described in any of the above.
(10) The adhesive cell derived from mesenchymal tissue is one type selected from the group consisting of ADSC, ADMPC, cells derived from alveolar tissue, cells derived from bone marrow tissue, and cells derived from synovial tissue, or The method according to any one of (6) to (8), wherein the cells are larger than that.
(11) A method for examining the effectiveness of the pharmaceutical composition according to any one of (1) to (5), comprising administering the pharmaceutical composition according to any one of (1) to (5) to cancer cells. and determining the survival rate or mortality rate of cancer cells.
(12) A method for predicting the effectiveness of cancer treatment using the pharmaceutical composition according to any one of (1) to (5), comprising examining whether cancer cells have death receptors. , and/or determining the amount of death receptors.
(13) A pharmaceutical composition for the treatment of cancer, comprising a bioactive polypeptide, LPS, dsRNA or poly(I:C) and a pharmaceutically acceptable carrier or excipient, the composition comprising: A pharmaceutical composition administered to cells (CAF).
(14) The physiologically active polypeptide is interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), interleukin 1α (IL-1α). ),
(15) The description in (13), wherein the physiologically active polypeptide is one or more polypeptides selected from the group consisting of interferon beta (IFNβ), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα). Pharmaceutical composition.
(16) Production of mesenchymal tissue-derived adherent cells that produce TRAIL, which comprises treating the mesenchymal tissue-derived adherent cells with a physiologically active polypeptide, LPS, dsRNA, or poly(I:C). Method.
(17) The physiologically active polypeptide is interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), interleukin 1α (IL-1α). ),
(18) The method according to (16), wherein the physiologically active polypeptide is one or more polypeptides selected from the group consisting of IFNβ, IFNγ, and TNFα.
(19) Adhesive cells derived from mesenchymal tissue include mesenchymal tissue-derived stem cells (MSCs), adipose tissue-derived stem cells (ADSCs), adipose tissue-derived multilineage progenitor cells (ADMPCs), Muse cells, bone marrow tissues, and cysts. One or more cells selected from the group consisting of cells derived from tissue, cartilage tissue, periosteal tissue, synovial tissue, skeletal muscle tissue, stem cells and stromal cells, and menstrual blood cells, (16) The method according to any one of (18).
(20) The adhesive cell derived from mesenchymal tissue is one type selected from the group consisting of ADSC, ADMPC, cells derived from alveolar tissue, cells derived from bone marrow tissue, and cells derived from synovial tissue, or The method according to any one of (16) to (18), wherein the cell is larger than that.
ＴＲＡＩＬは、がん細胞上のデスレセプターに結合し、がん細胞のアポトーシスを誘発する。しかし、ＴＲＡＩＬは血中での分解が早く、がん細胞を効果的に死滅させることができないという問題がある。そこで、本発明においては、生理活性ポリペプチドで処理された、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞を用いる。かかる細胞を用いると、ＴＲＡＩＬが患者体内で分解されても上記細胞から補充されるので、がん細胞を効果的に死滅させることができ、高い治療効果が得られる。 TRAIL binds to death receptors on cancer cells and induces cancer cell apoptosis. However, TRAIL has the problem that it degrades quickly in the blood and cannot effectively kill cancer cells. Therefore, in the present invention, adherent cells derived from mesenchymal tissue that produce TRAIL and are treated with a physiologically active polypeptide are used. When such cells are used, even if TRAIL is degraded within the patient's body, it is replenished from the cells, so cancer cells can be effectively killed and a high therapeutic effect can be obtained.
本発明は、１の態様において、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞および医薬上許容される担体および／または賦形剤を含む、がんの治療のための医薬組成物を提供する。 In one aspect, the present invention provides TRAIL-producing mesenchymal tissue-derived adherent cells treated with a bioactive polypeptide, LPS, dsRNA or poly(I:C) and a pharmaceutically acceptable carrier and Provided are pharmaceutical compositions for the treatment of cancer, including/or excipients.
生理活性ポリペプチドは、生体の特定の生理的調節機能に対して作用するポリペプチドである。ポリペプチドは、２つ以上のアミノ酸残基がペプチド結合を介して結合した物質をいう。 A physiologically active polypeptide is a polypeptide that acts on a specific physiological regulatory function of an organism. A polypeptide refers to a substance in which two or more amino acid residues are linked via a peptide bond.
本発明で用いられる生理活性ポリペプチドは、間葉系組織由来の接着性細胞に作用して、そのＴＲＡＩＬの産生を増加させるもの、あるいはＴＲＡＩＬの産生が認められない間葉系組織由来の接着性細胞にＴＲＡＩＬを産生させるものであれば、いずれのポリペプチドであってもよい。本発明で用いられる好ましい生理活性ポリペプチドとしては、サイトカイン、ケモカイン、成長因子、ホルモン等が例示されるが、これらに限定されない。本発明において好ましく用いられる生理活性ポリペプチドはサイトカインである。好ましいサイトカインとしてはインターフェロン、腫瘍壊死因子、インターロイキン、オンコスタチンＭなどが例示される。より好ましいサイトカインとしてはインターフェロンアルファ（ＩＦＮα）、インターフェロンベータ（ＩＦＮβ）、インターフェロンガンマ（ＩＦＮγ）、腫瘍壊死因子アルファ（ＴＮＦα）、腫瘍壊死因子ベータ（ＴＮＦβ）、インターロイキン１α（ＩＬ－１α）、インターロイキン１ベータ（ＩＬ－１β）およびオンコスタチンＭなどが例示される。さらに好ましいサイトカインとしてはＩＦＮβ、ＩＦＮγおよびＴＮＦαなどが例示される。 The bioactive polypeptide used in the present invention is one that acts on adhesive cells derived from mesenchymal tissues and increases their production of TRAIL, or adhesive cells derived from mesenchymal tissues in which no TRAIL production is observed. Any polypeptide may be used as long as it causes cells to produce TRAIL. Preferred physiologically active polypeptides used in the present invention include, but are not limited to, cytokines, chemokines, growth factors, hormones, and the like. The physiologically active polypeptide preferably used in the present invention is a cytokine. Preferred cytokines include interferon, tumor necrosis factor, interleukin, oncostatin M, and the like. More preferred cytokines include interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), interleukin 1α (IL-1α), and interleukin. Examples include IL-1beta (IL-1β) and oncostatin M. More preferred cytokines include IFNβ, IFNγ, and TNFα.
本発明で用いられる生理活性ポリペプチドは、その変異体も包含する。本発明において、変異体は、元の生理活性ポリペプチドと同様の生物学的活性を有するものである。変異体は、元のポリペプチドと比較して、ポリペプチドを構成するアミノ酸残基が置換、欠失、挿入または付加されているポリペプチドであってもよい。置換、欠失、挿入または付加されるアミノ酸残基の数は特に限定されない。例えば、置換、欠失、挿入または付加されるアミノ酸残基は１個～数個であってもよい。また例えば、変異体ポリペプチドは、元のポリペプチドに対してアミノ酸配列同一性が８０％以上、好ましくは９０％以上、例えば９５％以上９７％以上または９９％以上のものであってもよい。さらに変異体は、ポリペプチドを構成するアミノ酸残基が修飾されているもの、あるいはβ－アラニンやＤ－アミノ酸のような非天然アミノ酸を含むものであってもよい。修飾はあらゆる種類の標識であってよい。メチル化、ハロゲン化、配糖体化などの化学修飾であってもよく、蛍光標識や放射性標識などの標識付加であってもよい。また、変異体は、一部のアミノ酸残基がペプチド結合以外によって結合されているものであってもよい。 The physiologically active polypeptide used in the present invention also includes variants thereof. In the present invention, a variant is one that has the same biological activity as the original bioactive polypeptide. A variant may be a polypeptide in which amino acid residues constituting the polypeptide have been substituted, deleted, inserted, or added compared to the original polypeptide. The number of amino acid residues to be substituted, deleted, inserted or added is not particularly limited. For example, one to several amino acid residues may be substituted, deleted, inserted, or added. For example, the variant polypeptide may have an amino acid sequence identity of 80% or more, preferably 90% or more, such as 95% or more, 97% or more, or 99% or more, with respect to the original polypeptide. Furthermore, the variants may be those in which the amino acid residues constituting the polypeptide are modified, or those containing unnatural amino acids such as β-alanine or D-amino acids. The modification may be a label of any kind. It may be a chemical modification such as methylation, halogenation, or glycosylation, or it may be addition of a label such as a fluorescent label or a radioactive label. Furthermore, the variant may have some amino acid residues bonded through a bond other than a peptide bond.
ＬＰＳは様々な種類のものが公知であり、いずれのＬＰＳを本発明に用いてもよい。 Various types of LPS are known, and any of them may be used in the present invention.
ｄｓＲＮＡは、二本の相補的な配列を持つＲＮＡ鎖が二重鎖を形成したものである。二本のＲＮＡ鎖の相補性は１００％でなくてもよい。ｄｓＲＮＡはアデニン、グアニン、シトシン、ウラシル以外の塩基を含んでいてもよく、修飾塩基、人工塩基を含んでいてもよい。本発明に使用可能なｄｓＲＮＡの配列、長さは特に限定されず、いずれのｄｓＲＮＡを用いてもよい。 dsRNA is a duplex made up of two RNA strands with complementary sequences. The two RNA strands do not have to be 100% complementary. dsRNA may contain bases other than adenine, guanine, cytosine, and uracil, and may contain modified bases and artificial bases. The sequence and length of dsRNA that can be used in the present invention are not particularly limited, and any dsRNA may be used.
ｐｏｌｙ（Ｉ：Ｃ）は、イノシン酸のポリマーとシチジル酸のポリマーから構成されるミスマッチ二本鎖ＲＮＡである。ｐｏｌｙ（Ｉ：Ｃ）中の一部のイノシン酸および／またはシチジル酸が他の塩基に置き換わっていてもよい。本発明に使用可能なｐｏｌｙ（Ｉ：Ｃ）の長さは特に限定されない。 Poly(I:C) is a mismatched double-stranded RNA composed of a polymer of inosinic acid and a polymer of cytidylic acid. Some of the inosinic acid and/or cytidylic acid in poly(I:C) may be replaced with other bases. The length of poly(I:C) that can be used in the present invention is not particularly limited.
本発明の医薬組成物の有効成分は、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞である。ここで、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞とは、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）での処理によりＴＲＡＩＬの産生を増加させた細胞、あるいは元々はＴＲＡＩＬの産生が認められなかった細胞であるが、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）での処理によりＴＲＡＩＬを産生するようになった細胞である。細胞からのＴＲＡＩＬの産生量は、がん細胞を死滅させるに十分な量であればよい。ＴＲＡＩＬの産生の増加は、処理前と比較して例えば１０倍以上、好ましくは３０倍以上、より好ましくは５０倍以上、さらに好ましくは１００倍以上である。 The active ingredient of the pharmaceutical composition of the present invention is TRAIL-producing mesenchymal tissue-derived adherent cells treated with a bioactive polypeptide, LPS, dsRNA or poly(I:C). Here, adherent cells derived from mesenchymal tissue that produce TRAIL are cells whose production of TRAIL has been increased by treatment with a physiologically active polypeptide, LPS, dsRNA, or poly(I:C), or cells that have been originally These are cells in which TRAIL production was not observed, but came to produce TRAIL by treatment with a physiologically active polypeptide, LPS, dsRNA, or poly(I:C). The amount of TRAIL produced by cells may be sufficient to kill cancer cells. The increase in TRAIL production is, for example, 10 times or more, preferably 30 times or more, more preferably 50 times or more, even more preferably 100 times or more, compared to before treatment.
間葉系組織由来の接着性細胞であればいずれの細胞であっても本発明に用いることができる。細胞が由来する動物はいずれの動物であってもよく、好ましくは哺乳動物であり、例えばヒトが挙げられる。間葉系組織からの接着性細胞の取得手段・方法は公知である。間葉系組織由来の接着性細胞は市販されているものであってもよく、アメリカン・タイプ・カルチャー・コレクション（ＡＴＣＣ）（米国）、ＮＩＴＥ（日本）などの機関から分譲されるものであってもよい。あるいは、間葉系組織由来の接着性細胞を間葉系組織から得てもよい。 Any adherent cells derived from mesenchymal tissue can be used in the present invention. The animal from which the cells are derived may be any animal, preferably a mammal, such as a human. Means and methods for obtaining adherent cells from mesenchymal tissue are known. Adhesive cells derived from mesenchymal tissues may be commercially available, or may be distributed from institutions such as the American Type Culture Collection (ATCC) (USA), NITE (Japan), etc. Good too. Alternatively, mesenchymal tissue-derived adherent cells may be obtained from mesenchymal tissue.
好ましい間葉系組織由来の接着性細胞としては、間葉系組織由来幹細胞（ＭＳＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、Ｍｕｓｅ細胞、骨髄組織、胞衣組織（胎盤組織、臍帯組織、羊膜組織などを含む）、軟骨組織、骨膜組織、滑膜組織、骨格筋組織に由来する細胞、幹細胞および間質細胞、ならびに経血細胞などが例示されるが、これらに限定されない。さらに好ましい間葉系組織由来の接着性細胞としては、ＡＤＳＣ、ＡＤＭＰＣ、胞衣組織に由来する細胞、骨髄組織または滑膜組織に由来する細胞などが例示されるが、これらに限定されない。 Preferred mesenchymal tissue-derived adhesive cells include mesenchymal tissue-derived stem cells (MSCs), adipose tissue-derived stem cells (ADSCs), adipose tissue-derived multilineage progenitor cells (ADMPCs), Muse cells, bone marrow tissue, and alveolar tissue. (including placental tissue, umbilical cord tissue, amniotic tissue, etc.), cartilage tissue, periosteal tissue, synovial tissue, cells derived from skeletal muscle tissue, stem cells and stromal cells, and menstrual blood cells. Not limited. More preferable adhesive cells derived from mesenchymal tissue include, but are not limited to, ADSC, ADMPC, cells derived from alveolar tissue, cells derived from bone marrow tissue, or synovial tissue.
細胞を間葉系組織から得る場合は、いずれの間葉系組織から単離してもよい。間葉系組織としては、脂肪組織、骨髄組織、胞衣組織、軟骨組織、骨膜組織、滑膜組織、骨格筋組織、経血などが例示されるが、これらに限定されない。好ましい間葉系組織としては脂肪組織、骨髄組織、滑膜組織、胞衣組織が挙げられる。特に脂肪組織は体内に含まれる量が多く、細胞を多く取り出せる点で好ましい。かかる点においてＡＤＳＣおよびＡＤＭＰＣは有利である。 If the cells are obtained from a mesenchymal tissue, they may be isolated from any mesenchymal tissue. Examples of mesenchymal tissues include, but are not limited to, adipose tissue, bone marrow tissue, alveolar tissue, cartilage tissue, periosteum tissue, synovial tissue, skeletal muscle tissue, menstrual blood, and the like. Preferred mesenchymal tissues include adipose tissue, bone marrow tissue, synovial tissue, and alveolar tissue. In particular, adipose tissue is preferable because it is contained in a large amount in the body and a large number of cells can be extracted from it. ADSC and ADMPC are advantageous in this respect.
体内から間葉系組織を取り出して、培養容器中に組織を入れて培養し、容器に付着する細胞を選択的に取得することにより、接着性細胞を得ることができる。間葉系組織は、切除や吸引といった公知の手段、方法を用いて取り出すことができる。取り出した間葉系組織をそのまま培養してもよく、必要に応じて、取り出した間葉系組織を刻んだり、解したりした後、赤血球を除去し、得られた細胞集団を培養してもよい。これらの処理方法および手段、ならびに細胞培養手段、方法は公知であり、適宜選択することができる。間葉系組織由来の接着性細胞は、例えば培養容器に付着した細胞をトリプシンなどの酵素で処理することによって得てもよい。 Adhesive cells can be obtained by removing mesenchymal tissue from the body, culturing the tissue in a culture container, and selectively obtaining cells that adhere to the container. Mesenchymal tissue can be removed using known means and methods such as excision and aspiration. The removed mesenchymal tissue may be cultured as is, or if necessary, the removed mesenchymal tissue may be chopped or disintegrated, red blood cells removed, and the resulting cell population cultured. good. These processing methods and means as well as cell culture means and methods are known and can be selected as appropriate. Adherent cells derived from mesenchymal tissue may be obtained, for example, by treating cells attached to a culture vessel with an enzyme such as trypsin.
生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）での間葉系組織由来の接着性細胞の処理は、細胞と生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）を公知の方法にて接触させることによって行うことができる。典型的には、適当濃度の生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）を含む培地にて、間葉系組織由来の接着性細胞を一定時間培養することにより、この処理を行うことができる。通常は、数ピコグラム／ｍｌ～数百ナノグラム／ｍｌの生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）を添加した培地にて間葉系組織由来の接着性細胞を培養する。培養に使用する培地は公知のものでよい。培養時間、培養温度も適宜選択することができる。必要に応じて、間葉系組織由来の接着性細胞を培養して細胞数を増加させた後、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理してもよい。間葉系組織由来の接着性細胞の集団から所望の亜集団を得て、必要に応じて亜集団を培養して細胞数を増加させた後、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理してもよい。 Treatment of adherent cells derived from mesenchymal tissue with a bioactive polypeptide, LPS, dsRNA or poly(I:C) can be carried out by combining the cells with a bioactive polypeptide, LPS, dsRNA or poly(I:C) using a known method. This can be carried out by contacting the patient by a method. Typically, this treatment is performed by culturing adherent cells derived from mesenchymal tissue for a certain period of time in a medium containing an appropriate concentration of bioactive polypeptide, LPS, dsRNA, or poly(I:C). be able to. Usually, adherent cells derived from mesenchymal tissue are cultured in a medium supplemented with several picograms/ml to several hundred nanograms/ml of physiologically active polypeptide, LPS, dsRNA, or poly(I:C). The culture medium used for culture may be any known medium. The culture time and culture temperature can also be selected as appropriate. If necessary, after culturing adherent cells derived from mesenchymal tissue to increase the number of cells, they may be treated with a physiologically active polypeptide, LPS, dsRNA, or poly(I:C). After obtaining a desired subpopulation from a population of adherent cells derived from mesenchymal tissue and culturing the subpopulation as necessary to increase cell number, bioactive polypeptide, LPS, dsRNA or poly(I) :C) may be used.
間葉系組織由来の接着性細胞を処理するために用いられる生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）は１種類であってもよく、２種類以上であってもよい。 The number of physiologically active polypeptides, LPS, dsRNA, or poly(I:C) used to treat adherent cells derived from mesenchymal tissue may be one or more than one.
生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理される間葉系組織由来の接着性細胞は１種類であってもよく、２種類以上であってもよい。 The number of mesenchymal tissue-derived adherent cells treated with the physiologically active polypeptide, LPS, dsRNA, or poly(I:C) may be one or two or more.
間葉系組織由来の接着性細胞をＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理する際に、リポソームに封入されたＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）を用いてもよい。 When treating adherent cells derived from mesenchymal tissues with LPS, dsRNA, or poly(I:C), LPS, dsRNA, or poly(I:C) encapsulated in liposomes may be used.
がんの治療は、がんの症状を緩和、改善または消失させること包含する。また、がんの治療は、がん細胞の増殖を抑制すること、がん細胞数を減少させること、がん細胞を消失させることを包含する。 Cancer treatment includes alleviating, improving or eliminating cancer symptoms. Furthermore, cancer treatment includes suppressing the proliferation of cancer cells, reducing the number of cancer cells, and eliminating cancer cells.
本発明の医薬組成物はあらゆる種類のがんの治療に用いることができる。本発明の医薬組成物を用いて、例えば、胃がん、陰茎がん、各骨軟部腫瘍、肝臓がん、甲状腺がん、後腹膜腫瘍、骨転移、子宮がん、食道がん、腎がん、腎盂尿管がん、膵臓がん、精巣がん、前立腺がん、大腸がん、胆道がん、頭頸部がん、乳がん、肺がん、皮膚がん、副腎がん、膀胱がん、卵巣がん、悪性リンパ腫、白血病などを治療することができるが、これらのがんに限定されない。 The pharmaceutical composition of the present invention can be used to treat all types of cancer. Using the pharmaceutical composition of the present invention, for example, gastric cancer, penile cancer, bone and soft tissue tumors, liver cancer, thyroid cancer, retroperitoneal tumors, bone metastasis, uterine cancer, esophageal cancer, renal cancer, Renal pelvic and ureteral cancer, pancreatic cancer, testicular cancer, prostate cancer, colon cancer, biliary tract cancer, head and neck cancer, breast cancer, lung cancer, skin cancer, adrenal gland cancer, bladder cancer, ovarian cancer , malignant lymphoma, leukemia, etc., but are not limited to these cancers.
本発明の医薬組成物を、有効成分である生理活性ポリペプチドまたはＬＰＳで処理された間葉系組織由来の接着性細胞が由来する動物種と同種の対象に投与してもよく、異種の対象に投与してもよい。例えば、ヒト由来の細胞を含む本発明の医薬組成物をヒト対象に投与してもよい。本発明の医薬組成物中の細胞は、投与される対象と同一人に由来するものであってもよく、投与される対象とは異なる人に由来するものであってもよい。 The pharmaceutical composition of the present invention may be administered to a subject of the same species as the animal species from which the mesenchymal tissue-derived adhesive cells treated with the physiologically active polypeptide or LPS as an active ingredient are derived, or may be administered to a subject of a different species. It may be administered to For example, a pharmaceutical composition of the invention comprising cells of human origin may be administered to a human subject. The cells in the pharmaceutical composition of the present invention may be derived from the same person as the subject to be administered, or may be derived from a different person from the subject to be administered.
本発明の医薬組成物は、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された間葉系組織由来の接着性細胞を医薬上許容される担体および／または賦形剤と混合することにより製造することができる。医薬上許容される担体や賦形剤は様々なものが公知であり、適宜選択して使用できる。例えば、本発明の医薬組成物を注射剤として用いる場合は、精製水、生理食塩水、リン酸緩衝生理食塩水などの担体や賦形剤に細胞を懸濁してもよい。細胞と担体および／または賦形剤との混合方法、混合手段についても公知である。 The pharmaceutical composition of the present invention comprises mesenchymal tissue-derived adherent cells treated with a physiologically active polypeptide, LPS, dsRNA or poly(I:C) in a pharmaceutically acceptable carrier and/or excipient. It can be manufactured by mixing. Various pharmaceutically acceptable carriers and excipients are known, and can be appropriately selected and used. For example, when the pharmaceutical composition of the present invention is used as an injection, cells may be suspended in a carrier or excipient such as purified water, physiological saline, or phosphate buffered saline. Methods and means for mixing cells with carriers and/or excipients are also known.
本発明の医薬組成物の剤形は特に限定されず、液剤、半固形剤であってもよい。本発明の医薬組成物の投与方法も限定されず、局所注射、静脈注射、輸液などにより行ってもよく、患部に塗布や噴霧する等であってもよく、カテーテルにより患部に投与してもよく、外科的手法により組織に直接移植してもよい。本発明の医薬組成物を細胞シート、細胞塊、重層化細胞シート、スポンジ担持細胞などの形態として移植してもよい。 The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and may be a liquid or a semisolid dosage form. The method of administering the pharmaceutical composition of the present invention is not limited either, and it may be administered by local injection, intravenous injection, infusion, etc., it may be applied or sprayed on the affected area, or it may be administered to the affected area using a catheter. , may be implanted directly into tissue by surgical techniques. The pharmaceutical composition of the present invention may be transplanted in the form of a cell sheet, cell mass, stratified cell sheet, sponge-supported cell, or the like.
本発明の医薬組成物の投与経路および投与量は、治癒すべきがんの種類や部位、症状の重さ、対象の状態などを考慮して適宜決定することができる。 The administration route and dosage of the pharmaceutical composition of the present invention can be appropriately determined in consideration of the type and site of cancer to be cured, the severity of symptoms, the condition of the subject, and the like.
本発明の医薬組成物は、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された間葉系組織由来の接着性細胞以外の成分を含んでいてもよい。このような成分としては公知の抗がん剤、鎮痛剤、解熱剤、栄養剤などが例示されるが、これに限定されない。また、本発明の医薬組成物を、他の医薬組成物および／または他の療法と併用してもよい。 The pharmaceutical composition of the present invention may contain components other than bioactive polypeptide, LPS, dsRNA, or adherent cells derived from mesenchymal tissue treated with poly(I:C). Examples of such ingredients include, but are not limited to, known anticancer drugs, analgesics, antipyretics, nutritional supplements, and the like. The pharmaceutical compositions of the invention may also be used in combination with other pharmaceutical compositions and/or other therapies.
本発明は、さらなる態様において、がんの治療のための医薬組成物の製造のための、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された間葉系組織由来の接着性細胞の使用を提供する。 In a further aspect, the present invention provides adhesives derived from mesenchymal tissue treated with bioactive polypeptides, LPS, dsRNA or poly(I:C) for the manufacture of pharmaceutical compositions for the treatment of cancer. Provides for the use of sex cells.
本発明は、さらなる態様において、がんの治療に使用される、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された間葉系組織由来の接着性細胞を提供する。 In a further aspect, the invention provides adherent cells derived from mesenchymal tissue treated with bioactive polypeptides, LPS, dsRNA or poly(I:C) for use in the treatment of cancer.
本発明は、さらなる態様において、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された間葉系組織由来の接着性細胞を対象に投与することを含む、がんの治療を必要とする対象におけるがんの治療方法を提供する。 In a further aspect, the present invention provides a treatment for cancer comprising administering to a subject adherent cells derived from mesenchymal tissue treated with a bioactive polypeptide, LPS, dsRNA or poly(I:C). Provides methods for treating cancer in subjects in need.
本発明は、さらにもう１つの態様において、
がんの治療のための医薬組成物の製造方法であって、下記工程：
（ａ）間葉系組織由来の接着性細胞を生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理し、次いで、
（ｂ）工程（ａ）にて処理された、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞を医薬上許容される担体および／または賦形剤と混合する
を含む方法、を提供する。
In yet another aspect, the present invention provides:
A method for producing a pharmaceutical composition for the treatment of cancer, comprising the following steps:
(a) Adherent cells derived from mesenchymal tissue are treated with bioactive polypeptide, LPS, dsRNA or poly(I:C), and then
(b) mixing the TRAIL-producing mesenchymal tissue-derived adherent cells treated in step (a) with a pharmaceutically acceptable carrier and/or excipient.
本発明は、さらなる態様において、本発明の医薬組成物の有効性を調べる方法を提供する。該方法は、がん細胞に請求項１～３のいずれか１項記載の医薬組成物を添加し、がん細胞の生存率または死滅率を調べることを含む。該方法は、がん患者から得られたがん細胞を用いてもよく、公知のがん細胞株を用いてもよい。培養がん細胞に本発明の医薬組成物を添加し、一定時間後に、例えばトリパンブルー染色等の公知の方法を用いてがん細胞の生存率または死滅率を調べることにより、該方法を実施してもよい。
The invention provides, in a further aspect, a method of testing the effectiveness of a pharmaceutical composition of the invention. The method includes adding the pharmaceutical composition according to any one of
本発明は、さらなる態様において、本発明の医薬組成物の有効性を調べるためのキットを提供する。該キットは、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理された、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞を含んでいてもよい。該キットはがん細胞の生存率または死滅率を調べるための手段を含んでいてもよい。 In a further aspect, the invention provides a kit for testing the effectiveness of the pharmaceutical composition of the invention. The kit may include adherent cells from mesenchymal tissue producing TRAIL treated with a bioactive polypeptide, LPS, dsRNA or poly(I:C). The kit may include means for determining survival or mortality of cancer cells.
本発明は、さらなる態様において、本発明の医薬組成物を用いるがんの治療の有効性を予測する方法を提供する。該方法は、がん細胞がデスレセプターを有しているかどうかを調べること、および／またはデスレセプターの量を調べることを含む。該方法において、デスレセプター４および／またはデスレセプター５の有無および／または量について調べることが好ましい。これらのレセプターの有無や量を調べる手段は公知であり、リアルタイムＰＣＲや免疫染色などが例示される。がん細胞は、がん患者から取得されたものであってもよく、公知のがん細胞株であってもよい。がん患者からのがん細胞の取得方法は公知であり、生検、採血等が例示される。がん細胞におけるデスレセプター、特にデスレセプター４および／またはデスレセプター５の量が多いほど、本発明の医薬組成物を用いるがんの治療の有効性は高いと予測することができる。
In a further aspect, the invention provides a method of predicting the effectiveness of cancer treatment using a pharmaceutical composition of the invention. The method includes determining whether the cancer cell has death receptors and/or determining the amount of death receptors. In this method, it is preferable to examine the presence and/or amount of
本発明は、さらなる態様において、本発明の医薬組成物を用いるがんの治療の有効性を予測するためのキットを提供する。該キットは、デスレセプター、特にデスレセプター４および／またはデスレセプター５の有無および／または量を調べるための手段を含む。例えば、該キットは、デスレセプター、特にデスレセプター４および／またはデスレセプター５遺伝子の発現を調べるためのＲＴ－ＰＣＲ用の試薬類および／または器具を含んでいてもよく、あるいはデスレセプター、特にデスレセプター４および／またはデスレセプター５に対する標識された抗体を含んでいてもよい。
In a further aspect, the invention provides a kit for predicting the effectiveness of cancer treatment using the pharmaceutical composition of the invention. The kit comprises means for determining the presence and/or amount of death receptors, in
本発明は、さらなる態様において、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）および医薬上許容される担体または賦形剤を含むがんの治療のための医薬組成物であって、がん関連線維芽細胞（ＣＡＦ）に投与される医薬組成物を提供する。ＣＡＦは間葉系組織由来の接着性細胞の１種であり、がん微小環境の構成成分の１つである。ＣＡＦは、生理活性ポリペプチド等の上記因子が投与されるとＴＲＡＩＬの産生を増加させ、あるいはＴＲＡＩＬを産生するようになる。ＣＡＦはがん細胞に隣接しているか、あるいはその近傍に存在しているので、ＣＡＦがＴＲＡＩＬを産生すればがん細胞は効果的に死滅する。 The invention provides, in a further aspect, a pharmaceutical composition for the treatment of cancer, comprising a bioactive polypeptide, LPS, dsRNA or poly(I:C) and a pharmaceutically acceptable carrier or excipient, comprising: Pharmaceutical compositions administered to cancer-associated fibroblasts (CAFs) are provided. CAF is a type of adherent cell derived from mesenchymal tissue and is one of the components of the cancer microenvironment. CAF increases the production of TRAIL or comes to produce TRAIL when the above-mentioned factors such as physiologically active polypeptides are administered. Since CAFs are adjacent to or in the vicinity of cancer cells, when CAFs produce TRAIL, cancer cells are effectively killed.
上記態様の医薬組成物をＣＡＦに直接送達することが好ましい。該医薬組成物をがん患部に直接注射してもよい。また、ＣＡＦが有している細胞表面マーカーに特異的な抗体と生理活性ポリペプチドとの複合体を用いてもよく、あるいはＬＰＳ、二本鎖ＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）を含有するリポソームを該医薬組成物に用いてもよい。該複合体の例として、上記抗体と生理活性ポリペプチドとの融合タンパク質が挙げられる。 Preferably, the pharmaceutical composition of the above embodiments is delivered directly to the CAF. The pharmaceutical composition may be directly injected into the cancer affected area. Alternatively, a complex of an antibody specific to a cell surface marker possessed by CAF and a physiologically active polypeptide may be used, or a liposome containing LPS, double-stranded RNA, or poly(I:C) may be used. It may be used in the pharmaceutical composition. An example of such a complex is a fusion protein of the above antibody and a physiologically active polypeptide.
本発明は、さらなる態様において、ＣＡＦに投与されるがんの治療のための医薬組成物の製造のための、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）の使用を提供する。 The invention provides in a further aspect the use of a bioactive polypeptide, LPS, dsRNA or poly(I:C) for the manufacture of a pharmaceutical composition for the treatment of cancer administered to CAF.
本発明は、さらなる態様において、がんの治療に使用され、使用に際してＣＡＦに投与される、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）を提供する。 The invention provides, in a further aspect, a bioactive polypeptide, LPS, dsRNA or poly(I:C) for use in the treatment of cancer and administered to CAFs in use.
本発明は、さらなる態様において、生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）を対象のＣＡＦに投与することを含む、がんの治療を必要とする対象におけるがんの治療方法を提供する。 In a further aspect, the present invention provides a method for treating cancer in a subject in need of cancer treatment, comprising administering a bioactive polypeptide, LPS, dsRNA or poly(I:C) to CAF of the subject. provide.
本発明は、さらなる態様において、ＴＲＡＩＬを産生する間葉系組織由来の接着性細胞の製造方法を提供する。該方法は、間葉系組織由来の接着性細胞を生理活性ポリペプチド、ＬＰＳ、ｄｓＲＮＡまたはｐｏｌｙ（Ｉ：Ｃ）で処理することを含む。該方法で得られた細胞は、がんの治療に使用することができる。 In a further aspect, the present invention provides a method for producing TRAIL-producing mesenchymal tissue-derived adherent cells. The method involves treating adherent cells derived from mesenchymal tissue with a bioactive polypeptide, LPS, dsRNA or poly(I:C). Cells obtained by this method can be used for cancer treatment.
本明細書中の用語は、特に断らない限り、医学、薬学、生物学、化学等の分野において通常に理解されている意味を有する。 Terms used herein have meanings commonly understood in the fields of medicine, pharmacy, biology, chemistry, etc., unless otherwise specified.
以下に実施例を示して本発明をさらに詳細かつ具体的に説明するが、実施例は本発明を限定するものではない。 EXAMPLES The present invention will be explained in more detail and concretely with reference to Examples below, but the Examples are not intended to limit the present invention.
実施例１．生理活性ポリペプチドで処理された間葉系組織由来の接着性細胞のＴＲＡＩＬ発現増強効果
（１）実験方法
（ｉ）試供細胞
試供細胞として、脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、骨髄由来間葉系幹細胞（ＢＭ－ＭＳＣ）、胎盤由来細胞（Ｐｌａｃｅｎｔａｌ ｄｅｒｉｖｅｄ Ｃｅｌｌｓ）および臍帯由来幹細胞（ＵＣ－ＭＳＣ）を用いた。
（ｉｉ）ヒト対象からの脂肪組織の採取
インフォームドコンセントを受けた女性６人から脂肪吸引手術中に廃棄される余分な脂肪組織の分譲を受けた（米国ＣｕｒｅＬｉｎｅ社）。
（ｉｉｉ）脂肪組織由来幹細胞（ＡＤＳＣ）、骨髄由来間葉系幹細胞（ＢＭ－ＭＳＣ）、胎盤由来細胞（Ｐｌａｃｅｎｔａｌ ｄｅｒｉｖｅｄ Ｃｅｌｌｓ）および臍帯由来幹細胞（ＵＣ－ＭＳＣ）の入手
これらの細胞はＬｏｎｚａ社より購入した。
Example 1. Effect of enhancing TRAIL expression in mesenchymal tissue-derived adherent cells treated with bioactive polypeptide (1) Experimental method (i) Sample cells As sample cells, adipose tissue-derived multilineage progenitor cells (ADMPC), adipose tissue-derived Stem cells (ADSC), bone marrow-derived mesenchymal stem cells (BM-MSC), placenta-derived cells (placenta-derived cells), and umbilical cord-derived stem cells (UC-MSC) were used.
(ii) Collection of adipose tissue from human subjects Excess adipose tissue discarded during liposuction surgery was donated from six women who gave informed consent (CureLine, Inc., USA).
(iii) Obtaining adipose tissue-derived stem cells (ADSC), bone marrow-derived mesenchymal stem cells (BM-MSC), placenta-derived cells (placenta-derived cells), and umbilical cord-derived stem cells (UC-MSC) These cells were purchased from Lonza. did.
（ｉｖ） ＡＤＭＰＣの単離
脂肪組織を刻み、次に、０．００８％ リベレース（Ｒｏｃｈ Ｌｉｆｅｓｃｉｅｎｃｅ）含有ハンクス緩衝塩類溶液（ＨＢＳＳ）中、３７℃のウォーターバスにて振盪しながら１時間消化した。消化産物をＣｅｌｌ Ｓｔｒａｉｎｅｒ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ）で濾過し、８００ｘｇにて１０分間遠心した。リンパ球分離液（ｄ＝１．０７７）（Ｎａｃａｌａｉ ｔｅｓｑｕｅ）を用い、比重法により赤血球を除去し、得られたＡＤＭＰＣを含む細胞集団を、１０％ ウシ胎児血清（Ｈｙｃｌｏｎｅ）を含むＤＭＥＭ中に細胞を播種して細胞を付着させた後、洗浄し、ＥＤＴＡで処理して、ＡＤＭＰＣを得た。
（ｖ）脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、骨髄由来間葉系幹細胞（ＢＭ－ＭＳＣ）、胎盤由来細胞（Ｐｌａｃｅｎｔａｌ ｄｅｒｉｖｅｄ Ｃｅｌｌｓ）および臍帯由来幹細胞（ＵＣ－ＭＳＣ）の培養
これらの細胞を、培地（６０％ ＤＭＥＭ－低グルコース、４０％ ＭＣＤＢ２０１、１０μｇ／ｍＬ ＥＧＦ、１ｎＭ デキサメサゾン、１００μＭ アスコルビン酸、および５％ＦＢＳ）にてヒトフィブロネクチンコートディッシュに播種し、３から８継代し、培養細胞を得た。
(iv) Isolation of ADMPC Adipose tissue was minced and then digested in Hank's buffered saline solution (HBSS) containing 0.008% Liberace (Roch Lifescience) for 1 hour with shaking in a water bath at 37°C. Digestion products were filtered through a Cell Strainer (BD Bioscience) and centrifuged at 800xg for 10 minutes. Red blood cells were removed by a specific gravity method using a lymphocyte separation solution (d=1.077) (Nacalai tesque), and the resulting cell population containing ADMPC was placed in DMEM containing 10% fetal bovine serum (Hyclone). After seeding and adhering cells, the cells were washed and treated with EDTA to obtain ADMPCs.
(v) Adipose tissue-derived multilineage progenitor cells (ADMPCs), adipose tissue-derived stem cells (ADSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), placenta-derived cells (placenta-derived cells), and umbilical cord-derived stem cells (UC-MSCs) ) These cells were seeded on human fibronectin-coated dishes in medium (60% DMEM-low glucose, 40% MCDB201, 10 μg/mL EGF, 1 nM dexamethasone, 100 μM ascorbic acid, and 5% FBS) and cultured from 3 to After 8 passages, cultured cells were obtained.
（ｖｉ）ＩＦＮ－β処理およびＴＲＡＩＬ発現強度の測定
ＩＦＮ－βを、０ｎｇ／ｍｌ、２．５ｎｇ／ｍｌ、５ｎｇ／ｍｌ、１０ｎｇ／ｍｌ、２０ｎｇ／ｍｌ、４０ｎｇ／ｍｌとなるように、培地（６０％ ＤＭＥＭ－低グルコース、４０％ ＭＣＤＢ２０１、１０μｇ／ｍＬ ＥＧＦ、１ｎＭ デキサメサゾン、１００μＭ アスコルビン酸、および５％ＦＢＳ）に添加した。上で得られた培養細胞を、上記ＩＦＮ－β含有培地にて７２時間培養した。培地交換７２時間後にＲＬＴ Ｂｕｆｆｅｒ ６００ｕＬを添加して回収、ＲＮＡ抽出した。
ＲＬＴ Ｂｕｆｆｅｒサンプルは、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ ／ ＱＩＡＧＥＮを用いて全ＲＮＡを抽出し、全ＲＮＡを１００ｎｇ／ｕＬになるように調製後、１アレイ当たり全ＲＮＡ １５０ｎｇからラベルされたｃＲＮＡを合成した。合成したラベルされたｃＲＮＡは濃度、収量およびＣｙ３取込率を算出し、合成サイズ（２００－２０００ｎｔを増幅）を測定した。ラベルされたｃＲＮＡ ６００ｎｇを６０℃でフラグメンテーションを形成させ、６５℃、１７時間でハイブリダイゼーションを行い、アレイを洗浄してスキャンし、ＴＲＡＩＬのプローブであるＡ＿２３＿Ｐ１２１２５３）のｎｏｒｍａｌｉｚｅｄ ｉｎｔｅｎｓｉｔｙをプロットした結果を図１に示す。
(vi) IFN-β treatment and measurement of TRAIL expression intensity IFN-β was added to the medium ( 60% DMEM-low glucose, 40% MCDB201, 10 μg/mL EGF, 1 nM dexamethasone, 100 μM ascorbic acid, and 5% FBS). The cultured cells obtained above were cultured in the above IFN-β-containing medium for 72 hours. 72 hours after the medium was replaced, 600 μL of RLT Buffer was added and recovered, and RNA was extracted.
For the RLT Buffer sample, total RNA was extracted using RNeasy Mini Kit/QIAGEN, total RNA was adjusted to 100 ng/uL, and labeled cRNA was synthesized from 150 ng of total RNA per array. The concentration, yield, and Cy3 uptake rate of the synthesized labeled cRNA were calculated, and the synthesized size (amplified from 200 to 2000 nt) was measured. 600 ng of labeled cRNA was subjected to fragmentation at 60°C, hybridization was performed at 65°C for 17 hours, the array was washed and scanned, and the normalized intensity of TRAIL probe A_23_P121253) was plotted in Figure 1. Shown below.
（２）実験結果
図１からわかるように、脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、骨髄由来間葉系幹細胞（ＢＭ－ＭＳＣ）、胎盤由来細胞（Ｐｌａｃｅｎｔａｌ ｄｅｒｉｖｅｄ Ｃｅｌｌｓ）および臍帯由来幹細胞（ＵＣ－ＭＳＣ）において、ＩＦＮβ処理によって濃度依存的にＴＲＡＩＬ発現が増加していた。ＩＦＮβ処理によるＴＲＡＩＬ発現増加は広く間葉系幹細胞および間葉系細胞に認められることが確認された。
(2) Experimental results As can be seen from Figure 1, adipose tissue-derived multilineage progenitor cells (ADMPCs), adipose tissue-derived stem cells (ADSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and placenta-derived cells (placenta-derived cells) ) and umbilical cord-derived stem cells (UC-MSCs), TRAIL expression was increased in a concentration-dependent manner by IFNβ treatment. It was confirmed that the increase in TRAIL expression due to IFNβ treatment was widely observed in mesenchymal stem cells and mesenchymal cells.
実施例２．生理活性ポリペプチド処理による間葉系組織由来の接着性細胞のＴＲＡＩＬ産生
（１）実験方法
（ｉ）試供細胞
試供細胞として、脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、骨髄由来間葉系幹細胞（ＢＭ－ＭＳＣ）および胎盤由来細胞（Ｐｌａｃｅｎｔａｌ ｄｅｒｉｖｅｄ Ｃｅｌｌｓ）を用いた。
（ｉｉ）ヒト対象からの脂肪組織の採取
インフォームドコンセントを受けた女性６人から脂肪吸引手術中に廃棄される余分な脂肪組織の分譲を受けた（米国ＣｕｒｅＬｉｎｅ社）。
（ｉｉｉ）脂肪組織由来幹細胞（ＡＤＳＣ）、骨髄由来間葉系幹細胞（ＢＭ－ＭＳＣ）および胎盤由来細胞（Ｐｌａｃｅｎｔａｌ ｄｅｒｉｖｅｄ Ｃｅｌｌｓ）の入手
これらの細胞はＬｏｎｚａ社より購入した。
Example 2. TRAIL production of mesenchymal tissue-derived adhesive cells by bioactive polypeptide treatment (1) Experimental method (i) Sample cells Sample cells include adipose tissue-derived multilineage progenitor cells (ADMPC) and adipose tissue-derived stem cells (ADSC). , bone marrow-derived mesenchymal stem cells (BM-MSCs) and placenta-derived cells (BM-MSCs) were used.
(ii) Collection of adipose tissue from human subjects Excess adipose tissue discarded during liposuction surgery was donated from six women who gave informed consent (CureLine, Inc., USA).
(iii) Obtaining adipose tissue-derived stem cells (ADSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and placenta-derived cells These cells were purchased from Lonza.
（ｉｖ） ＡＤＭＰＣの単離
脂肪組織を刻み、次に、０．００８％ リベレース（Ｒｏｃｈｅ Ｌｉｆｅｓｃｉｅｎｃｅ）含有ハンクス緩衝塩類溶液（ＨＢＳＳ）中、３７℃のウォーターバスにて振盪しながら１時間消化した。消化産物をＣｅｌｌ Ｓｔｒａｉｎｅｒ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ）で濾過し、８００ｘｇにて１０分間遠心した。リンパ球分離液（ｄ＝１．０７７）（Ｎａｃａｌａｉ ｔｅｓｑｕｅ）を用い、比重法により赤血球を除去し、得られたＡＤＭＰＣを含む細胞集団を、１０％ ウシ胎児血清（Ｈｙｃｌｏｎｅ）を含むＤＭＥＭ中に細胞を播種して細胞を付着させた後、洗浄し、ＥＤＴＡで処理して、ＡＤＭＰＣを得た。
（ｖ）脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、骨髄由来間葉系幹細胞（ＢＭ－ＭＳＣ）および胎盤由来細胞（Ｐｌａｃｅｎｔａｌ ｄｅｒｉｖｅｄ Ｃｅｌｌｓ）の培養
これらの細胞を、培地（６０％ ＤＭＥＭ－低グルコース、４０％ ＭＣＤＢ２０１、１０μｇ／ｍＬ ＥＧＦ、１ｎＭ デキサメサゾン、１００μＭ アスコルビン酸、および５％ＦＢＳ）にてヒトフィブロネクチンコートディッシュに播種し、３から８継代し、培養細胞を得た。
(iv) Isolation of ADMPC Adipose tissue was minced and then digested in Hank's Buffered Salt Solution (HBSS) containing 0.008% Liberace (Roche Lifescience) for 1 hour with shaking in a water bath at 37°C. Digestion products were filtered through a Cell Strainer (BD Bioscience) and centrifuged at 800xg for 10 minutes. Red blood cells were removed by a specific gravity method using a lymphocyte separation solution (d=1.077) (Nacalai tesque), and the resulting cell population containing ADMPC was placed in DMEM containing 10% fetal bovine serum (Hyclone). After seeding and adhering cells, the cells were washed and treated with EDTA to obtain ADMPCs.
(v) Culture of adipose tissue-derived multilineage progenitor cells (ADMPCs), adipose tissue-derived stem cells (ADSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and placenta-derived cells (placenta-derived cells). (60% DMEM-low glucose, 40% MCDB201, 10 μg/mL EGF, 1 nM dexamethasone, 100 μM ascorbic acid, and 5% FBS) on human fibronectin-coated dishes and passaged 3 to 8 times to obtain cultured cells. Ta.
（ｖｉ）ＩＦＮ－β処理およびＴＲＡＩＬ産生量の測定
ＩＦＮ－βを１０ｎｇ／ｍｌとなるように、培地（６０％ ＤＭＥＭ－低グルコース、４０％ ＭＣＤＢ２０１、１０μｇ／ｍＬ ＥＧＦ、１ｎＭ デキサメサゾン、１００μＭ アスコルビン酸、および５％ＦＢＳ）に添加した。上の（ｖ）で得られた培養細胞を、上記ＩＦＮ－β含有培地にて７２時間培養した。無血清培地（６０％ ＤＭＥＭ－低グルコース、４０％ ＭＣＤＢ２０１、１０μｇ／ｍＬ ＥＧＦ、１ｎＭ デキサメサゾン、１００μＭ アスコルビン酸、および２ｍｇ／ｍＬ ＢＳＡ）にて２４時間追加培養し、培地中に産生されたＴＲＡＩＬを測定した。コントロール系では、ＩＦＮ－βを添加しない上記無血清培地で上記培養細胞を培養し、培地中のＴＲＡＩＬを測定した。ＴＲＡＩＬの測定はＲ＆Ｄ ＳＹＳＴＥＭＳ社のＥＬＩＳＡキット（ＴＲＡＩＬ／ＴＮＦＳＦ１０ Ｑｕａｎｔｉｋｉｎｅ ＥＬＩＳＡ Ｋｉｔ）を用いて行った。
(vi) IFN-β treatment and measurement of TRAIL production amount IFN-β was added to the culture medium (60% DMEM-low glucose, 40% MCDB201, 10 μg/mL EGF, 1 nM dexamethasone, 100 μM ascorbic acid, and 5% FBS). The cultured cells obtained in (v) above were cultured in the above IFN-β-containing medium for 72 hours. Additional culture was performed for 24 hours in serum-free medium (60% DMEM-low glucose, 40% MCDB201, 10 μg/mL EGF, 1 nM dexamethasone, 100 μM ascorbic acid, and 2 mg/mL BSA), and TRAIL produced in the medium was measured. did. In a control system, the above-mentioned cultured cells were cultured in the above-mentioned serum-free medium to which IFN-β was not added, and TRAIL in the medium was measured. TRAIL was measured using an ELISA kit (TRAIL/TNFSF10 Quantikine ELISA Kit) from R&D SYSTEMS.
（２）実験結果
ＴＲＡＩＬ産生量の測定結果を図２に示す。ＩＦＮ－β処理された脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）、脂肪組織由来幹細胞（ＡＤＳＣ）、骨髄由来間葉系幹細胞（ＢＭ－ＭＳＣ）および胎盤由来細胞（Ｐｌａｃｅｎｔａｌ ｄｅｒｉｖｅｄ Ｃｅｌｌｓ）からＴＲＡＩＬが産生されることが確認された。臍帯由来幹細胞（ＵＣ－ＭＳＣ）についても同様の結果を得た。一方、コントロール系では、これらの細胞からのＴＲＡＩＬ産生は確認されなかった。
(2) Experimental results The measurement results of TRAIL production amount are shown in FIG. 2. TRAIL is produced from IFN-β-treated adipose tissue-derived multilineage progenitor cells (ADMPCs), adipose tissue-derived stem cells (ADSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and placenta-derived cells. It was confirmed that Similar results were obtained for umbilical cord-derived stem cells (UC-MSCs). On the other hand, in the control system, TRAIL production from these cells was not confirmed.
実施例３．各種生理活性ポリペプチドによる間葉系組織由来の接着性細胞の処理効果
（１）実験方法
（ｉ）試供細胞
試供細胞として、脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）を用いた。生理活性ポリペプチドとしては各種のサイトカインおよびケモカインを用いた。
Example 3. Effects of treating mesenchymal tissue-derived adherent cells with various physiologically active polypeptides (1) Experimental method (i) Sample cells Adipose tissue-derived multilineage progenitor cells (ADMPCs) were used as sample cells. Various cytokines and chemokines were used as physiologically active polypeptides.
（ｉｉ）生理活性ペプチド
生理活性ポリペプチドとしてサイトカイン（ＩＬ－１α、ＩＬ－１β、ＩＬ３～ＩＬ３５、オンコスタチンＭ、ＬＩＦ、ＣＮＴＦ、ＣＴ－１、ＴＮＦα、ＴＮＦβ、ＢＡＦＦ、ＦａｓＬ、ＲＡＮＫＬ、ＴＲＡＩＬ、ＩＮＦ－α、ＩＦＮ－β、ＩＦＮ－γ）およびケモカイン（ＣＣＬ１～ＣＣＬ２８、ＣＸＣＬ１～ＣＸＣＬ１０）を用いた。
(ii) Physiologically active peptides Bioactive polypeptides include cytokines (IL-1α, IL-1β, IL3 to IL35, Oncostatin M, LIF, CNTF, CT-1, TNFα, TNFβ, BAFF, FasL, RANKL, TRAIL, INF). -α, IFN-β, IFN-γ) and chemokines (CCL1 to CCL28, CXCL1 to CXCL10) were used.
（ｉｉｉ）生理活性ペプチドでの処理およびＴＲＡＩＬ発現強度の測定
試供細胞を、生理活性ペプチド添加培地（最終濃度１００ｎｇ／ｍＬ）および生理活性ペプチド非添加培地（コントロール）にて培地交換を行い、更に３日間（７２時間）継続培養を行った。培地交換７２時間後にＲＬＴ Ｂｕｆｆｅｒ ６００ｕＬを添加して回収、ＲＮＡ抽出した。
ＲＬＴ Ｂｕｆｆｅｒサンプルは、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ ／ ＱＩＡＧＥＮを用いて全ＲＮＡを抽出し、全ＲＮＡを１００ｎｇ／ｕＬになるように調製後、１アレイ当たり全ＲＮＡ １５０ｎｇからラベルされたｃＲＮＡを合成した。合成したラベルされたｃＲＮＡは濃度、収量およびＣｙ３取込率を算出し、合成サイズ（２００－２０００ｎｔを増幅）を測定した。ラベルされたｃＲＮＡ ６００ｎｇを６０℃でフラグメンテーションを形成させ、６５℃、１７時間でハイブリダイゼーションを行い、アレイを洗浄してスキャンし、ＴＲＡＩＬのプローブであるＡ＿２３＿Ｐ１２１２５３）のｎｏｒｍａｌｉｚｅｄ ｉｎｔｅｎｓｉｔｙをプロットした。
(iii) Treatment with bioactive peptide and measurement of TRAIL expression intensity Sample cells were subjected to culture medium exchange with a bioactive peptide-added medium (final concentration 100 ng/mL) and a bioactive peptide-free medium (control), and further 3 Continuous culture was performed for days (72 hours). 72 hours after the medium was replaced, 600 μL of RLT Buffer was added and recovered, and RNA was extracted.
For the RLT Buffer sample, total RNA was extracted using RNeasy Mini Kit/QIAGEN, total RNA was adjusted to 100 ng/uL, and labeled cRNA was synthesized from 150 ng of total RNA per array. The concentration, yield, and Cy3 uptake rate of the synthesized labeled cRNA were calculated, and the synthesized size (amplified from 200 to 2000 nt) was measured. 600 ng of labeled cRNA was subjected to fragmentation at 60°C, hybridization was performed at 65°C for 17 hours, the array was washed and scanned, and the normalized intensity of TRAIL probe A_23_P121253) was plotted.
（２）実験結果
結果を表１に示す。
試験した大部分の生理活性ポリペプチドが細胞のＴＲＡＩＬ発現を増強することがわかった。ＩＦＮα、ＩＦＮβ、ＩＦＮγ、ＴＮＦα、ＴＮＦβ、ＩＬ－１α、ＩＬ－１βおよびオンコスタチンＭのＴＲＡＩＬ発現増強効果が大きかった。なかでもＩＦＮβ、ＩＦＮγ、ＴＮＦαのＴＲＡＩＬ発現増強効果が大きく、ＩＦＮβのＴＲＡＩＬ発現増強効果が最も大きかった。 Most bioactive polypeptides tested were found to enhance cellular TRAIL expression. The effects of IFNα, IFNβ, IFNγ, TNFα, TNFβ, IL-1α, IL-1β, and oncostatin M on enhancing TRAIL expression were significant. Among them, IFNβ, IFNγ, and TNFα had the greatest effect of enhancing TRAIL expression, and IFNβ had the greatest effect of enhancing TRAIL expression.
実施例４．生理活性ポリペプチドで処理された間葉系組織由来の接着性細胞のインビボでの抗腫瘍効果
（１）実験方法
（ｉ）ＩＦＮ－β活性化ＡＤＭＰＣの調製
実施例２に記載した方法にて脂肪組織からＡＤＭＰＣを単離、培養し、ＩＦＮ－βにて処理して、ＴＲＡＩＬを産生するＩＦＮ－β活性化ＡＤＭＰＣを得た。
Example 4. In vivo antitumor effect of mesenchymal tissue-derived adherent cells treated with bioactive polypeptides (1) Experimental method (i) Preparation of IFN-β-activated ADMPC ADMPCs were isolated from the tissues, cultured, and treated with IFN-β to obtain IFN-β-activated ADMPCs that produce TRAIL.
（ｉｉ）インビボ実験
肺がん患者から同意を得て入手した肺がん細胞をヌードマウス腰背部に移植し、患者由来腫瘍移植マウス（ＰＤＸマウス）を作出した。肺がん細胞移植後２４日目には肺がん細胞は腫瘤を形成した。形成した腫瘤を摘出し、３ｍｍ角にサイコロ状として、新たなヌードマウス腰背部に植え継ぎを行った。動物をＡ－Ｆの６群に分け、ＩＦＮ－β活性化ＡＤＭＰＣを投与した。Ａ群のマウスには、患者由来腫瘍移植片（ＰＤＸ）植え継ぎと同時にＩＦＮ－β活性化ＡＤＭＰＣを経血管的に投与した。Ｂ群のマウスには、ＰＤＸ植え継ぎ１週間後にＩＦＮ－β活性化ＡＤＭＰＣを経血管的に投与した。Ｃ群のマウスには、ＰＤＸ植え継ぎと同時にＩＦＮ－β活性化ＡＤＭＰＣを腹腔内投与した。Ｄ群のマウスには、ＰＤＸ植え継ぎ１週間後にＩＦＮ－β活性化ＡＤＭＰＣを腹腔内投与した。Ｅ群のマウスには、ＰＤＸ植え継ぎ１週間後にＩＦＮ－β活性化ＡＤＭＰＣを腫瘤に直接投与した。Ｆ群のマウスは、ＰＤＸ植え継ぎのみのコントロール群マウスであった。Ａ群－Ｅ群における投与細胞数は、動物１匹あたり１ｘ１０６個であった。ＰＤＸ植え継ぎ後４週にて各マウスから腫瘤を摘出し、容量を測定し、写真を撮った。
(ii) In vivo experiment Lung cancer cells obtained with consent from a lung cancer patient were transplanted into the lumbar region of a nude mouse to create a patient-derived tumor-grafted mouse (PDX mouse). On the 24th day after lung cancer cell transplantation, the lung cancer cells formed a mass. The formed tumor was removed, diced into 3 mm cubes, and transplanted to the back of a new nude mouse. Animals were divided into 6 groups AF and administered IFN-β activated ADMPC. Mice in group A were transvascularly administered with IFN-β-activated ADMPC at the same time as patient-derived tumor graft (PDX) implantation. Mice in group B were transvascularly administered with IFN-β-activated ADMPC one week after PDX transplantation. Mice in group C were intraperitoneally administered with IFN-β-activated ADMPC at the same time as PDX transplantation. Mice in group D were intraperitoneally administered with IFN-β-activated ADMPC one week after PDX transplantation. For mice in group E, IFN-β-activated ADMPC was directly administered to the tumor mass one week after PDX transplantation. Group F mice were control group mice that received only PDX transplantation. The number of cells administered in groups A-E was 1 x 10 6 cells per animal. Four weeks after PDX transplantation, the tumor mass was excised from each mouse, the volume was measured, and a photograph was taken.
（２）実験結果
ＰＤＸ植え継ぎ後４週にて各マウスから摘出した腫瘤の写真を図３に示す。摘出した腫瘤の容量の平均値を図４に示す。コントロール群（Ｆ群）と比較し、ＩＦＮβ活性化ＡＤＭＰＣを投与した群では、経血管的投与・腹腔内投与あるいは腫瘤直接投与のいずれでも腫瘤の縮小を認めた。これは、ＩＦＮ－β活性化ＡＤＭＰＣが投与経路を選ばずに有効であることを示している。ＰＤＸ植え継ぎ後１週間後に当該細胞を投与することで腫瘤の縮小を認めたことは、腫瘤治療を目的として当該細胞の投与が有用であることを示している。また、ＰＤＸ植え継ぎと同時にＩＦＮ－β活性化ＡＤＭＰＣを投与しても縮小を認めたことは、周術期に当該細胞を投与することの有用性を示すものである。
(2) Experimental Results Figure 3 shows photographs of tumors excised from each
本発明は、がんの治療薬の分野、がんの研究の分野等において利用可能である。 INDUSTRIAL APPLICABILITY The present invention can be used in the field of cancer therapeutics, cancer research, and the like.
Claims (3)
（ａ）脂肪組織由来幹細胞（ＡＤＳＣ）または脂肪組織由来多系統前駆細胞（ＡＤＭＰＣ）をＩＦＮβで処理し、次いで、
（ｂ）工程（ａ）にて処理された、ＴＲＡＩＬを産生するＡＤＳＣまたはＡＤＭＰＣを医薬上許容される担体および／または賦形剤と混合する
を含む方法。 A method for producing a pharmaceutical composition for the treatment of cancer, comprising the following steps:
(a) Treating adipose tissue-derived stem cells (ADSCs) or adipose tissue-derived multilineage progenitor cells (ADMPCs) with IFNβ, and then
(b) A method comprising mixing the TRAIL-producing ADSCs or ADMPCs treated in step (a) with a pharmaceutically acceptable carrier and/or excipient.
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| JP2018160842 | 2018-08-29 | ||
| JP2018160842 | 2018-08-29 | ||
| PCT/JP2019/033448 WO2020045399A1 (en) | 2018-08-29 | 2019-08-27 | Medicinal composition for treating cancer |
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| JPWO2020045399A1 JPWO2020045399A1 (en) | 2021-08-12 |
| JP7430914B2 true JP7430914B2 (en) | 2024-02-14 |
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| WO2025093747A1 (en) * | 2023-11-02 | 2025-05-08 | Belgian Volition Srl | Novel rapid high-throughput method of netosis induction and inhibition with physiologically relevant molecules |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150272992A1 (en) | 2012-10-24 | 2015-10-01 | The Texas A&M University System | Treatment of Tumors with Activated Mesenchymal Stem Cells |
| CN105779386A (en) | 2016-04-06 | 2016-07-20 | 南京大学 | Application of mesenchymal stem cell in preparation of medicine used for treating M5 type leukaemia |
| JP2017531425A (en) | 2014-10-01 | 2017-10-26 | コメンス バイオ，インク． | Induction media and methods for stem cell culture and therapy |
| WO2018110686A1 (en) | 2016-12-16 | 2018-06-21 | 有限会社大阪空気機械サービス | Tissue healing agent |
Family Cites Families (1)
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| WO2017135800A1 (en) * | 2016-02-05 | 2017-08-10 | 주식회사 에스엘바이젠 | Mesenchymal stem cell expressing trail and cd, and use thereof |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150272992A1 (en) | 2012-10-24 | 2015-10-01 | The Texas A&M University System | Treatment of Tumors with Activated Mesenchymal Stem Cells |
| JP2017531425A (en) | 2014-10-01 | 2017-10-26 | コメンス バイオ，インク． | Induction media and methods for stem cell culture and therapy |
| CN105779386A (en) | 2016-04-06 | 2016-07-20 | 南京大学 | Application of mesenchymal stem cell in preparation of medicine used for treating M5 type leukaemia |
| WO2018110686A1 (en) | 2016-12-16 | 2018-06-21 | 有限会社大阪空気機械サービス | Tissue healing agent |
Non-Patent Citations (5)
| Title |
|---|
| Anticancer Research，2015年，35，pp.4749-4756 |
| Cancer Letters，2019年，440-441，pp.202-210 |
| Cell stem cell，2012年，Vol.11，p.825-835 |
| Int J Biochem Cell Biol，2012年，Vol.44，p.1305-1314 |
| PLOS ONE，2013年，8(9)，e74897, page 1-11 |
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| JPWO2020045399A1 (en) | 2021-08-12 |
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