JP6695795B2 - Hematopoietic cell growth peptide and uses thereof - Google Patents
Hematopoietic cell growth peptide and uses thereof Download PDFInfo
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- JP6695795B2 JP6695795B2 JP2016511969A JP2016511969A JP6695795B2 JP 6695795 B2 JP6695795 B2 JP 6695795B2 JP 2016511969 A JP2016511969 A JP 2016511969A JP 2016511969 A JP2016511969 A JP 2016511969A JP 6695795 B2 JP6695795 B2 JP 6695795B2
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Description
本発明は、造血細胞(造血幹細胞、造血前駆細胞、および成熟造血細胞)を生体外で増殖させるため、またはこれらを製造するために有効に利用される新規ペプチドに関する。より具体的には、既存の細胞刺激因子の存在下で、造血幹細胞または造血前駆細胞を培養し、上記造血細胞を増殖させるために有効に使用できるペプチドに関する。 The present invention relates to a novel peptide that is effectively used for proliferating hematopoietic cells (hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells) in vitro or for producing them. More specifically, it relates to a peptide that can be effectively used to grow hematopoietic cells by culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of existing cell stimulating factors.
また本発明は、当該ペプチドの用途、詳細には造血細胞を生体外で増殖させるため、またはこれらを製造するために用いられる試薬(または製剤)としての用途、具体的には、既存の細胞刺激因子の存在下、造血細胞の増殖を補助するために使用される増殖助剤としての用途に関する。さらに本発明は、当該ペプチドの用途として、当該ペプチドを用いた造血細胞の生体外での増殖方法、言い換えると造血細胞の生体外での製造方法に関する。 The present invention also relates to the use of the peptide, in particular to grow hematopoietic cells in vitro, or as a reagent (or preparation) used for producing these, specifically, existing cell stimulators. The present invention relates to the use as a growth auxiliary agent used for supporting the growth of hematopoietic cells in the presence of a factor. Furthermore, the present invention relates to a method for proliferating hematopoietic cells in vitro using the peptide, in other words, a method for producing hematopoietic cells in vitro, using the peptide.
さらにまた本発明は、当該ペプチドに対する抗体に関する。 Furthermore, the present invention relates to an antibody against the peptide.
造血幹細胞を多能性幹細胞から生体外で製造し、または造血幹細胞を生体外に取り出して培養し、腫瘍化させることなく造血幹細胞だけを増幅することができれば、移植関連問題の多くは解決し、造血幹細胞移植療法の適応が広がるだけではなく、他の臓器特異的幹細胞移植療法の開発など、新しい再生医療を開拓することができる。しかし、現在まで開発されてきた造血幹細胞の製造法および増幅法では、以下の問題点が指摘されている。
1.HoxB4遺伝子を多能性幹細胞へ導入すると、造血幹細胞を製造できるものの、急性白血病が誘導される。
2.HoxB4遺伝子を造血幹細胞に導入すると、造血幹細胞は増幅するものの、急性白血病が誘導される。
3.サイトカイン添加により成熟血球の分化が誘導され、造血幹細胞の未分化性を維持することが難しい。
4.脱メチル化剤などの低分子化合物の添加で造血幹細胞の増幅効率は上がるが、エピジェネティクスに影響を与えるため安全性の確認が必要である。If hematopoietic stem cells are produced in vitro from pluripotent stem cells, or if hematopoietic stem cells are taken out in vitro and cultured, and only hematopoietic stem cells can be amplified without tumor formation, many transplant-related problems will be solved, Not only will the application of hematopoietic stem cell transplantation therapy expand, but it will also open up new regenerative medicine such as the development of other organ-specific stem cell transplantation therapy. However, the following problems have been pointed out in the methods for producing and expanding hematopoietic stem cells that have been developed to date.
1. When the HoxB4 gene is introduced into pluripotent stem cells, hematopoietic stem cells can be produced, but acute leukemia is induced.
2. When the HoxB4 gene is introduced into hematopoietic stem cells, hematopoietic stem cells are expanded but acute leukemia is induced.
3. The addition of cytokine induces differentiation of mature blood cells, and it is difficult to maintain the undifferentiated state of hematopoietic stem cells.
4. Addition of low molecular weight compounds such as demethylating agents increases the efficiency of hematopoietic stem cell expansion, but it is necessary to confirm the safety as it affects epigenetics.
多能性幹細胞はすべての細胞系譜へ理論上分化が可能であるが、生体外においてその分化の運命性を決定する技術は発展途上であり、臨床応用可能な造血幹細胞を多能性幹細胞から作製する技術も発展途上である。多分化能を有する細胞は、ある一定の方向へ分化すると他の系統へ分化する能力を失う性質を保持しており、造血幹細胞以外への分化を抑制すれば、多能性幹細胞から造血幹細胞の製造が期待できる。造血幹細胞は自己複製能と多分化能という一見相反する能力を併せ持ち、サイトカインなどの刺激を受けて成熟血球へと分化する。つまり造血幹細胞を増幅させるためには、自己複製にアクセルをかけながら、様々な細胞系譜への分化にブレーキをかける必要がある。 Although pluripotent stem cells can theoretically differentiate into all cell lineages, the technology to determine the fate of their differentiation in vitro is still under development, and hematopoietic stem cells that can be applied clinically are produced from pluripotent stem cells. The technology to do so is also developing. Pluripotent cells retain the property of losing the ability to differentiate into other lineages when differentiated in a certain direction, and by suppressing the differentiation into cells other than hematopoietic stem cells, pluripotent stem cells are transformed into hematopoietic stem cells. Manufacturing can be expected. Hematopoietic stem cells have the seemingly contradictory abilities of self-renewal and pluripotency, and differentiate into mature blood cells upon stimulation with cytokines. In other words, in order to expand hematopoietic stem cells, it is necessary to accelerate self-renewal and brake differentiation into various cell lineages.
上記問題点を回避しながら新しい造血幹細胞の製造法および増幅法を開発するためには、造血幹細胞以外への細胞分化を抑制する因子、造血幹細胞の自己複製のみを促す因子、または造血幹細胞からの分化にブレーキをかける因子の探索および開発が重要になる。造血幹細胞の生体内での増幅場所は主として胎生期肝臓であり、新規因子の探索および開発にはこの肝臓における造血幹細胞の増幅メカニズムを明らかにし、その成果を試験管内で再現する正攻法をとることが近道である。 In order to develop a new method for producing and expanding hematopoietic stem cells while avoiding the above problems, a factor that suppresses cell differentiation other than hematopoietic stem cells, a factor that promotes only self-renewal of hematopoietic stem cells, or from hematopoietic stem cells The search and development of factors that brake differentiation are important. The place of expansion of hematopoietic stem cells in vivo is mainly in the embryonic liver, and for the search and development of new factors, the mechanism of expansion of hematopoietic stem cells in this liver should be clarified, and a straightforward method to reproduce the results in vitro can be taken. It's a shortcut.
特許文献1は、胎盤構成細胞粉砕物を有効成分として含有する造血幹細胞の増殖剤を開示しているが、このような生物由来の材料は感染や、材料の調製が難しいなどの問題を有する。
特許文献2は、膜タンパク質Thsd1/Tmtspからなる造血幹細胞の維持因子または増幅促進因子を開示しているが、このような膜タンパク質は、活性な形態で調製するのが困難である。
造血幹細胞移植療法は造血器疾患のみならず、自己免疫疾患、悪性固形腫瘍、および再生医療などに幅広く応用されている。現在、移植用造血幹細胞の供給源としては、自家骨髄、ドナー骨髄、および臍帯血が使用されている。ドナー骨髄や臍帯血を使用する場合、移植造血幹細胞の絶対数の不足、ドナー不足、更に免疫応答による生着不全、および移植片対宿主病が問題になっている。 Hematopoietic stem cell transplantation therapy is widely applied not only to hematopoietic diseases but also to autoimmune diseases, solid tumors, and regenerative medicine. Currently, autologous bone marrow, donor bone marrow, and cord blood are used as sources of hematopoietic stem cells for transplantation. When using donor bone marrow or umbilical cord blood, insufficient absolute numbers of transplanted hematopoietic stem cells, insufficient donor, engraftment failure due to immune response, and graft-versus-host disease have become problems.
造血幹細胞を多能性幹細胞から生体外で製造できれば、骨髄、末梢血、臍帯血などの供給源に依存しない新たな治療用ツールとなり得る。また、造血幹細胞を生体外で培養し増幅させることができれば、患者自身の骨髄などから少量の造血幹細胞を採取するだけで移植治療に充分な量の造血幹細胞がいつでも供給可能となり、HLA適合のドナーを待つことなく安全に移植が可能となる。つまり、移植に必要な造血幹細胞数も確保できる上に拒絶反応のない自家移植ができるようになる。自家移植が不可能な場合も、増幅技術が確立できれば、ドナーの骨髄から少量の造血幹細胞を採取するだけで移植治療が可能となり、ドナーのリスクもなくなり骨髄バンクへのドナー登録者数も増え、ドナーが見つかるチャンスの増大が期待できる。臍帯血も生体外で培養して、造血幹細胞を増幅させることができれば、成人への治療が可能となる。 The ability to produce hematopoietic stem cells in vitro from pluripotent stem cells could provide a new therapeutic tool independent of sources such as bone marrow, peripheral blood, and cord blood. Also, if hematopoietic stem cells can be cultured and expanded in vitro, it will be possible to supply a sufficient amount of hematopoietic stem cells for transplantation treatment at any time simply by collecting a small amount of hematopoietic stem cells from the bone marrow of the patient's own, etc. It can be safely ported without waiting. In other words, the number of hematopoietic stem cells required for transplantation can be secured, and autologous transplantation without rejection can be performed. Even if autologous transplantation is not possible, if amplification technology can be established, transplantation treatment will be possible by simply collecting a small amount of hematopoietic stem cells from the bone marrow of the donor, the risk of the donor will disappear, and the number of donors registered in the bone marrow bank will increase, The chances of finding a donor can be expected to increase. If cord blood can also be cultured in vitro to expand hematopoietic stem cells, it will be possible to treat adults.
このように造血幹細胞の生体外での培養および増幅技術の確立は、多くの難治性血液疾患患者の生命を救うことを可能にする。また、遺伝子治療に必要な造血幹細胞を簡単に入手ができるようになり、レトロウイルスを用いない安全な遺伝子導入が可能となり、遺伝子治療の普及も期待できる。造血幹細胞の生体外での培養および増幅技術の開発は、難治性血液疾患患者への移植治療、再生医療遺伝子治療などにとっても緊急に解決すべき重要な技術課題となっている。 Thus, establishment of in vitro culture and expansion technology of hematopoietic stem cells makes it possible to save the lives of many patients with intractable blood diseases. In addition, hematopoietic stem cells required for gene therapy can be easily obtained, safe gene transfer can be performed without using retrovirus, and the spread of gene therapy can be expected. The development of in vitro culture and expansion of hematopoietic stem cells has become an important technical issue to be urgently solved for transplantation therapy for patients with intractable blood diseases, gene therapy for regenerative medicine, and the like.
かかる観点から、本発明は、脊椎動物、特に哺乳動物の造血幹細胞に対して増幅作用を有し、造血幹細胞およびその分化した細胞である造血前駆細胞を生体外で増殖させ、これらを製造するうえで有用な新規なペプチドを提供することを目的とする。また本発明は、造血前駆細胞に対しても増殖作用を有し、成熟造血細胞を生体外で増殖させ、これらを製造するうえで有用な新規なペプチドを提供することを目的とする。 From such a viewpoint, the present invention has a proliferative effect on hematopoietic stem cells of vertebrates, particularly mammals, and proliferates hematopoietic progenitor cells which are hematopoietic stem cells and their differentiated cells in vitro to produce them. The purpose of the present invention is to provide a novel peptide useful in. Another object of the present invention is to provide a novel peptide that has a proliferative effect also on hematopoietic progenitor cells, proliferates mature hematopoietic cells in vitro, and is useful for producing them.
さらに本発明は、当該ペプチドの(i)造血細胞(造血幹細胞、造血前駆細胞、および成熟造血細胞)の増殖助剤としての用途、並びに当該ペプチドを用いた生体外での造血細胞の増殖方法、言い換えると造血細胞の製造方法を提供することを目的とする。 Furthermore, the present invention uses the peptide as (i) a hematopoietic cell (hematopoietic stem cell, hematopoietic progenitor cell, and mature hematopoietic cell) as a growth aid, and a method for proliferating the hematopoietic cell in vitro using the peptide, In other words, it aims to provide a method for producing hematopoietic cells.
本発明者は、造血細胞の増殖場所であるヒト胎仔肝臓の肝芽細胞に発現する各種の膜表面タンパクの膜外ドメインをベースにデザインした複数のペプチドの中に、造血幹細胞の分化増殖を抑制する作用を有するペプチド;骨芽細胞、脂肪細胞、筋細胞、および軟骨細胞などの間葉細胞系譜に分化する能力をもつとされる間葉系幹細胞の自律増殖(増幅)を促進する作用を有するペプチド;並びにES細胞などの多能性幹細胞から造血幹細胞を特異的に誘導する作用を有するペプチドが存在することを見出した(特許文献3参照)。 The present inventor suppresses the differentiation and proliferation of hematopoietic stem cells in a plurality of peptides designed based on the transmembrane domains of various membrane surface proteins expressed in hepatoblasts of human fetal liver, which is the place of proliferation of hematopoietic cells. A peptide having the action of promoting the autonomous proliferation (amplification) of mesenchymal stem cells, which are said to have the ability to differentiate into mesenchymal cell lineages such as osteoblasts, adipocytes, myocytes, and chondrocytes It was found that there are peptides; and peptides having an action of specifically inducing hematopoietic stem cells from pluripotent stem cells such as ES cells (see Patent Document 3).
そして、更なる研究により、上記ペプチドの特定の改変物に、サイトカインなどの細胞刺激因子の存在下、生体外である例えば試験管内で、造血幹細胞を自律増殖(増幅)および分化増殖させる作用があることを確認し、当該ペプチドが造血幹細胞およびその分化した細胞である造血前駆細胞の増殖を補助する「造血幹細胞および造血前駆細胞の増殖助因子」として極めて有効であることを確認した。また当該ペプチドには、細胞刺激因子の存在下、生体外で、造血前駆幹細胞を分化増殖させ、成熟造血細胞を生成する作用があることも確認した。また、生体外で造血幹細胞を維持させる、すなわち造血幹細胞から何らかの細胞に分化させることなく造血幹細胞としての形質を維持させる作用を有することも確認した。 Further studies have shown that specific modifications of the above peptides have the effect of autonomously proliferating (amplifying) and differentiating and proliferating hematopoietic stem cells in the presence of cell stimulating factors such as cytokines in vitro, for example, in vitro. It was confirmed that the peptide was extremely effective as a "proliferation cofactor for hematopoietic stem cells and hematopoietic progenitor cells" that assists the proliferation of hematopoietic stem cells and hematopoietic progenitor cells which are differentiated cells thereof. It was also confirmed that the peptide has an action of differentiating and proliferating hematopoietic progenitor stem cells in vitro in the presence of a cell stimulating factor to generate mature hematopoietic cells. In addition, it was also confirmed that it has an action of maintaining hematopoietic stem cells in vitro, that is, maintaining a trait as a hematopoietic stem cell without differentiating the hematopoietic stem cell into any cell.
このことから、細胞刺激因子と共に、本発明者が開発したペプチドの存在下で造血幹細胞または造血前駆細胞を培養することで、生体外で造血幹細胞、造血前駆細胞、および成熟造血細胞(本発明では、これらを総称して「造血細胞」という。)を増殖させることができると考えられる。つまり、本発明者が開発したペプチド、および細胞刺激因子の存在下で、造血幹細胞または造血前駆細胞を培養することで、生体外で高効率に造血細胞(造血幹細胞、造血前駆細胞、および成熟造血細胞)を増殖させ、これらを培養することができる。このように、本発明のペプチドによれば造血幹細胞を増殖させ、これらを製造することができることから、造血幹細胞移植療法における移植用造血幹細胞の供給源の問題を解消しえる材料として大いに期待される。 From this, by culturing the hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide developed by the present inventor together with the cell stimulating factor, hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells in vitro (in the present invention, , Collectively referred to as "hematopoietic cells"). That is, by culturing the hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide developed by the present inventor, and a cell stimulating factor, the hematopoietic cells (hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic progenitor cells) can be efficiently produced in vitro. Cells) and these can be cultured. As described above, since the peptide of the present invention can proliferate hematopoietic stem cells and produce them, it is highly expected as a material that can solve the problem of the source of hematopoietic stem cells for transplantation in hematopoietic stem cell transplantation therapy. ..
本発明はこれらの知見に基づいて完成したものであり、下記の実施形態を有するものである。
(I)新規ペプチドおよびその修飾物
(I-1)下記一般式(I)で示される(配列番号6)ペプチドまたはその修飾物:The present invention has been completed based on these findings and has the following embodiments.
(I) Novel peptide and modified product thereof (I-1) Peptide represented by the following general formula (I) (SEQ ID NO: 6) or modified product thereof:
〔式中、nは3〜15の整数、xおよびyは0または1(但し、xおよびyの一方が1であるとき、他方yおよびxは0それぞれである)、mおよびpは1〜3の整数、ならびにqは0または1である(但し、yが1であるとき、qは1である)〕。
(I-2)上記一般式(I)で示されるペプチドが、下式のアミノ酸配列(配列番号1)からなるペプチドである(I-1)記載のペプチドまたはその修飾物:[In the formula, n is an integer of 3 to 15, x and y are 0 or 1 (provided that when one of x and y is 1, the other y and x are 0 respectively), m and p are 1 An integer of 3, and q is 0 or 1 (provided that when y is 1, q is 1)].
(I-2) The peptide of (I-1) or a modified product thereof, wherein the peptide represented by the general formula (I) is a peptide having the amino acid sequence of the following formula (SEQ ID NO: 1):
(II)造血細胞の増殖助剤、およびその用法
(II-1)(I-1)または(I-2)に記載するペプチド、その修飾物、これらの薬学的に許容される塩、およびこれらの溶媒和物からなる群から選択される少なくとも1種を有効成分とする、造血細胞の増殖助剤。
(II-2)(I-1)または(I-2)に記載するペプチド、その修飾物、これらの薬学的に許容される塩、およびこれらの溶媒和物からなる群から選択される少なくとも1種、ならびに細胞刺激因子を含有する培地中で、造血幹細胞または造血前駆細胞を培養する工程を有する、造血細胞の増殖方法。
(II-3)上記細胞刺激因子が、幹細胞刺激因子、トロンボポエチン、インターロイキン−6、可溶性インターロイキン−6受容体、顆粒球コロニー刺激因子、インターロイキン−3、インターロイキン−11、およびFlt3リガンドからなる群から選択される少なくとも1種である、(II-2)に記載する造血細胞の増殖方法。
(III)造血細胞の生体外増殖方法
(III-1)(I-1)または(I-2)に記載するペプチド、その修飾物、これらの薬学的に許容される塩、およびこれらの溶媒和物からなる群から選択される少なくとも1種、ならびに細胞刺激因子を含有する培地中で、造血幹細胞または造血前駆細胞を培養する工程を有する、造血細胞の生体外増殖方法。
(III-2)上記細胞刺激因子が、幹細胞刺激因子、トロンボポエチン、インターロイキン−6、可溶性インターロイキン−6受容体、顆粒球コロニー刺激因子、インターロイキン−3、インターロイキン−11、およびFlt3リガンドからなる群から選択される少なくとも1種である、(III-1)に記載する造血細胞の生体外増殖方法。
(IV)造血細胞の生体外製造方法
(IV-1)(I-1)または(I-2)に記載するペプチド、その修飾物、これらの薬学的に許容される塩、およびこれらの溶媒和物からなる群から選択される少なくとも1種、ならびに細胞刺激因子を含有する培地中で、造血幹細胞または造血前駆細胞を培養する工程を有する、造血細胞の生体外製造方法。
(IV-2)上記細胞刺激因子が、幹細胞刺激因子、トロンボポエチン、インターロイキン−6、可溶性インターロイキン−6受容体、顆粒球コロニー刺激因子、インターロイキン−3、インターロイキン−11、およびFlt3リガンドからなる群から選択される少なくとも1種である、(IV-1)に記載する造血細胞の生体外製造方法。
(V)造血細胞を含む細胞集団、およびその用途
(V-1)(III-1)または(III-2)に記載する方法によって得られる、造血細胞を含む細胞集団。
(V-2)(V-1)に記載する細胞集団を有する医薬組成物。
(V-3)造血機能改善剤である(V-2)に記載する医薬組成物。 (II) Hemopoietic cell growth auxiliary agent, peptide described in its usage (II-1) (I-1) or (I-2), modified product thereof, pharmaceutically acceptable salt thereof, and these An agent for proliferating hematopoietic cells, which comprises as an active ingredient at least one selected from the group consisting of solvates of.
(II-2) At least one selected from the group consisting of the peptide described in (I-1) or (I-2), a modified product thereof, a pharmaceutically acceptable salt thereof, and a solvate thereof. A method for proliferating hematopoietic cells, which comprises a step of culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing a seed and a cell stimulating factor.
(II-3) The cell stimulating factor is derived from a stem cell stimulating factor, thrombopoietin, interleukin-6, soluble interleukin-6 receptor, granulocyte colony stimulating factor, interleukin-3, interleukin-11, and Flt3 ligand. The method for proliferating hematopoietic cells according to (II-2), which is at least one selected from the group consisting of:
(III) Peptide described in the method for proliferating hematopoietic cells in vitro (III-1) (I-1) or (I-2), a modified product thereof, a pharmaceutically acceptable salt thereof, and a solvate thereof. A method for proliferating hematopoietic cells in vitro, comprising the step of culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing at least one selected from the group consisting of the following: and a cell stimulating factor.
(III-2) The cell stimulating factor is derived from a stem cell stimulating factor, thrombopoietin, interleukin-6, soluble interleukin-6 receptor, granulocyte colony stimulating factor, interleukin-3, interleukin-11, and Flt3 ligand. The method for proliferating hematopoietic cells in vitro according to (III-1), which is at least one selected from the group consisting of:
(IV) In vitro production method of hematopoietic cells (IV-1) Peptides described in (I-1) or (I-2), modified products thereof, pharmaceutically acceptable salts thereof, and solvates thereof. A method for in vitro production of hematopoietic cells, which comprises a step of culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing at least one selected from the group consisting of the following: and a cell stimulating factor.
(IV-2) The cell stimulating factor is selected from stem cell stimulating factor, thrombopoietin, interleukin-6, soluble interleukin-6 receptor, granulocyte colony stimulating factor, interleukin-3, interleukin-11, and Flt3 ligand. The method for producing a hematopoietic cell in vitro according to ( IV- 1), which is at least one selected from the group consisting of:
(V) A cell population containing hematopoietic cells, and a cell population containing hematopoietic cells obtained by the method described in (V-1) (III-1) or (III-2).
(V-2) A pharmaceutical composition having the cell population described in (V-1).
(V-3) The pharmaceutical composition according to (V-2), which is a hematopoietic function improving agent.
(VI)抗体
(VI-1)(I-1)または(I-2)に記載する少なくとも1種のペプチドに対する抗体。
(VI-2)モノクローナル抗体である(VI-1)に記載する抗体。 (VI) Antibody (VI-1) An antibody against at least one peptide described in (I-1) or (I-2).
(VI-2) The antibody described in (VI-1) which is a monoclonal antibody.
本発明のペプチドの存在下で、好ましくはサイトカインなどの細胞刺激因子とともに、造血幹細胞(例えば自己造血幹細胞または免疫反応が比較的少ない臍帯血造血幹細胞など)を培養することで、生体外の例えば試験管などの培養容器内で、造血幹細胞を自律増殖させてその絶対数を増やしたり、また分化増殖させることで造血前駆細胞および成熟造血細胞の数を増やすことが可能になる。これにより、本発明の課題を解決し、従来の造血幹細胞移植療法における移植用造血幹細胞や造血前駆細胞の供給源の問題を解消することが可能になる。 For example, in vitro by culturing hematopoietic stem cells (such as autologous hematopoietic stem cells or cord blood hematopoietic stem cells with relatively few immune reactions), preferably in the presence of the peptide of the present invention, together with cell stimulating factors such as cytokines. In a culture vessel such as a tube, hematopoietic stem cells can be autonomously proliferated to increase their absolute number, or they can be differentiated and proliferated to increase the number of hematopoietic progenitor cells and mature hematopoietic cells. This makes it possible to solve the problems of the present invention and solve the problem of the supply source of hematopoietic stem cells for transplantation and hematopoietic progenitor cells in conventional hematopoietic stem cell transplantation therapy.
また、本発明のペプチドの存在下で、好ましくは細胞刺激因子とともに、造血前駆細胞を培養することで、生体外の例えば試験管などの培養容器内で、造血幹細胞を分化増殖させて造血成熟細胞数を増やすことが可能になる。 Further, by culturing hematopoietic progenitor cells in the presence of the peptide of the present invention, preferably together with a cell stimulating factor, in a culture container such as a test tube in vitro, the hematopoietic stem cells are differentiated and proliferated to generate hematopoietic mature cells. It is possible to increase the number.
さらに本発明のペプチドおよびその抗体を用いることにより、造血幹細胞の分化増殖および自律増殖のメカニズムを解析することが可能になる。 Furthermore, by using the peptide of the present invention and its antibody, it becomes possible to analyze the mechanism of differentiation and proliferation of hematopoietic stem cells and autonomous proliferation.
本発明で用いる用語の定義
本明細書におけるアミノ酸配列などの略号による表示は、IUPAC−IUBの規定〔IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)〕、「塩基配列またはアミノ酸配列を含む明細書などの作製のためのガイドライン」(特許庁編)および当該分野における慣用記号に従うものとする。 Definitions of terms used in the present invention The abbreviations of amino acid sequences and the like in the present specification refer to IUPAC-IUB rules [IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)], Guidelines for preparation of specifications including base sequences or amino acid sequences "(edited by the Patent Office) and symbols conventionally used in the art.
本発明において「造血幹細胞」とは、未分化性を維持したまま増殖する能力である「自己複製能」と、あらゆる血球系細胞およびリンパ球系細胞に分化する能力である「多分化能」を併せ持ち、数ヶ月以上の長期間にわたり造血を再構築する能力をもった細胞をいう。 In the present invention, "hematopoietic stem cells" means "self-renewal ability", which is the ability to proliferate while maintaining undifferentiated state, and "multipotency", which is the ability to differentiate into all hematopoietic cells and lymphoid cells. In addition, it refers to cells that have the ability to reconstitute hematopoiesis over a long period of several months or more.
ヒト造血幹細胞は、幹細胞マーカーであるCD34を発現していることから、CD34(+)として特徴づけることができる。このため、ヒト造血幹細胞としてCD34陽性細胞を用いることができる。また、ヒト造血幹細胞は、造血幹細胞に発現する細胞表面の抗原に基づいて、CD34に加えて、他の造血幹細胞マーカーで特徴付けることもできる。例えば、CD34を含む造血幹細胞マーカーとしては、Lin(-)、CD34(+)、CD38(-)、DR(-)、CD45(+)、CD90(+)、CD117(+)、CD123(+)、およびCD133(+)を挙げることができる。これらは、1種単独で、または複数組み合わせて用いることができ、組み合わせの態様としては、Lin(-)CD34(+)CD38(-)、およびCD45(+)CD34(+)CD38(-)などを挙げることができる。 Human hematopoietic stem cells express CD34, which is a stem cell marker, and thus can be characterized as CD34 (+). Therefore, CD34-positive cells can be used as human hematopoietic stem cells. Human hematopoietic stem cells can also be characterized by other hematopoietic stem cell markers, in addition to CD34, based on cell surface antigens expressed on hematopoietic stem cells. For example, hematopoietic stem cell markers including CD34, Lin (-), CD34 (+), CD38 (-), DR (-), CD45 (+), CD90 (+), CD117 (+), CD123 (+) , And CD133 (+). These can be used singly or in combination of two or more, and as a combination mode, Lin (-) CD34 (+) CD38 (-), CD45 (+) CD34 (+) CD38 (-), etc. Can be mentioned.
一方マウス造血幹細胞は、胎生期と成体で細胞表面抗原の発現が変化することが知られており、胎生期造血幹細胞のマーカーとして、Lin(-)、CD31(+)、CD34(+)、CD41(+)、c-Kit(+)、成体造血幹細胞のマーカーとして、Lin(-)、CD31(+)、CD34(-/+)、CD45(+)、c-Kit(+)、Sca-1(+)、CD150(+)、EPCR(+)を挙げることができる。これらは、1種単独で、または複数組み合わせて用いることができ、組み合わせの態様としては、Lin(-)c-Kit(+)Sca-1(+)、およびCD45(+)c-Kit(+)Sca-1(+)などを挙げることができる。 On the other hand, mouse hematopoietic stem cells are known to have altered expression of cell surface antigens during the fetal and adult stages, and as markers for fetal hematopoietic stem cells, Lin (-), CD31 (+), CD34 (+), CD41 (+), C-Kit (+), as a marker of adult hematopoietic stem cells, Lin (-), CD31 (+), CD34 (-/ +), CD45 (+), c-Kit (+), Sca-1 (+), CD150 (+), EPCR (+) can be mentioned. These can be used alone or in combination of two or more, and as an embodiment of the combination, Lin (-) c-Kit (+) Sca-1 (+) and CD45 (+) c-Kit (+ ) Sca-1 (+) and the like.
造血幹細胞は、骨髄、末梢血、および臍帯血にごく微量含まれていることが明らかとなっており、これらから上記の幹細胞マーカーを指標としてFACS(fluorescence activated cell sorting)などの慣用方法を用いて採取することができる。 It has been clarified that hematopoietic stem cells are contained in a very small amount in bone marrow, peripheral blood, and umbilical cord blood. From these, using the above-mentioned stem cell marker as an index, a conventional method such as FACS (fluorescence activated cell sorting) is used. Can be collected.
「造血前駆細胞」は、造血幹細胞に由来する細胞であって、終末分化していない細胞をいう。造血前駆細胞は、2〜3系統の血球へと分化できる多能性造血前駆細胞、または1つの血球への分化が限定された単能性造血前駆細胞に分類することができる。造血前駆細胞からは骨髄球系前駆細胞、リンパ球系前駆細胞、または赤血球・巨核球形前駆細胞の3種類の前駆細胞へと分化する。骨髄球系前駆細胞からは、顆粒球(好中球、好酸球、好塩基球)、単球などに最終分化する前駆細胞へと分化する。なお、造血前駆細胞は、上記の系統に関わらず、成熟造血細胞へと分化する細胞であればよい。 “Hematopoietic progenitor cells” are cells that are derived from hematopoietic stem cells and are not terminally differentiated. Hematopoietic progenitor cells can be classified into pluripotent hematopoietic progenitor cells that can differentiate into 2-3 blood cells, or unipotent hematopoietic progenitor cells that have limited differentiation into one blood cell. Hematopoietic progenitor cells are differentiated into three types of progenitor cells: myeloid progenitor cells, lymphoid progenitor cells, or erythroid / megakaryocytic progenitor cells. The myeloid progenitor cells differentiate into progenitor cells that undergo terminal differentiation into granulocytes (neutrophils, eosinophils, basophils), monocytes and the like. The hematopoietic progenitor cells may be cells that differentiate into mature hematopoietic cells regardless of the above lineage.
またリンパ球系前駆細胞からは、T細胞、B細胞やNK細胞に最終分化する前駆細胞へと分化する。このため、造血前駆細胞は、骨髄球系細胞〔顆粒球(好酸球、好中球、好塩基球)、単球、マクロファージ、肥満細胞〕、赤血球系細胞〔赤血球〕、巨核球系細胞〔巨核球、血小板〕、並びにリンパ球系細胞〔T細胞、B細胞、形質細胞〕の前駆細胞であり得る。 In addition, the lymphoid progenitor cells differentiate into progenitor cells that undergo terminal differentiation into T cells, B cells, and NK cells. Therefore, hematopoietic progenitor cells include myeloid cells [granulocytes (eosinophils, neutrophils, basophils), monocytes, macrophages, mast cells], erythroid cells [erythrocytes], megakaryocyte cells [ Megakaryocytes, platelets] as well as progenitor cells of lymphoid cells [T cells, B cells, plasma cells].
「成熟造血細胞」は、上記「造血幹細胞」および「造血前駆細胞」が終末分化した細胞であり、上記するように骨髄球系細胞〔顆粒球(好酸球、好中球、好塩基球)、単球、マクロファージ、肥満細胞〕、赤血球系細胞〔赤血球〕、巨核球系細胞〔巨核球、血小板〕、並びにリンパ球系細胞〔T細胞、B細胞、形質細胞〕を挙げることができる。 “Mature hematopoietic cells” are cells obtained by terminally differentiating the above “hematopoietic stem cells” and “hematopoietic progenitor cells”, and as described above, myelocytic cells [granulocytes (eosinophils, neutrophils, basophils) , Monocytes, macrophages, mast cells], erythroid cells [erythrocytes], megakaryocytes (megakaryocytes, platelets), and lymphoid cells [T cells, B cells, plasma cells].
本発明において「造血細胞」は、前述する造血幹細胞、造血前駆細胞、および成熟造血細胞を、区別することなく、包括的に総称する用語として使用される。 In the present invention, the “hematopoietic cell” is used as a generic term for the above-mentioned hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells, without any distinction.
本発明が対象とする造血細胞は、脊椎動物に由来するものであることが好ましく、より好ましくは鳥類(ニワトリなど)または哺乳類(ヒト、マウス、ラット、ウサギ、サル、チンパンジー、ブタ、ウマ、ヤギ、ヒツジ、ウシ、イヌ、ネコ、ワラビー、カンガルーなど)に由来するものである。好ましくは哺乳類に由来する細胞である。哺乳類の中でも特に好ましくはヒトまたは実験動物として汎用されるげっ歯動物(マウス、ラット、ウサギなど)である。 The hematopoietic cells targeted by the present invention are preferably derived from vertebrates, more preferably birds (such as chicken) or mammals (human, mouse, rat, rabbit, monkey, chimpanzee, pig, horse, goat). , Sheep, cattle, dogs, cats, wallabies, kangaroos, etc.). Preferred are cells derived from mammals. Among mammals, humans or rodents (mouse, rat, rabbit, etc.) commonly used as experimental animals are particularly preferable.
「増殖」とは、終末分化していない細胞と終末分化した細胞の総数を増加させることをいう。ここで終末分化していない細胞には、造血幹細胞および造血前駆細胞が含まれ、終末分化した細胞には造血成熟細胞が含まれる。したがって「造血細胞の増殖」には、造血幹細胞、造血前駆細胞、および造血成熟細胞を区別することなく、これらの細胞の総数を増加させることが含まれる。 “Proliferation” refers to increasing the total number of cells that have not been terminally differentiated and cells that have been terminally differentiated. Here, cells that have not been terminally differentiated include hematopoietic stem cells and hematopoietic progenitor cells, and cells that have been terminally differentiated include hematopoietic mature cells. Thus, "hematopoietic cell proliferation" includes increasing the total number of hematopoietic stem cells, hematopoietic progenitor cells, and hematopoietic mature cells without distinguishing them.
なお、使用する細胞の種類によって増殖する細胞の種類も相違する。例えば細胞として造血幹細胞を使用する場合は、造血幹細胞の増幅(造血幹細胞の未分化性を維持しながら、造血幹細胞が自律増殖すること)に加えて、造血幹細胞から分化した造血前駆細胞が終末分化しない状態で増殖するため、造血幹細胞と造血前駆細胞の総数が増加する。また細胞として造血前駆細胞を使用する場合は、造血前駆細胞が分化増殖するため、造血前駆細胞から終末分化した細胞(造血成熟細胞)の総数が増加する。 The type of cells that grow depends on the type of cells used. For example, when hematopoietic stem cells are used as cells, hematopoietic progenitor cells differentiated from hematopoietic stem cells are subjected to terminal differentiation in addition to expansion of hematopoietic stem cells (autonomous proliferation of hematopoietic stem cells while maintaining undifferentiated state of hematopoietic stem cells). The total number of hematopoietic stem cells and hematopoietic progenitor cells increases because they grow in the absence of hematopoietic stem cells. When hematopoietic progenitor cells are used as cells, the hematopoietic progenitor cells differentiate and proliferate, and thus the total number of cells terminally differentiated from the hematopoietic progenitor cells (hematopoietic mature cells) increases.
細胞数が増加する現象(増殖)には、分化または成熟を伴う「分化増殖」と、分化または成熟を伴わない「自律増殖」の2つがあり、前者の現象を「分化増殖」、後者の現象を「自律増殖」(本発明でいう「増幅」に相当する)と称することがある。なお、成熟とは最終分化の如何に関わらず分化した状態のことを言う。 There are two phenomena (proliferation) in which the number of cells increases (differentiation / proliferation) that involves differentiation or maturation, and “autonomous growth” that does not involve differentiation or maturation. May be referred to as "autonomous growth" (corresponding to "amplification" in the present invention). Note that maturation refers to a state of differentiation regardless of the final differentiation.
本発明において「造血細胞の増殖助剤」とは、造血幹細胞、造血前駆細胞、および造血成熟細胞の区別することなく、これらの造血細胞の総数を増加させるために有効な試薬(製剤)を意味する。これを、例えば、造血幹細胞に対して適用すると、前述するように、造血幹細胞と造血前駆細胞の総数を増加させることができ、また造血前駆細胞に対して適用すると、造血成熟駆細胞の総数を増加させることができる。 In the present invention, the "proliferation aid for hematopoietic cells" means a reagent (formulation) effective for increasing the total number of these hematopoietic cells without distinguishing between hematopoietic stem cells, hematopoietic progenitor cells, and hematopoietic mature cells. To do. When this is applied to, for example, hematopoietic stem cells, the total number of hematopoietic stem cells and hematopoietic progenitor cells can be increased as described above, and when applied to hematopoietic progenitor cells, the total number of hematopoietic mature progenitor cells is increased. Can be increased.
なお、造血幹細胞、造血前駆細胞、および造血成熟細胞の増殖を個別に評価するには、造血細胞マーカーの解析(例えば、FACSによるCD34(+)に対応する細胞の計数)、コロニーアッセイ法に基づく定量的な解析など採用すればよい。 In order to individually evaluate the proliferation of hematopoietic stem cells, hematopoietic progenitor cells, and hematopoietic mature cells, analysis of hematopoietic cell markers (for example, counting of cells corresponding to CD34 (+) by FACS) and colony assay method are used. A quantitative analysis or the like may be adopted.
「臍帯血」とは、哺乳類、好ましくはヒトの臍帯から取得できる血液のことをいう。「骨髄由来血液」とは、哺乳類、好ましくはヒトの骨髄中に存在する髄液に含まれる血液をいう。臍帯血および骨髄は、それぞれ臍帯血バンクおよび骨髄バンクから取得することができる。また、ボランティアから取得する事も可能である。 "Umbilical cord blood" refers to blood that can be obtained from umbilical cords of mammals, preferably humans. "Bone marrow-derived blood" refers to blood contained in cerebrospinal fluid present in the bone marrow of mammals, preferably humans. Cord blood and bone marrow can be obtained from a cord blood bank and a bone marrow bank, respectively. It can also be obtained from volunteers.
(I)新規ペプチド(本発明ペプチド)
本発明が対象とするペプチド(以下、「本発明ペプチド」と称する)は、造血幹細胞および造血前駆細胞に作用して、造血幹細胞、造血前駆細胞および成熟造血細胞を増殖させる作用を有することを特徴とする。 (I) Novel peptide (peptide of the present invention)
The peptide targeted by the present invention (hereinafter referred to as "the peptide of the present invention") has an action of acting on hematopoietic stem cells and hematopoietic progenitor cells to proliferate hematopoietic stem cells, hematopoietic progenitor cells and mature hematopoietic cells. And
当該作用は、具体的には、造血幹細胞に作用して造血幹細胞を増幅させる作用、造血前駆細胞を分化増殖させる作用、および造血前駆細胞に作用して成熟造血細胞を分化増殖させる作用に分類することができる。 Specifically, the action is classified into an action that acts on hematopoietic stem cells to amplify hematopoietic stem cells, an action that differentiates and proliferates hematopoietic progenitor cells, and an action that acts on hematopoietic progenitor cells to differentiate and proliferate mature hematopoietic cells. be able to.
かかるペプチドの一態様として、下記のアミノ酸配列(配列番号1)からなるペプチドを挙げることができる: One aspect of such a peptide is a peptide consisting of the following amino acid sequence (SEQ ID NO: 1):
本発明において、上記ペプチドは最も好ましいペプチドであるが、下記の一般式(I)に示すように、当該アミノ酸配列のN末端側のArgリッチ領域のアルギニン残基の数(n)は、上記の8個に限定されず、例えば3〜15個の範囲から選択することができる。好ましくは7〜15個、より好ましくは7〜11個、特に好ましくは7〜8個である。 In the present invention, the above-mentioned peptide is the most preferable peptide, but as shown in the following general formula (I), the number (n) of arginine residues in the Arg-rich region on the N-terminal side of the amino acid sequence is The number is not limited to eight and can be selected from the range of 3 to 15, for example. It is preferably 7 to 15, more preferably 7 to 11, and particularly preferably 7 to 8.
〔式中、nは3〜15の整数、xおよびyは0または1(但し、xおよびyの一方が1であるとき、他方yおよびxはそれぞれ0である)、mおよびpは1〜3の整数、ならびにqは0または1である(但し、yが1であるとき、qは1である)〕。 [In the formula, n is an integer of 3 to 15, x and y are 0 or 1 (provided that when one of x and y is 1, the other y and x are 0 respectively), m and p are 1 to An integer of 3, and q is 0 or 1 (provided that when y is 1, q is 1)].
また、上記式(1)で示すように、本発明ペプチドは、3〜15個のアルギニン残基からなるArgリッチ領域と、本発明ペプチドのコアになるアミノ酸配列[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser](配列番号2;以下、これを「コア配列」と称する)との間(以下、この部位を「コア配列N側」と称する)に、ペプチド結合を介してポリエチレングリコール鎖(上記一般式(I)中、「(NH-[CH2-CH2-O]m-CO)x」で示す。以下「PEG鎖」とも称する。)を有するものであっても、また当該部位に代えて、コア配列のC末端側(以下、この部位を「コア配列C側」と称する。)にペプチド結合を介してポリエチレングリコール鎖(上記一般式(I)中、「(NH-[CH2-CH2-O]p-CO)y」で示す。以下「PEG鎖」とも称する。)を有するものであってもよい。Further, as shown in the above formula (1), the peptide of the present invention has an Arg-rich region consisting of 3 to 15 arginine residues and an amino acid sequence [Cys Gln Lys Lys Asp Gly Pro Cys] which is a core of the peptide of the present invention. Val Ile Asn Gly Ser] (SEQ ID NO: 2; hereinafter referred to as “core sequence”) (hereinafter, this site is referred to as “core sequence N side”) via a polyethylene glycol chain. (In the above general formula (I), represented by “(NH— [CH 2 —CH 2 —O] m—CO) x”, hereinafter also referred to as “PEG chain”). Instead of the site, a polyethylene glycol chain (in the general formula (I) above, “(NH- [ CH 2 —CH 2 —O] p-CO) y ”, which may hereinafter be referred to as“ PEG chain ”).
なお、コア配列N側またはコア配列C側のいずれか一方にPEG鎖を有する場合、当該PEG鎖は、1単位の[CH2-CH2-O]を有するものであってもよいし(mまたはpが1の場合)、また当該単位が2以上繰り返してなるものであってもよい(重合)。PEG鎖の重合度(m、p)としては、好ましくは2〜3であり、より好ましくは3である。In addition, when having a PEG chain on either the core sequence N side or the core sequence C side, the PEG chain may have one unit of [CH 2 —CH 2 —O] (m Or when p is 1), the unit may be repeated two or more (polymerization). The polymerization degree (m, p) of the PEG chain is preferably 2 to 3, and more preferably 3.
コア配列N側にPEG鎖を有する場合(x=1)、コア配列C側はPEG鎖を有さず(y=0)、また逆にコア配列C側にPEG鎖を有する場合(y=1)、コア配列N側はPEG鎖を有さない(x=0)。また、ポリエチレングリコール鎖(-NH-[CH2-CH2-O]m-CO-、または-NH-[CH2-CH2-O]p-CO-)に代えて、アミノカプロン酸残基(-NH-[CH2]5-CO-)を有するものであってもよい。When the PEG chain is present on the core sequence N side (x = 1), the PEG chain is not present on the core sequence C side (y = 0), and conversely, when the PEG chain is present on the core sequence C side (y = 1). ), The core sequence N side does not have a PEG chain (x = 0). Further, a polyethylene glycol chain (-NH- [CH 2 -CH 2 -O ] m-CO-, or -NH- [CH 2 -CH 2 -O] p-CO-) in place, aminocaproic acid residue ( -NH- [CH 2] 5 -CO-) may have a.
本発明ペプチドは、そのC末端にアミノ基を有するものであってもよいし(q=1)、アミノ基を有さないものであってもよい(q=0)。但し、本発明ペプチドがコア配列C側にPEG鎖を有するものである場合(x=0およびy=1)、そのC末端はアミノ基である(q=1)。また、本発明ペプチドがコア配列C側にPEG鎖を有さないものであって、C末端にアミノ基を有する場合(y=0およびq=1)、C末端のセリンのカルボキシル基はアミド化されている。 The peptide of the present invention may have an amino group at its C terminus (q = 1) or may have no amino group (q = 0). However, when the peptide of the present invention has a PEG chain on the C side of the core sequence (x = 0 and y = 1), its C-terminal is an amino group (q = 1). When the peptide of the present invention does not have a PEG chain on the C side of the core sequence and has an amino group at the C terminus (y = 0 and q = 1), the carboxyl group of serine at the C terminus is amidated. Has been done.
本発明ペプチドは、上記式に基づいて、ペプチド合成機を用いて固相合成法または液体合成法により合成することができ、アミノ酸の置換、付加または欠失は、ペプチド合成機を用いる場合には保護アミノ酸の種類を変えることにより容易に行うことができる。また、ペプチドにPEG鎖を挿入することも、公知の技術に従って行うことができる。PEG鎖の挿入には、例えば、Peptides Internationalから商業的に入手できる「mini-PEG(登録商標)」(Fmoc-mini-PEGTM、Fmoc-mini-PEG-3TM、Boc-mini-PEGTM、Boc-mini-PEG-3TM)を使用することができる。The peptide of the present invention can be synthesized by a solid phase synthesis method or a liquid synthesis method using a peptide synthesizer based on the above formula, and amino acid substitution, addition or deletion can be achieved by using a peptide synthesizer. This can be easily performed by changing the type of protected amino acid. In addition, inserting a PEG chain into a peptide can also be performed according to known techniques. For the insertion of the PEG chain, for example, “mini-PEG (registered trademark)” (Fmoc-mini-PEG ™ , Fmoc-mini-PEG-3 ™ , Boc-mini-PEG ™ , commercially available from Peptides International, Boc-mini-PEG-3 ™ ) can be used.
本発明ペプチドは、フリーの状態であってもよいし、また塩の形態であってもよいし、また水和物を含む溶媒和物の形態を有していてもよい。塩としては、生理学的に許容される、例えば、細胞生物学的または薬学的に許容される酸付加塩や塩基塩を挙げることができる。かかる酸付加塩としては、塩酸塩、臭化水素酸塩、硝酸塩、硫酸塩などの無機酸塩;またはメタンスルホン酸、トルエンスルホン酸などのスルホン酸塩、トリフルオロ酢酸、コハク酸、酢酸などの有機酸塩などが挙げられる。塩基塩としては、ナトリウム、カリウム、リチウムなどのアルカリ金属塩;またはカルシウム、マグネシウムなどのアルカリ土類金属塩などが挙げられる。なお、本発明ペプチドは、L体、D体、またはその両者ののアミノ酸残基から構成されていてもよい。 The peptide of the present invention may be in a free state, a salt form, or a solvate form including a hydrate. Examples of the salt include physiologically acceptable acid addition salts and basic salts that are cell biologically or pharmaceutically acceptable. Such acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, nitrate and sulfate; or sulfonates such as methanesulfonic acid and toluenesulfonic acid; trifluoroacetic acid, succinic acid and acetic acid. Examples thereof include organic acid salts. Examples of the base salt include alkali metal salts such as sodium, potassium and lithium; or alkaline earth metal salts such as calcium and magnesium. The peptide of the present invention may be composed of amino acid residues of L-form, D-form, or both.
上記式(I)で示される本発明ペプチド(配列番号1に示すペプチドを含む。以下、同じ。)は、ペプチド分野で慣用の修飾基を有するものであってもよい。これを本発明ではペプチド修飾物という。 The peptide of the present invention represented by the above formula (I) (including the peptide shown in SEQ ID NO: 1, hereinafter the same) may have a modifying group commonly used in the field of peptides. This is referred to as a peptide modified product in the present invention.
当該修飾物には、一般式(I)で示される本発明ペプチドのC末端がカルボキシル基(-COOH)、カルボキシレート(-COO-)、アミド(−CONH2)、またはエステル(−COOR)であるペプチドが含まれる。ここでエステルにおける基Rとしては、例えばメチル、エチル、n−プロピル、イソプロピル、n−ブチルなどの炭素数1〜6のアルキル基;シクロペンチル、シクロヘキシルなどの炭素数3〜8のシクロアルキル基;C7-14アラルキル基(例えば、フェニル、α-ナフチルなどの炭素数6〜12のアリール基;ベンジル、フェネチルなどのフェニル−C1-2アルキル基;α−ナフチルメチルなどのα-ナフチル−C1-2アルキル基など);ピバロイルオキシメチル基などを例示することができる。本発明ペプチドがC末端以外にカルボキシル基(またはカルボキシレート)を有している場合、そのカルボキシル基がアミド化またはエステル化されているものも本発明のペプチド修飾物に含まれる。The said modifications, in the C-terminal peptide of the invention represented by the general formula (I) is a carboxyl group (-COOH), a carboxylate (-COO-), an amide (-CONH 2), or an ester (-COOR) Certain peptides are included. Here, the group R in the ester includes, for example, an alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl and n-butyl; a cycloalkyl group having 3 to 8 carbon atoms such as cyclopentyl and cyclohexyl; C 7-14 Aralkyl group (for example, aryl group having 6 to 12 carbon atoms such as phenyl and α-naphthyl; phenyl-C 1-2 alkyl group such as benzyl and phenethyl; α-naphthyl-C 1 such as α-naphthylmethyl) -2 alkyl group, etc.); pivaloyloxymethyl group and the like. When the peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the peptide modified product of the present invention.
また本発明ペプチドの修飾物には、N末端のアミノ酸残基(Arg)のアミノ基がC1-6アシル基などの保護基(例えば、ホルミル基、アセチル基などのC1-6アルカノイルなど。)で保護されているもの、脂肪酸(C8-18の飽和脂肪酸)で修飾されているもの、分子内のアミノ酸の側鎖上の置換基(例えば-OH、-SH、アミノ基、イミダゾール基、インドール基、グアニジノ基など)が、C1-6アシル基などの適当な保護基(例えば、ホルミル基、アセチル基などのC1-6アルカノイル基など。)で保護されているものなど、あるいは糖鎖が結合したいわゆる糖ペプチドなどの複合ペプチドなども含まれる。In the modified product of the peptide of the present invention, the amino group of the N-terminal amino acid residue (Arg) is a protecting group such as C 1-6 acyl group (for example, C 1-6 alkanoyl such as formyl group, acetyl group, etc.). ), A fatty acid (C 8-18 saturated fatty acid) -modified, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, an amino group, an imidazole group, Indole group, guanidino group, etc.) protected by a suitable protecting group such as C 1-6 acyl group (eg, C 1-6 alkanoyl group such as formyl group, acetyl group, etc.), or sugar Also included are complex peptides such as so-called glycopeptides in which chains are linked.
さらに本発明ペプチドの修飾物には、イミダゾリル基またはSH基がアルキル化(例えばメチル化)、アラルキル化(例えばベンジル化)、アシル化(例えばアセチル化、ベンゾイル化)されたものも含まれる。なお、上記脂肪酸によるペプチド修飾物としては、N末端のアルギニン残基の、ペプチド主鎖のアミノ基がミリスチン酸で修飾されたミリストイル化ペプチド;またはN末端のアルギニン残基の、側鎖のグアニジル基(グアニジン基またはグアニジノ基ともいう。)を構成するアミノ基がミリスチン酸で修飾された(当該アミノ基とミリスチン酸のカルボキシル基がアミド結合する。)ミリストイル化ペプチドが含まれる。 Further, the modified products of the peptide of the present invention include those in which an imidazolyl group or an SH group is alkylated (eg methylated), aralkylated (eg benzylated) or acylated (eg acetylated, benzoylated). The modified fatty acid peptide includes a myristoylated peptide in which the amino group of the peptide main chain of the N-terminal arginine residue is modified with myristic acid; or the side chain guanidyl group of the N-terminal arginine residue. A myristoylated peptide in which an amino group constituting (also referred to as a guanidine group or a guanidino group) is modified with myristic acid (the amino group and a carboxyl group of myristic acid form an amide bond) is included.
なお、上述するN末端がアセチル化されたペプチドとは、N末端のアルギニン残基のペプチド主鎖のアミノ基がアセチル化(エタノイル化ともいう。)されたもの;またはN末端のアルギニン残基の側鎖のグアニジル基を構成するアミノ基がアセチル化されたものである。 In addition, the above-mentioned N-terminal acetylated peptide means that the amino group of the peptide main chain of the N-terminal arginine residue is acetylated (also referred to as ethanoylation); or the N-terminal arginine residue The amino group constituting the guanidyl group of the side chain is acetylated.
(II)造血細胞の増殖助剤
本発明ペプチドは、そのまま、あるいは必要に応じて生理学的に許容される、例えば細胞生理学的または薬学的に許容し得る担体とともに混合して組成物とした後に、造血細胞の増殖助剤として用いることができる。なお、本発明ペプチド類は1種単独からなるものであっても、2種以上のものを任意に組み合わせて用いることもできる。 (II) hematopoietic cell growth auxiliary agent peptide of the present invention, as it is, or after physiologically acceptable if necessary, for example, after mixing with a physiologically or pharmaceutically acceptable carrier to form a composition, It can be used as a growth aid for hematopoietic cells. The peptides of the present invention may be composed of one kind alone, or may be used in any combination of two or more kinds.
ここで増殖助剤とは、造血幹細胞または造血前駆細胞の増殖に際して、細胞刺激因子とともに用いられる増殖補助剤であり、造血幹細胞または造血前駆細胞の増殖を促す役割を担う。具体的には、造血幹細胞の増殖に際して、細胞刺激因子とともに、かかる増殖助剤を用いることで、造血幹細胞を増幅させ、または造血前駆細胞を分化増殖させ、これらを製造することが可能になる。また造血前駆細胞の増殖に際して、細胞刺激因子とともに、かかる増殖助剤を用いることで、成熟造血細胞を分化増殖させ、これを製造することが可能になる。 Here, the growth auxiliary agent is a growth auxiliary agent used together with a cell stimulating factor in the growth of hematopoietic stem cells or hematopoietic progenitor cells, and has a role of promoting the growth of hematopoietic stem cells or hematopoietic progenitor cells. Specifically, when proliferating hematopoietic stem cells, by using such a growth auxiliary together with a cell stimulating factor, hematopoietic stem cells can be amplified or hematopoietic progenitor cells can be differentiated and proliferated to produce them. Further, when the hematopoietic progenitor cells are proliferated, by using such a growth auxiliary together with the cell stimulating factor, it becomes possible to differentiate and proliferate mature hematopoietic cells and to produce them.
当該増殖助剤は、例えば、本発明ペプチドを、水もしくは適当な緩衝液(例、リン酸緩衝液、PBS、トリス塩酸緩衝液など)中に適当な濃度となるように溶解することにより調製することができる。また、必要に応じて、通常使用される保存剤、安定剤、還元剤、等張化剤などを配合させてもよい。 The growth auxiliary agent is prepared, for example, by dissolving the peptide of the present invention in water or an appropriate buffer solution (eg, phosphate buffer solution, PBS, Tris-HCl buffer solution, etc.) so as to have an appropriate concentration. be able to. Further, if necessary, a preservative, a stabilizer, a reducing agent, an isotonicity agent and the like which are commonly used may be added.
本発明の増殖助剤は、例えば、本発明ペプチドの有効量を培地に添加して、細胞刺激因子の存在下で、造血幹細胞または造血細胞を培養することにより、造血細胞(造血幹細胞、造血前駆細胞および/または成熟造血細胞)を培養して増殖させ、これらを製造するために用いることができる。したがって、本発明はまた、本発明ペプチド(または本発明の増殖助剤)の存在下で、造血幹細胞または造血前駆細胞を培養することを含む、造血細胞の増殖方法を提供する。これについては後述する。 The growth-promoting agent of the present invention is obtained by, for example, adding an effective amount of the peptide of the present invention to a medium and culturing the hematopoietic stem cells or hematopoietic cells in the presence of a cell stimulating factor to give hematopoietic cells (hematopoietic stem cells, hematopoietic precursors Cells and / or mature hematopoietic cells) can be cultured and expanded and used to produce them. Therefore, the present invention also provides a method for expanding hematopoietic cells, which comprises culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide of the present invention (or the growth auxiliary agent of the present invention). This will be described later.
このように本発明ポリペプチド(または本発明の増殖助剤)および細胞刺激因子の存在下で、造血幹細胞または造血前駆細胞を培養することによって、造血細胞の増殖を促進することができる。このため、本発明ペプチドは、造血細胞の生体外増殖用試薬キットの一成分としても利用することができる。かかるキットとは、造血幹細胞を生体外で維持することを目的に用いてもよい。 Thus, by culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of the polypeptide of the present invention (or the growth auxiliary agent of the present invention) and a cell stimulating factor, the proliferation of hematopoietic cells can be promoted. Therefore, the peptide of the present invention can be used as a component of a reagent kit for in vitro proliferation of hematopoietic cells. Such a kit may be used for the purpose of maintaining hematopoietic stem cells in vitro.
(III)造血細胞の生体外増殖方法(造血細胞試験管内増殖法)
(IV)造血細胞の生体外製造方法(造血細胞試験管内製造法)
本発明の造血細胞の増殖方法は、本発明ペプチドの存在下で、造血幹細胞または/および造血前駆細胞を培養する工程を含む。また当該増殖方法は、造血幹細胞または/および造血前駆細胞を培養することで、造血細胞を製造する方法であるといえる。このため、以下の「造血細胞の増殖方法」は「造血細胞の製造方法」と言い換えることができる。 (III) In vitro proliferation method of hematopoietic cells (hematopoietic cell in vitro proliferation method)
(IV) In vitro production method of hematopoietic cells (in vitro production method of hematopoietic cells)
The method for proliferating hematopoietic cells of the present invention includes a step of culturing hematopoietic stem cells or / and hematopoietic progenitor cells in the presence of the peptide of the present invention. It can be said that the proliferation method is a method for producing hematopoietic cells by culturing hematopoietic stem cells or / and hematopoietic progenitor cells. Therefore, the following “method for proliferating hematopoietic cells” can be restated as “method for producing hematopoietic cells”.
本発明の方法に用いる造血幹細胞としては、少なくとも造血幹細胞を含む細胞群が挙げられ、造血幹細胞が単離されたものであってもよい。上記細胞群は、造血幹細胞を含む細胞群から分画された造血幹細胞を含む分画であってもよい。また本発明の方法に用いる造血前駆細胞としても、少なくとも造血前駆細胞を含む細胞群が挙げられ、造血前駆細胞が単離されたものであってもよい。上記細胞群は、造血前駆細胞を含む細胞群から分画された造血前駆細胞を含む分画であってもよい。 Examples of the hematopoietic stem cells used in the method of the present invention include cell groups containing at least hematopoietic stem cells, and hematopoietic stem cells may be isolated. The cell group may be a hematopoietic stem cell-containing fraction that is fractionated from a hematopoietic stem cell-containing cell group. The hematopoietic progenitor cells used in the method of the present invention also include a cell group containing at least hematopoietic progenitor cells, and may be those in which hematopoietic progenitor cells have been isolated. The cell group may be a fraction containing hematopoietic progenitor cells, which is fractionated from a cell group containing hematopoietic progenitor cells.
造血幹細胞や造血前駆細胞の採取源としては、鳥類や哺乳類などの脊椎動物、好ましくはヒトやげっ歯類に属するマウスおよびラットなどの哺乳類の幹細胞を含む組織であればいずれでもよい。例えば、造血幹細胞は、造血幹細胞を含む胎児肝臓、胎児骨髄、骨髄、末梢血、臍帯血、あるいはサイトカインおよび/または抗癌剤の投与によって幹細胞がリクルートした末梢血などから採取することができる。 The source of hematopoietic stem cells or hematopoietic progenitor cells may be any tissue containing vertebrates such as birds and mammals, preferably mammalian stem cells such as mice and rats belonging to humans and rodents. For example, hematopoietic stem cells can be collected from fetal liver, fetal bone marrow, bone marrow, peripheral blood, umbilical cord blood containing hematopoietic stem cells, or peripheral blood to which stem cells have been recruited by administration of cytokines and / or anticancer agents.
本発明ペプチドの存在下で造血幹細胞または造血前駆細胞を培養するにあたっては、いわゆる培養用のプレート、シャーレまたはフラスコを用いた培養法が可能であるが、培地組成、pHなどを機械的に制御し、高密度での培養が可能なバイオリアクターによって、その培養系を改善することもできる(Schwartz, Proc.Natl.Acad.Sci.U.S.A., 88:6760, 1991; Koller, M.R., Bio/Technology, 11:358, 1993; Koller, M.R.,Blood, 82: 378, 1993; Palsson, B.O., Bio/Technology, 11:368,1993)。 In culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide of the present invention, a so-called culturing plate, a culturing method using a petri dish or a flask is possible, but the medium composition, pH, etc. are mechanically controlled. , The culture system can be improved by a bioreactor capable of culturing at high density (Schwartz, Proc. Natl. Acad. Sci. USA, 88: 6760, 1991; Koller, MR, Bio / Technology, 11 : 358, 1993; Koller, MR, Blood, 82: 378, 1993; Palsson, BO, Bio / Technology, 11: 368, 1993).
培養に用いる培地としては、造血幹細胞または造血前駆細胞の増殖、生存が害されない限り特に制限されない。例えば、約5〜約20%の胎児ウシ血清を含む最小必須培地(MEM)、ダルベッコ改変イーグル培地(DMEM)、IMDM培地、RPMI1640培地、199培地、SF-02培地(三光純薬)、Opti-MEM培地(GIBCO BRL)および造血幹細胞培養用培地であるX-VIVO 10(Lonza社)、StemSpan SFEM(無血清増殖培地)およびStemSpan H3000(アニマルコンポーネントフリー培地)(以上、Veritas社)などが好ましいものとして挙げられる。培地のpHとしては、好ましくは6〜8程度を挙げることができる。 The medium used for culturing is not particularly limited as long as the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells are not impaired. For example, minimal essential medium (MEM) containing about 5 to about 20% fetal bovine serum, Dulbecco's modified Eagle medium (DMEM), IMDM medium, RPMI1640 medium, 199 medium, SF-02 medium (Sanko Junyaku), Opti- MEM medium (GIBCO BRL) and hematopoietic stem cell culture medium X-VIVO 10 (Lonza), StemSpan SFEM (serum-free growth medium) and StemSpan H3000 (animal component-free medium) (above, Veritas) are preferable. As. The pH of the medium is preferably about 6-8.
培地には、必要に応じて細胞刺激因子(サイトカイン類やEPO(エリスロポエチン)のような造血ホルモンなど)、インスリンなどのホルモン類、Wnt(Thimoth, A. W., Blood, 89:3624-3635,1997)遺伝子産物のような分化増殖調節因子、トランスフェリンなどの輸送タンパク質、5azaDやTSAなどの脱メチル化剤(Exp. Hematol. 34:140, 2006)、Fibronectin、Collagenなどの細胞外マトリックスタンパク質(Curr Opin Biotechnol. 2008 October ; 19(5): 534-540.; Cell 2007 June; 129(7): 1377-1388.)などをさらに含有させることができる。特に、本発明ペプチド類とともに培地に細胞刺激因子を配合し、本発明ペプチドと細胞刺激因子の存在下で造血幹細胞または造血前駆細胞を培養することによって、造血細胞をより効率的に増殖増幅させることができる。 If necessary, cell stimulating factors (cytokines and hematopoietic hormones such as EPO (erythropoietin)), hormones such as insulin, Wnt (Thimoth, AW, Blood, 89: 3624-3635, 1997) gene may be added to the medium. Differentiation and growth regulators such as products, transport proteins such as transferrin, demethylating agents such as 5azaD and TSA (Exp. Hematol. 34: 140, 2006), extracellular matrix proteins such as Fibronectin and Collagen (Curr Opin Biotechnol. 2008 October; 19 (5): 534-540 .; Cell 2007 June; 129 (7): 1377-1388.) And the like can be further contained. In particular, by adding a cell stimulating factor to a medium together with the peptides of the present invention and culturing the hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptides of the present invention and the cell stimulating factors, the hematopoietic cells can be more efficiently proliferated and amplified. You can
細胞刺激因子とは造血幹細胞や造血前駆細胞などの組織特異的細胞に増殖、分化、生存、遊走などの刺激を与える因子である。このような細胞刺激因子は、造血幹細胞や造血前駆細胞の増殖を妨げないものであれば特に制限されないが、具体的には、SCF(幹細胞成長因子(stem cellfactor))、IL-3(インターロイキン−3)、GM-CSF(顆粒球マクロファージ・コロニー刺激因子(granulocyte/macrophage colony-stimulating factor))、IL-6(インターロイキン−6)、可溶性IL-6受容体(sIL-6R)、IL-11(インターロイキン−11)、Flt-3L(fms様チロシンキナーゼ−3(Flt-3)リガンド)、EPO(エリスロポエチン)、TPO(トロンボポエチン)、G-CSF(顆粒球コロニー刺激因子)、TGF-β(トランスフォーミング成長因子−β)、MIP-1α(George, D., J. Exp. Med. 167:1939-1944, 1988)、Flt3/Flk2-ligand、FGF(繊維芽細胞増殖因子)などが挙げられる。これらの刺激因子などはGallard, R.E., The cytokine facts book, AcademicPress, 1994などに詳しい。 The cell stimulating factor is a factor that gives stimulation to tissue-specific cells such as hematopoietic stem cells and hematopoietic progenitor cells such as proliferation, differentiation, survival and migration. Such a cell stimulating factor is not particularly limited as long as it does not prevent proliferation of hematopoietic stem cells or hematopoietic progenitor cells, and specifically, SCF (stem cell factor), IL-3 (interleukin). -3), GM-CSF (granulocyte / macrophage colony-stimulating factor), IL-6 (interleukin-6), soluble IL-6 receptor (sIL-6R), IL- 11 (interleukin-11), Flt-3L (fms-like tyrosine kinase-3 (Flt-3) ligand), EPO (erythropoietin), TPO (thrombopoietin), G-CSF (granulocyte colony stimulating factor), TGF-β. (Transforming growth factor-β), MIP-1α (George, D., J. Exp. Med. 167: 1939-1944, 1988), Flt3 / Flk2-ligand, FGF (fibroblast growth factor) and the like. Be done. Details of these stimulating factors are described in Gallard, R.E., The cytokine facts book, Academic Press, 1994, etc.
培地に配合する細胞刺激因子は、1種類であっても、2種類以上であってもよい。好ましくは、SCF、G-CSF、IL-3、IL-6、IL-11、Flt-3L、sIL-6R、およびTPOを挙げることができ、とりわけSCFは必須である。培地に添加される細胞刺激因子の濃度としては、1〜500ng/mL、好ましくは5〜300ng/mL、さらに好ましくは10〜100ng/mLである。 The cell stimulating factor to be mixed in the medium may be one type or two or more types. Preferable examples include SCF, G-CSF, IL-3, IL-6, IL-11, Flt-3L, sIL-6R, and TPO, and SCF is essential. The concentration of the cell stimulating factor added to the medium is 1 to 500 ng / mL, preferably 5 to 300 ng / mL, and more preferably 10 to 100 ng / mL.
培養に際して、本発明ペプチドは、培地中の最終濃度が1〜500μg/mL、好ましくは5〜300μg/mL、さらに好ましくは10〜100μg/mLとなるように、上記培地に添加することができる。また、造血幹細胞または造血前駆細胞は、当分野で通常用いられる細胞密度となるように、上記培地に添加することができる。培養は、通常約30〜40℃、約5〜10% CO2の雰囲気下で、所望の増殖が達成される時間で行なわれる。必要に応じて通気や撹拌を行ってもよい。Upon culturing, the peptide of the present invention can be added to the above medium so that the final concentration in the medium is 1 to 500 µg / mL, preferably 5 to 300 µg / mL, and more preferably 10 to 100 µg / mL. In addition, hematopoietic stem cells or hematopoietic progenitor cells can be added to the above-mentioned medium so that the cell density is the one usually used in the art. Culturing is usually performed under an atmosphere of about 30 to 40 ° C. and about 5 to 10% CO 2 for a time at which desired growth is achieved. Aeration and agitation may be performed as necessary.
(V)本発明の増殖方法(製造方法)により得られた造血細胞を含有する細胞集団、およびその用途
前述する本発明の増殖方法(III)または製造方法(IV)により得られた「造血細胞を含有する細胞集団」は、従来の骨髄移植や臍帯血移植に代わる血液細胞移植用または再生医療用の組成物(移植片)として用いることができる。 (V) Cell population containing hematopoietic cells obtained by the proliferation method (production method) of the present invention , and use thereof "Hematopoietic cells obtained by the proliferation method (III) or production method (IV) of the present invention described above Can be used as a composition (graft) for blood cell transplantation or regenerative medicine in place of conventional bone marrow transplantation or cord blood transplantation.
ここで「造血細胞を含有する細胞集団」(以下、単に「細胞集団」ともいう。)とは、前述する本発明の増殖方法(III)または製造方法(IV)によって得られる造血細胞(造血幹細胞、造血前駆細胞、および/または成熟造血細胞)を含む細胞集団を意味する。なお、本発明の増殖方法(III)または製造方法(IV)で得られる細胞集団を、さらにFlow cytometryなどに供し、慣用の技術を用いて造血幹細胞群、造血前駆細胞群、または成熟造血細胞を純化および採取することもできる。 Here, the “cell population containing hematopoietic cells” (hereinafter, also simply referred to as “cell population”) means hematopoietic cells (hematopoietic stem cells) obtained by the above-described proliferation method (III) or production method (IV) of the present invention. , Hematopoietic progenitor cells, and / or mature hematopoietic cells). The cell population obtained by the proliferation method (III) or the production method (IV) of the present invention is further subjected to Flow cytometry and the like, and a hematopoietic stem cell group, a hematopoietic progenitor cell group, or a mature hematopoietic cell is subjected to a conventional technique. It can also be purified and harvested.
本発明の方法により製造および増殖させた細胞集団は、白血病に対する全身X線療法や高度化学療法を行う際に、これらの治療と組み合わせる他、種々の疾患に用いることができる。例えば、固形癌患者の化学療法、放射線療法などの骨髄での造血機能の抑制が副作用として生じる治療を実施する際に、施術前に骨髄を採取しておき、造血幹細胞を試験管内で増幅させ、施術後に患者に戻すことで、副作用による造血系の障害から早期に回復させることができ、より強力な化学療法を行えるようになり、化学療法の治療効果を改善することができる。 The cell population produced and expanded by the method of the present invention can be used for various diseases in addition to these treatments when performing systemic X-ray therapy or advanced chemotherapy for leukemia. For example, when performing a treatment such as chemotherapy of solid cancer patients, suppression of hematopoietic function in bone marrow such as radiation therapy, the bone marrow is collected before the treatment, and hematopoietic stem cells are amplified in vitro, By returning to the patient after the treatment, the hematopoietic system damage due to side effects can be recovered early, more powerful chemotherapy can be performed, and the therapeutic effect of chemotherapy can be improved.
また本発明の細胞集団は、造血機能の障害を伴う疾患、例えば、再生不良性貧血、先天性免疫不全症、先天性代謝異常症、骨髄異形成症候群、白血病、悪性リンパ腫、多発性骨髄腫、骨髄線維症、慢性肉芽腫症、重複免疫不全症候群、無ガンマグロブリン血症、Wiskott-Aldrich症候群、後天性免疫不全症候群(AIDS)などの免疫不全症候群、サラセミア、酵素欠損による溶血性貧血、鎌状赤血球症などの先天性貧血、Gaucher病、ムコ多糖症などのリソゾーム蓄積症、副腎白質変性症などの予防または治療剤として使用され得る。 Further, the cell population of the present invention is a disease associated with impairment of hematopoietic function, for example, aplastic anemia, congenital immunodeficiency, congenital metabolic disorder, myelodysplastic syndrome, leukemia, malignant lymphoma, multiple myeloma, Myelofibrosis, chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, immunodeficiency syndromes such as acquired immunodeficiency syndrome (AIDS), thalassemia, hemolytic anemia due to enzyme deficiency, sickle It can be used as a prophylactic or therapeutic agent for congenital anemia such as erythrocytosis, Gaucher disease, lysosomal storage diseases such as mucopolysaccharidosis, and adrenoleukodystrophy.
かかる細胞集団は、本発明の方法によって増殖または製造した造血細胞の他に、必要に応じて薬理学的に許容し得る担体や緩衝液と共に混合して医薬組成物とした状態で用いることもできる。 Such a cell population can be used in a state of being mixed with a pharmacologically acceptable carrier or a buffer, if necessary, in addition to the hematopoietic cells grown or produced by the method of the present invention to prepare a pharmaceutical composition. ..
ここで、薬理学的に許容される担体としては、製剤素材として慣用の各種有機あるいは無機担体物質が用いられ、例えば、懸濁液剤における懸濁化剤、等張化剤、緩衝剤、無痛化剤などとして配合される。また必要に応じて、防腐剤、抗酸化剤、増粘剤、安定化剤などの製剤添加物を用いることもできる。 Here, as the pharmacologically acceptable carrier, various organic or inorganic carrier substances that are commonly used as pharmaceutical materials are used, and examples thereof include suspending agents in suspensions, isotonic agents, buffers, and soothing agents. It is mixed as an agent. If necessary, formulation additives such as antiseptics, antioxidants, thickeners and stabilizers can also be used.
細胞集団またはそれから調製した医薬組成物の移植(投与)は、従来行われている骨髄移植や臍帯血移植と同様に行えばよく、例えば、非経口的な投与(例、静脈注射、局所注入など)を挙げることができる。医薬組成物の好適な製剤としては、水性および非水性の等張な無菌の注射液剤が挙げられる。 Transplantation (administration) of the cell population or a pharmaceutical composition prepared therefrom may be performed in the same manner as conventional bone marrow transplantation or cord blood transplantation, for example, parenteral administration (eg, intravenous injection, local infusion, etc.). ) Can be mentioned. Suitable formulations of the pharmaceutical composition include aqueous and non-aqueous isotonic sterile injection solutions.
本発明の細胞集団またはそれから調製した医薬組成物の投与量は、本発明ペプチドの活性、病気の重篤度、投与対象となる動物種、投与対象の薬物受容性、性別、体重、年齢などによって異なり一概にいえないが、通常、成人1回あたり造血幹細胞の量として1×106細胞/kg以上、好ましくは1×106〜1×1010細胞/kg、さらに好ましくは、2×106〜1×109細胞/kgである。The dose of the cell population of the present invention or a pharmaceutical composition prepared from the cell population depends on the activity of the peptide of the present invention, the severity of disease, the animal species to be administered, the drug acceptability of the administration target, sex, body weight, age, etc. Although it cannot be said unequivocally, the amount of hematopoietic stem cells per adult is usually 1 × 10 6 cells / kg or more, preferably 1 × 10 6 to 1 × 10 10 cells / kg, more preferably 2 × 10 6 ~ 1 x 10 9 cells / kg.
(VI)本発明ペプチドに対する抗体
本発明はまた、前述する本発明ペプチドに対する抗体を提供する。 (VI) Antibodies Against Peptides of the Present Invention The present invention also provides antibodies to the peptides of the present invention described above.
本発明の抗体には、ポリクローナル抗体およびモノクローナル抗体が含まれる。好ましくはモノクローナル抗体である。 The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies. It is preferably a monoclonal antibody.
好ましい抗体として、本発明ペプチドのうち配列番号1で示すアミノ酸配列からなるペプチドに対する抗体を挙げることができる。本発明のモノクローナル抗体は、鳥類やげっ歯類を含む哺乳類などの脊椎動物、好ましくはヒト、マウスまたはラットなどの哺乳類に由来するイムノグロブリンであり、そのクラスおよびサブクラスは特に限定されない。好ましいクラスおよびサブクラスはイムノグロブリンM(IgM)であり、より好ましくはIgM(κ鎖)である。 Examples of preferred antibodies include antibodies against the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 among the peptides of the present invention. The monoclonal antibody of the present invention is an immunoglobulin derived from vertebrates such as mammals including birds and rodents, preferably mammals such as humans, mice or rats, and its class and subclass are not particularly limited. The preferred class and subclass is immunoglobulin M (IgM), more preferably IgM (kappa chain).
モノクローナル抗体は、Molecular Cloning, A Laboratory Manual, Second Edition(Cold Spring Harbor Laboratory Press, 1989)などに記載されるような公知の方法に従って作製することができる。本発明のモノクローナル抗体は、ヒトに対する抗原性を軽減するという観点から、ヒト化抗体であることが好ましい。ヒト化抗体とは、ヒト以外の動物抗体の可変領域(または超過変領域)以外の部分をヒト免疫グロブリンのアミノ酸配列に置き換えたキメラ抗体であり、本発明ペプチド、特に配列番号1で示すアミノ酸配列からなるペプチドに対する親和性を保持しつつ、ヒトに対する抗原性が軽減された抗体である。ヒト化モノクローナル抗体は、公知の手法に従って作製することができる。 The monoclonal antibody can be produced according to a known method as described in Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989) and the like. The monoclonal antibody of the present invention is preferably a humanized antibody from the viewpoint of reducing antigenicity to humans. The humanized antibody is a chimeric antibody in which a portion other than the variable region (or hypervariable region) of an animal antibody other than human is replaced with the amino acid sequence of human immunoglobulin, and the peptide of the present invention, particularly the amino acid sequence represented by SEQ ID NO: 1. It is an antibody with reduced antigenicity for humans, while retaining the affinity for the peptide consisting of. The humanized monoclonal antibody can be produced according to a known method.
以下、本発明を、実施例および実験例を用いてより詳細に説明する。但し、本発明はこれらの実験例によって何ら制限を受けるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the present invention is not limited to these experimental examples.
実施例1 ペプチドの作製
本発明のペプチドとして、下記の4種類のペプチドをデザイン(以下、ペプチドA〜Dとする。)し、株式会社スクラムに合成を依頼した。下記式中、以下の「PEG」とはエチレングリコール鎖(-CH2-CH2-O-)を意味する。
ペプチドA: [Arg]8-[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]-NH2(配列番号1)
ペプチドB: [Arg]8-[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]-NH-(PEG)3-CO-NH2(配列番号3)
ペプチドC: [Arg]8-NH-(PEG)3-CO-[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser](配列番号4)
ペプチドD: Ac-[Arg]8-NH-(PEG)3-CO-[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]-NH2(配列番号5)。 Example 1 Preparation of Peptide The following four types of peptides were designed as the peptides of the present invention (hereinafter referred to as peptides A to D), and synthesis was requested to Scrum Co., Ltd. In the following formulas, the following as "PEG" refers to ethylene glycol chain (-CH 2 -CH 2 -O-).
Peptide A: [Arg] 8 - [ Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH 2 ( SEQ ID NO: 1)
Peptide B: [Arg] 8 - [ Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH- (PEG) 3 -CO-NH 2 ( SEQ ID NO: 3)
Peptide C: [Arg] 8 -NH- (PEG) 3 -CO- [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] (SEQ ID NO: 4)
Peptide D: Ac- [Arg] 8 -NH- (PEG) 3 -CO- [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH 2 (SEQ ID NO: 5).
[Arg]8とは、8個アルギニンがその側鎖を介さずに直鎖状にペプチド結合したポリアルギニンであることを意味する。[Arg] 8 means that eight arginines are linear arginines that are linearly peptide-bonded without interposing the side chains.
[Arg]8にて示す右端の「-」および[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]の両端にて示す「-」は、それぞれ「-」を介して隣接する基またはアミノ酸残基(ペプチド残基)とアミド結合(またはペプチド結合)していることを意味する。"-" At the right end of [Arg] 8 and "-" at both ends of [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] are groups or amino acids adjacent to each other via "-". It means that it has an amide bond (or peptide bond) with a residue (peptide residue).
ここで、ペプチドAおよびペプチドBに示す[Arg]8-[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]における「-」について、これは[Arg]8内のC末端のアルギニン残基と、「-」を介して隣接する[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]内のN末端に存在するシステイン残基のアミノ基とがペプチド結合していることを示す。Here, shown in peptide A and peptide B [Arg] 8 - "-" in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] For this arginine residue at the C-terminus of the [Arg] 8 And the amino group of the cysteine residue existing at the N-terminal in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] that is adjacent via “-” is peptide-bonded.
ペプチドCおよびペプチドDに示すCO-[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]における「-」について、これは[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]内のN末端に位置するシステイン残基のアミド基と、「-」を介して隣接する「CO」(ケトン基)とがアミド結合を形成していることを意味する。 Regarding "-" in CO- [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] shown in peptide C and peptide D, this means N in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]. It means that the amide group of the cysteine residue located at the terminal and the “CO” (ketone group) adjacent via the “−” form an amide bond.
ペプチドAおよびペプチドDに示す[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]-NH2における「-」について、これは[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]内のC末端に位置するセリン残基のカルボキシル基と、「-」を介して隣接する「NH2」とがアミド結合を形成していることを意味する。すなわち、斯かるC末端のセリン残基はアミド基を有していることを意味する。For the "-" in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH 2 shown in Peptide A and Peptide D, this is within [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] the carboxyl group of a serine residue located at the C-terminus, "-" and "NH 2" adjacent through means that are to form an amide bond. That is, it means that such a C-terminal serine residue has an amide group.
ペプチドBに示す[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]-NHにおける「-」について、これは[Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]内のC末端に位置するセリン残基のカルボキシル基と、「-」を介して隣接する「NH」とがアミド結合を形成していることを意味する。 Regarding "-" in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH shown in peptide B, this is located at the C-terminal in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]. It means that the carboxyl group of the serine residue and the “NH” that is adjacent via the “-” form an amide bond.
ペプチドCおよびペプチドDに示す[Arg]8-NHにおける「-」について、これは[Arg]8内のC末端のアルギニン残基のカルボキシル基と、「-」を介して隣接する「NH」とがアミド結合を形成していることを意味する。Regarding "-" in [Arg] 8 -NH shown in Peptide C and Peptide D, this means that the carboxy group of the C-terminal arginine residue in [Arg] 8 and the adjacent "NH" via "-". Form an amide bond.
ペプチドDに示すAc-[Arg]8における「-」について、これは[Arg]8内のN末端のアルギニン残基のペプチド主鎖のアミド基および/または側鎖のグアニジル基を構成するアミド基がアセチル化(Ac)されていることを意味する。Regarding "-" in Ac- [Arg] 8 shown in Peptide D, this is an amide group constituting the amide group of the peptide main chain of the N-terminal arginine residue in [Arg] 8 and / or the guanidyl group of the side chain. Is acetylated (Ac).
これらのペプチドのうち、ペプチドAを用いて、以下の実験を行った。 The following experiment was performed using peptide A among these peptides.
実験例1 ペプチドAを用いたヒト臍帯血造血幹細胞の増殖
(1)実験方法
(1-1)ヒト臍帯血細胞からFACSで分画したヒト臍帯血CD34陽性造血幹細胞集団(造血幹細胞および分化がやや進んだ造血前駆細胞が混在した細胞集団)を、ペプチドA(10μg/mL)並びに各種細胞刺激因子の混合物(Full:hSCF 50ng/ml、hTPO 10ng/mL、hFIt3L 20ng/mL、hIL-6 20ng/mL、hsIL-6Rα 20ng/mL)を添加した無血清液体培地(StemSpan)で培養して(1.5×104 cells/well, 100μL media/well)、生体外増殖を試みた(本発明試験)。対照試験(コントロール)として、ペプチドAを添加せず、各種細胞刺激因子の混合物(Full:hSCF 50ng/ml、hTPO 10ng/mL、hFIt3L 20ng/mL、hIL-6 20ng/mL、hsIL-6Rα 20ng/mL)を添加した無血清液体培地(StemSpan)を用いて、同様にしてヒト臍帯血CD34陽性造血幹細胞集団を培養した。 Experimental Example 1 Proliferation of Human Cord Blood Hematopoietic Stem Cells Using Peptide A (1) Experimental Method (1-1) Human Cord Blood CD34-Positive Hematopoietic Stem Cell Population Fractionated from Human Cord Blood Cells by FACS Cell population in which hematopoietic progenitor cells are mixed), peptide A (10 μg / mL) and a mixture of various cell stimulating factors (Full: hSCF 50 ng / ml,
そして、培養開始から7日目および11日目に液体培養により得られた細胞について、総細胞数、生細胞の割合(生細胞率)、およびCD34陽性CD38陰性細胞数(造血幹細胞および造血前駆細胞の数)を測定した。生細胞率は、液体培養した細胞を対象としてTrypan Blue染色を行い、計測した生細胞および死細胞数から、生細胞の割合({(生細胞数/(生細胞数+死細胞数))を算出することで求めた。なお、上記で使用した細胞刺激因子は、既存のヒトCD34陽性造血幹細胞の増幅法で使用されている因子であり、これらはヒト造血幹細胞の増幅に作用することが報告されている(Sui X, et al., Proc Natl Acad Sci USA,1995,92,2859-2863; Ebihara Y, et al., Blood 1997, 90, 4363-4368)。 Then, regarding the cells obtained by liquid culture on the 7th and 11th days from the start of the culture, the total number of cells, the ratio of viable cells (viable cell rate), and the number of CD34 positive CD38 negative cells (hematopoietic stem cells and hematopoietic progenitor cells Was measured. The viable cell rate was determined by performing Trypan Blue staining on liquid-cultured cells and calculating the ratio of viable cells from the measured viable and dead cell numbers ({(viable cell number / (viable cell number + dead cell number))). The cell stimulating factors used above were those used in the existing method for expanding human CD34-positive hematopoietic stem cells, and it was reported that these act on the expansion of human hematopoietic stem cells. (Sui X, et al., Proc Natl Acad Sci USA, 1995,92,2859-2863; Ebihara Y, et al., Blood 1997, 90, 4363-4368).
(1-2)培養開始から11日目に液体培養により得られた細胞を、Flow cytometory法を用いて、系統特異的抗原マーカー(CD13、CD34、CD38、CD45、およびGPA)を細胞表面に発現している細胞を分画し、その総数と生細胞の総数に占める割合(%)を算出した(細胞表面マーカー試験)。なお、系統特異的抗原マーカーのうち、CD13は骨髄球系細胞(好中球、好酸球、好塩基球、単球)および骨髄系分化が運命づけられた前駆細胞に発現する糖タンパク質;CD34は、未分化な多能性幹細胞および全系統の造血前駆細胞に発現するリン酸化糖タンパク質;CD38は、活性化T細胞、B細胞、NK細胞、単球、形質細胞などに発現する糖タンパク質;CD45は、全ての白血球系統の細胞(リンパ球、好中球、好酸球、好塩基球、単球)に発現する糖タンパク質;GPA(CD235a)は、ヒト赤血球および赤芽球系細胞に発現する糖タンパク質である。 (1-2) Expression of lineage-specific antigen markers (CD13, CD34, CD38, CD45, and GPA) on the cell surface of cells obtained by liquid culture on the 11th day from the start of the culture using the flow cytometory method Cells were fractionated, and the total number and the ratio (%) to the total number of living cells were calculated (cell surface marker test). Among lineage-specific antigen markers, CD13 is a glycoprotein expressed on myeloid cells (neutrophils, eosinophils, basophils, monocytes) and progenitor cells destined for myeloid differentiation; CD34 Is a phosphorylated glycoprotein expressed on undifferentiated pluripotent stem cells and hematopoietic progenitor cells of all lineages; CD38 is a glycoprotein expressed on activated T cells, B cells, NK cells, monocytes, plasma cells, etc .; CD45 is a glycoprotein expressed on cells of all leukocyte lineages (lymphocytes, neutrophils, eosinophils, basophils, monocytes); GPA (CD235a) is expressed on human erythrocytes and erythroid cells It is a glycoprotein.
(2)実験結果
(2-1)総細胞数および生細胞率の経時的変化
図1(A)および(B)に、それぞれ細胞総数および生細胞率を示す。図1(A)に示すように、ペプチドA(10μg/mL)および細胞刺激因子の存在下で、ヒト臍帯血CD34陽性造血幹細胞を培養することで細胞総数が増加することが確認された。つまりペプチドAにより、ヒト臍帯血CD34陽性造血幹細胞の増殖性が向上することが判明した。一方、細胞生存率は、図1(B)に示すように、ペプチドAを用いた本発明試験と対照試験(コントロール)との結果に差異はみられなかった。このことから、ペプチドAは細胞毒性がないか(細胞死を誘導しない)、あっても極めて低いと考えられる。(2) Experimental results (2-1) Time-dependent changes in total cell number and viable cell rate FIGS. 1 (A) and 1 (B) show the total cell number and viable cell rate, respectively. As shown in FIG. 1 (A), it was confirmed that the total number of cells was increased by culturing human cord blood CD34-positive hematopoietic stem cells in the presence of peptide A (10 μg / mL) and a cell stimulating factor. That is, it was revealed that peptide A improves the proliferative property of human cord blood CD34-positive hematopoietic stem cells. On the other hand, regarding the cell viability, as shown in FIG. 1 (B), no difference was observed between the results of the test of the present invention using peptide A and the control test (control). From this, it is considered that peptide A has no cytotoxicity (does not induce cell death), or even if it is extremely low.
(2-2)CD34陽性CD38陰性細胞数の経時的変化
培養開始から7日目および11日目の培養物のそれぞれから、Flow cytometory法を用いて、造血幹細胞集団であるCD34陽性CD38陰性細胞の総数とその全体(培養によって得られる生細胞の総数)に占める割合を求めた。7日目の結果を図2、11日目の結果を図3にそれぞれ示す。各図において、(A)は培養によって得られる生細胞総数に占めるCD34陽性CD38陰性細胞の割合(%)、(B)はCD34陽性CD38陰性細胞の総数を示す。これらの図から、7〜11日の培養により、生細胞総数に占めるCD34陽性CD38陰性細胞の割合(%)と比較して、CD34陽性CD38陰性細胞の総数が格段に増加していることがわかる。このことから、ペプチドAの存在下でヒト臍帯血CD34陽性造血幹細胞集団を培養することで、CD34陽性CD38陰性細胞、つまり造血幹細胞が増幅し造血前駆細胞が増殖され、純度の高い造血細胞集団が得られることが示唆された。そして、臍帯血に含まれる造血幹細胞は、生体外にて長期間その形質を維持していることも示唆される。(2-2) Time-dependent change in the number of CD34-positive CD38-negative cells From each of the cultures on the 7th day and the 11th day after the start of culture, the flow cytometory method was used to analyze the CD34-positive CD38-negative cells which are hematopoietic stem cell populations. The total number and the ratio of the total number (the total number of living cells obtained by culture) were calculated. The results on the 7th day are shown in FIG. 2 and the results on the 11th day are shown in FIG. 3, respectively. In each figure, (A) shows the ratio (%) of CD34-positive CD38-negative cells to the total number of viable cells obtained by culture, and (B) shows the total number of CD34-positive CD38-negative cells. From these figures, it can be seen that the number of CD34-positive CD38-negative cells is significantly increased by the culture for 7 to 11 days as compared with the ratio (%) of the CD34-positive and CD38-negative cells to the total number of viable cells. .. From this, by culturing the human cord blood CD34-positive hematopoietic stem cell population in the presence of peptide A, CD34-positive CD38-negative cells, that is, hematopoietic stem cells are amplified and hematopoietic progenitor cells are proliferated, resulting in a highly pure hematopoietic cell population. It was suggested to be obtained. It is also suggested that the hematopoietic stem cells contained in cord blood maintain their trait for a long period of time in vitro.
なお、既存のヒトCD34陽性造血幹細胞増幅法で使用されている細胞刺激因子を用いたコントロールでは、CD34陽性細胞、つまり造血幹細胞や造血前駆細胞も増殖するものの、同時にCD34陰性細胞である血球細胞も顕著に増加することが認められ、造血幹細胞や造血前駆細胞の分化増殖を優位に促進していることが確認されている。つまり、上記試験に於いてペプチドAとともに培地に配合した細胞刺激因子は、ヒト造血幹細胞の分化増殖を促す作用があることから、ペプチドAは造血幹細胞の分化増殖をコントロールよりも更に促進する作用を有し、またさらに(可能性として)自律増殖(増幅)を促す作用が有するものと考えられる。そして、臍帯血に含まれる造血幹細胞は、生体外にて長期間その形質を維持していることも示唆される。 In the control using the cell stimulating factor used in the existing human CD34-positive hematopoietic stem cell expansion method, although CD34-positive cells, that is, hematopoietic stem cells and hematopoietic progenitor cells also proliferate, at the same time, hematopoietic cells that are CD34-negative cells also It has been confirmed that the amount is remarkably increased, and it has been confirmed that the differentiation and proliferation of hematopoietic stem cells and hematopoietic progenitor cells are predominantly promoted. That is, in the above test, the cell stimulating factor mixed with the peptide A in the medium has the action of promoting the differentiation and proliferation of human hematopoietic stem cells, and thus the peptide A has the action of further promoting the differentiation and proliferation of hematopoietic stem cells as compared with the control. In addition, it is considered to have an action of promoting (potentially) autonomous proliferation (amplification). It is also suggested that the hematopoietic stem cells contained in cord blood maintain their trait for a long period of time in vitro.
(2-3)細胞表面マーカー試験
細胞表面マーカー試験の結果のうち、各細胞表面マーカーを発現している細胞の総数を図4に、またその全体細胞数に占める割合(%)を図5に示す。(2-3) Cell surface marker test Among the results of the cell surface marker test, the total number of cells expressing each cell surface marker is shown in FIG. 4, and the ratio (%) to the total number of cells is shown in FIG. Show.
この結果から、ペプチドAの存在下でヒト臍帯血CD34陽性造血幹細胞集団を培養することで、CD13陽性(骨髄球系)細胞、CD34陽性(造血幹/前駆)細胞、CD45陽性(白血球系)細胞、GPA陽性(赤血球系)細胞群それぞれの総数が著しく増加しており、ペプチドAの添加により各系統の細胞の増殖が促進されることがわかる。したがって、ペプチドAは、ヒト臍帯血細胞からCD34陽性であることを指標にしてFACSで分画した、ヒト臍帯血CD34陽性造血幹細胞集団に対して造血幹細胞の増幅または分化増殖、造血前駆細胞の分化増殖、および成熟細胞の増殖作用(これらを合わせて造血細胞の増殖作用ともいう。)のみならず、骨髄球系、白血球系、および赤血球系の細胞をも増殖させていることが明らかとなった。 From these results, by culturing a human cord blood CD34-positive hematopoietic stem cell population in the presence of peptide A, CD13-positive (myelocytic) cells, CD34-positive (hematopoietic stem / progenitor) cells, CD45-positive (white blood cell) cells , The total number of each GPA-positive (erythroid) cell group is remarkably increased, and it can be seen that the addition of peptide A promotes the proliferation of cells of each line. Therefore, peptide A was used for the human cord blood CD34-positive hematopoietic stem cell population, which was fractionated by FACS using human umbilical cord blood cells as an index to be CD34-positive, to amplify or differentiate and proliferate hematopoietic stem cells, and to proliferate and differentiate hematopoietic progenitor cells. , And mature cells (also collectively referred to as hematopoietic cell proliferative effect), as well as proliferating myeloid, leukocyte, and erythroid cells.
実験例2 コロニー形成細胞アッセイ(造血前駆細胞数の計測)
実験例1の実験系(コントロール、ペプチドA[10μg/mL]添加)において11日間液体培養した細胞を用いて、コロニー形成細胞アッセイを行い、造血前駆細胞のコロニー〔CFU-G(colony forming unit-granulocyte)、CFU-M(colony forming unit- macrophage)、CFU-GM(colony forming unit-granulocyte/macrophage)、CFU-MK(colony forming unit- megakaryocyte)、BFU-E(burst forming unit-erythrocyte)、CFU-GEMM(colony forming unit-granulocyte/erythrocyte/macrophage/ megakaryocyte)〕の数を計測した。具体的には、11日間液体培養した細胞を、35mm径のペトリ皿に入れたヒト造血前駆細胞コロニー測定用培地(MethoCult H4435 Veritas社)に、1,000 cells/ dishの割合で混合し、マニュアルに従って14日間培養した。なお、上記培地には、細胞刺激因子として、SCF、IL-3、IL-6、G-CSF、GM-CSF、およびEpoが含まれている。結果を図6に示す。 Experimental Example 2 Colony forming cell assay (counting the number of hematopoietic progenitor cells)
A colony-forming cell assay was performed using cells cultured in liquid for 11 days in the experimental system of Experimental Example 1 (control, addition of peptide A [10 μg / mL]), and colonies of hematopoietic progenitor cells [CFU-G (colony forming unit- granulocyte), CFU-M (colony forming unit- macrophage), CFU-GM (colony forming unit-granulocyte / macrophage), CFU-MK (colony forming unit- megakaryocyte), BFU-E (burst forming unit-erythrocyte), CFU -GEMM (colony forming unit-granulocyte / erythrocyte / macrophage / megakaryocyte)] was counted. Specifically, cells cultured in liquid for 11 days are mixed with a medium for measuring human hematopoietic progenitor cell colonies (MethoCult H4435 Veritas) in a Petri dish having a diameter of 35 mm at a rate of 1,000 cells / dish, and then mixed according to the manual. Cultured for a day. The medium contains SCF, IL-3, IL-6, G-CSF, GM-CSF, and Epo as cell stimulating factors. Results are shown in FIG.
図6に示すように、ペプチドA[10μg/mL]を添加して11日間液体培養した細胞において、コントロールと比較して、比較的分化または成熟が進んだ骨髄球系前駆細胞であるCFU-GとCFU-GMの増殖が認められ、ヒト臍帯血CD34陽性造血幹細胞が分化増殖したことが伺えた。これらのことから、ペプチドAは、ヒト臍帯血CD34陽性造血幹細胞が骨髄球系細胞に分化または成熟する過程を促進することで、CFU-GとCFU-GMを増殖させることが明らかになった。 As shown in FIG. 6, CFU-G, which is a myeloid progenitor cell in which the peptide A [10 μg / mL] was added and which was liquid-cultured for 11 days, was relatively differentiated or matured compared with the control. And CFU-GM proliferation was observed, indicating that human cord blood CD34-positive hematopoietic stem cells were differentiated and proliferated. From these results, it was revealed that peptide A promotes the process of differentiation or maturation of human umbilical cord blood CD34-positive hematopoietic stem cells into myeloid cells, thereby proliferating CFU-G and CFU-GM.
図6(B)に、造血前駆細胞(CFU-G、CFU-M、CFU-GM、CFU-MK、BFU-E、およびCFU-GEMM)の総数を示す。これから、ヒト臍帯血CD34陽性造血幹細胞を、細胞刺激因子だけでなくペプチドAの存在下で培養することで、細胞刺激因子だけの存在下で培養した場合(コントロール)に比べて、造血前駆細胞の数が約1.4倍増加することがわかる。 The total number of hematopoietic progenitor cells (CFU-G, CFU-M, CFU-GM, CFU-MK, BFU-E, and CFU-GEMM) is shown in FIG. 6 (B). From this, by culturing human umbilical cord blood CD34-positive hematopoietic stem cells in the presence of not only the cell stimulating factor but also peptide A, compared with the case of culturing in the presence of only the cell stimulating factor (control), It can be seen that the number increases by about 1.4 times.
同様に、11日間液体培養した細胞に代えて7日間液体培養した細胞をコロニー形成細胞アッセイに供した。結果を図7に示す。 Similarly, cells cultured in liquid for 7 days instead of cells cultured in liquid for 11 days were subjected to a colony forming cell assay. The results are shown in Fig. 7.
図7に示すように、ペプチドA[10μg/mL]を添加して7日間液体培養した細胞において、コントロールと比較して、比較的分化または成熟が進んだ骨髄球系前駆細胞であるCFU-GM、BFU-E、およびCFU-GEMMの増殖が認められ、ヒト臍帯血CD34陽性造血幹細胞が分化増殖したことが伺えた。また、これらのことから、ペプチドAは、ヒト臍帯血CD34陽性造血幹細胞が骨髄球系細胞に分化または増殖する過程を促進することで、CFU-GとCFU-GMを分化増殖させることが明らかになった。 As shown in FIG. 7, CFU-GM, which is a myeloid progenitor cell in which the peptide A [10 μg / mL] was added and the liquid culture was carried out for 7 days, was relatively differentiated or matured as compared with the control. , BFU-E, and CFU-GEMM were observed to be proliferated, indicating that human cord blood CD34-positive hematopoietic stem cells were differentiated and proliferated. Further, from these facts, it is clear that peptide A promotes the process in which human cord blood CD34-positive hematopoietic stem cells are differentiated or proliferated into myeloid lineage cells to differentiate and proliferate CFU-G and CFU-GM. became.
以上、ヒト臍帯血CD34陽性造血幹細胞はペプチドAの存在下、11日目のみならず7日目においても骨髄球系細胞に分化または成熟する過程を促進することが明らかとなった。 As described above, it was revealed that human cord blood CD34-positive hematopoietic stem cells promote the process of differentiation or maturation into myeloid cells in the presence of peptide A not only on the 11th day but also on the 7th day.
実験例3 ペプチドAの細胞内の取り込み
ビオチン化したペプチドAを10μg/ML含有する実験例1で用いたものと同じ培地中でヒト臍帯血CD34陽性造血幹細胞を6時間または12時間培養し、培養細胞を回収してこれを標本とした。次いで、核を染色するための(TOTO-3iodide)およびAlexaFluor488標識されたストレプトアビジンで処理して、共焦点蛍光顕微鏡観察に供した。結果を図8に示す。なお、陰性対象としてビオチン化したペプチドAを含まない培地で培養したサンプルを準備した。 Experimental Example 3 Intracellular uptake of peptide A Human cord blood CD34-positive hematopoietic stem cells were cultured for 6 hours or 12 hours in the same medium as used in Experimental Example 1 containing 10 μg / ML of biotinylated peptide A and cultured. The cells were collected and used as a specimen. The cells were then treated with (TOTO-3iodide) for staining nuclei and with AlexaFluor488-labeled streptavidin and subjected to confocal fluorescence microscopy. The results are shown in Fig. 8. As a negative control, a sample cultured in a medium containing no biotinylated peptide A was prepared.
陰性対象では、NucleusおよびMergeともに蛍光顕微鏡写真像の輝度に変化が無いのに対して、ビオチン化したペプチドAでは核染色とペプチドAを合わせたMergeの方が輝度が高く、細胞内にペプチドAが取り込まれていることが明らかとなった。よって、ペプチドAは細胞内でその機能を発揮するものと考えられる。 In the case of the negative target, both Nucleus and Merge had no change in the brightness of the fluorescence micrograph image, whereas in the biotinylated peptide A, the merger with the nuclear staining and the peptide A had higher brightness, and the intracellular peptide A Has been revealed to have been incorporated. Therefore, peptide A is considered to exert its function intracellularly.
実験例4 発現遺伝子の解析
実験例1の実験系(コントロール、ペプチドA[10μg/mL]添加)において、液体培養の開始から1日後および2日後の細胞群からFACSを用いて臍帯血CD34陽性造血幹細胞を回収し、細胞内で発現する遺伝子を調べた。図9に細胞増殖関連遺伝子であるMYC、CCND1、およびCCND2の遺伝子発現量を示す。図10にはSOCSファミリー(SOCS1、SOCS2、SOCS3、SOCS4、SOCS5、SOCS6、およびSOCS7)の培養開始から1日目の遺伝子発現量、そして図11にはSOCSファミリーの培養開始から2日目の遺伝子発現量を示す。 Experimental Example 4 Analysis of Expressed Gene In the experimental system of Experimental Example 1 (control, addition of peptide A [10 μg / mL]), cord blood CD34-positive hematopoiesis using FACS from
ここで、ペプチドAの添加によりCCND1の発現量が低下していることに着目すべきであり、特にペプチドAは細胞周期に影響を及ぼしているものと考えられる。またSOCSファミリーは、JAK-STATシグナルカスケードの阻害因子と考えられているが、中でもSOCS5にペプチドAが影響しているところに着目すべきである。 Here, it should be noted that the expression level of CCND1 is decreased by the addition of peptide A, and it is considered that peptide A particularly affects the cell cycle. The SOCS family is considered to be an inhibitor of the JAK-STAT signal cascade, but it should be noted that peptide A affects SOCS5 among them.
配列番号1〜5は、本発明が対象とする一般式(I)で示されるペプチドまたはその修飾物の具体例を示す。 SEQ ID NOs: 1 to 5 show specific examples of the peptide represented by the general formula (I) or a modified product thereof which is the object of the present invention.
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