JP2016094458A - Methods for culturing dendritic cells - Google Patents
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Abstract
Description
本発明は、樹状細胞の培養方法と、該方法によって得られる樹状細胞を含む医薬品組成物とに関し、具体的には、患者から採取された末梢血から樹状細胞の前駆細胞を大量に選択的に増殖させる培養方法と、該方法によって調製される樹状細胞を含む医薬品組成物とに関する。 The present invention relates to a method for culturing dendritic cells and a pharmaceutical composition containing dendritic cells obtained by the method, and specifically, a large amount of precursor cells of dendritic cells from peripheral blood collected from a patient. The present invention relates to a culture method for selective growth and a pharmaceutical composition comprising dendritic cells prepared by the method.
樹状細胞は、抗原提示細胞として、獲得免疫及び自然免疫の両方を含む免疫系を刺激又は抑制することができる（非特許文献１）。そこで、がん、感染症、自己免疫疾患、アレルギー疾患等に関連する特定の抗原を提示させるように予め体外で処理された樹状細胞を患者に移植することによって、患者の免疫系を制御する、樹状細胞移植療法が試みられている（非特許文献２）。しかし、かかる樹状細胞移植療法には大量の樹状細胞が必要である。例えば、皮膚の腫瘍及び肺転移の実験動物モデル系を用いた研究によると、いずれのモデル系でも、移植する樹状細胞の１回投与の最適数は、実験動物の体重３０ｇあたり少なくとも１０６個であるとの結果が得られた（非特許文献３及び４）。これはヒトに換算すると、患者１人あたり１回投与で少なくとも約１０９個の樹状細胞に相当する。しかし臨床的には、１人の人間から回収できる樹状細胞は、アフェレーシスを行った場合でもせいぜい１０６ないし１０８個にすぎない。 Dendritic cells can stimulate or suppress the immune system including both acquired immunity and innate immunity as antigen-presenting cells (Non-patent Document 1). Therefore, the patient's immune system is controlled by transplanting dendritic cells previously treated outside the body to present specific antigens related to cancer, infectious diseases, autoimmune diseases, allergic diseases, etc. Dendritic cell transplantation therapy has been attempted (Non-patent Document 2). However, such dendritic cell transplantation therapy requires large amounts of dendritic cells. For example, according to studies using experimental animal model systems for skin tumors and lung metastases, the optimal number of dendritic cells to be transplanted in any model system is at least 10 6 per 30 g body weight of experimental animals. The result that it was was obtained (nonpatent literature 3 and 4). This corresponds to at least about 10 9 dendritic cells in a single dose per patient when converted to humans. However, clinically, only 10 6 to 10 8 dendritic cells can be recovered from one person even when apheresis is performed.
１９９０年代に確立された樹状細胞の調製方法では、末梢血、臍帯血、リンパ節等に由来する単球が、ＧＭ−ＣＳＦ及びＩＬ−４の存在下で５ないし７日培養され、その後、微生物又はＴ細胞由来の因子、例えば、ＴＮＦ−α、ＩＬ−１β、ＩＬ−６、ＰＧＥ２等の向炎症性メディエーター等で刺激されて、Ｔ細胞刺激能を獲得した成熟樹状細胞が得られた（特許文献１ないし３）。その後、ＧＭ−ＣＳＦ及びＩＬ−４存在下で１日培養後に、前記向炎症性メディエーター存在下で１日培養するだけの促成調製方法が開発された（特許文献４）。しかし、これらの調製方法では、樹状細胞の前駆細胞から、樹状細胞に分化させるだけであって、樹状細胞の前駆細胞自体の数を実質的に増加させるわけではない。 In the method for preparing dendritic cells established in the 1990s, monocytes derived from peripheral blood, umbilical cord blood, lymph nodes and the like are cultured in the presence of GM-CSF and IL-4 for 5 to 7 days. A mature dendritic cell that has been stimulated with a microbial or T cell-derived factor, for example, a pro-inflammatory mediator such as TNF-α, IL-1β, IL-6, PGE 2 or the like, and has acquired T cell stimulating ability is obtained. (Patent Documents 1 to 3). Thereafter, a forcing preparation method was developed in which culturing was carried out for 1 day in the presence of the pro-inflammatory mediator after culturing for 1 day in the presence of GM-CSF and IL-4 (Patent Document 4). However, these preparation methods only differentiate the dendritic cell progenitor cells into dendritic cells, and do not substantially increase the number of dendritic cell progenitor cells themselves.
樹状細胞の移植による免疫療法の対象となる患者の中には、疾患そのものか、あるいは、他の治療法の副作用かが原因で、造血能力が弱っている場合が多い。特に、現状では免疫療法は他の治療法で効果がない場合に試みられることが多いため、他の治療法の副作用により、アフェレーシスを利用しても、有効な治療に必要な１０９個のオーダーの樹状細胞又はその前駆細胞を用意できないことが治験拡大の妨げとなっている。 Many patients who are candidates for immunotherapy by dendritic cell transplantation have weak hematopoietic potential due to the disease itself or the side effects of other treatments. In particular, at present, immunotherapy is often tried when there is no effect on other treatments. Therefore, due to side effects of other treatments, the order of 10 9 necessary for effective treatment is possible even if apheresis is used. The inability to prepare dendritic cells or their progenitor cells prevents the clinical trial from expanding.
したがって、患者から採取された血液細胞から、試験管内で樹状細胞又はその前駆細胞を増殖させて、前記免疫療法の最適数の樹状細胞を調製する技術を開発する必要がある。 Therefore, it is necessary to develop a technique for preparing an optimal number of dendritic cells for the immunotherapy by growing dendritic cells or their precursor cells in vitro from blood cells collected from patients.
本発明は樹状細胞の調製方法を提供する。本発明の樹状細胞の調製方法は、被検者から採取された血球細胞から単核球を分離するステップと、Ｔ細胞を前記単核球から除去するステップとを含む。 The present invention provides a method for preparing dendritic cells. The method for preparing dendritic cells of the present invention comprises the steps of separating mononuclear cells from blood cells collected from a subject and removing T cells from the mononuclear cells.
本発明の樹状細胞の調製方法において、前記Ｔ細胞を前記単核球から除去するステップは、ＣＤ３を発現する細胞を除去することによって達成される場合がある。 In the method for preparing a dendritic cell of the present invention, the step of removing the T cell from the mononuclear cell may be achieved by removing a cell expressing CD3.
本発明の樹状細胞の調製方法は、前記Ｔ細胞が除去された残りの細胞を、ＧＭ−ＣＳＦ及びＳＣＦを含む培地で培養するステップを含む場合がある。 The method for preparing dendritic cells of the present invention may include a step of culturing the remaining cells from which the T cells have been removed in a medium containing GM-CSF and SCF.
本発明の樹状細胞の調製方法において、前記ＧＭ−ＣＳＦ及びＳＣＦを含む培地で培養するステップは、少なくとも３週間実行され、その後、ＧＭ−ＣＳＦ及びＩＬ−４を含む培地に切り替えて培養するステップを含む場合がある。 In the method for preparing a dendritic cell of the present invention, the step of culturing in a medium containing GM-CSF and SCF is performed for at least 3 weeks, and thereafter, the culture is performed by switching to a medium containing GM-CSF and IL-4. May be included.
本発明の樹状細胞の調製方法において、前記ＧＭ−ＣＳＦ及びＳＣＦを含む培地で培養するステップは、少なくとも４週間実行され、その後、ＧＭ−ＣＳＦ及びＩＬ−４を含む培地に切り替えて培養するステップを含む場合がある。 In the method for preparing a dendritic cell according to the present invention, the step of culturing in a medium containing GM-CSF and SCF is performed for at least 4 weeks, and then the culture is performed by switching to a medium containing GM-CSF and IL-4. May be included.
本発明の樹状細胞の調製方法において、前記血球細胞は、末梢血、臍帯血、骨髄及び／又はリンパ節から採取される場合がある。 In the method for preparing dendritic cells of the present invention, the blood cells may be collected from peripheral blood, umbilical cord blood, bone marrow and / or lymph nodes.
本発明の樹状細胞の調製方法において、前記血球細胞は末梢血からアフェレーシス法により採取される場合がある。 In the method for preparing dendritic cells of the present invention, the blood cells may be collected from peripheral blood by an apheresis method.
本発明は、本発明の樹状細胞の調製方法によって調製される樹状細胞を含む、樹状細胞移植療法用医薬品組成物を提供する。 The present invention provides a pharmaceutical composition for dendritic cell transplantation therapy comprising dendritic cells prepared by the method for preparing dendritic cells of the present invention.
本発明の樹状細胞移植療法用医薬品組成物は、がん又は感染症を治療するための場合がある。 The pharmaceutical composition for dendritic cell transplantation therapy of the present invention may be used for treating cancer or infectious diseases.
本発明の樹状細胞移植療法用医薬品組成物は、自己免疫疾患、アレルギー疾患又は１型糖尿病を治療するための場合がある。 The pharmaceutical composition for dendritic cell transplantation therapy of the present invention may be used to treat autoimmune diseases, allergic diseases or type 1 diabetes.
本発明の樹状細胞移植療法用医薬品組成物は、本発明の調製方法によって調製される樹状細胞と異なるＨＬＡ遺伝子型を有する患者に移植される場合がある。 The pharmaceutical composition for dendritic cell transplantation therapy of the present invention may be transplanted to a patient having an HLA genotype different from the dendritic cell prepared by the preparation method of the present invention.
本発明は、本発明の方法によって調製される樹状細胞を患者に移植するステップを含む、患者の疾患の治療方法を提供する。 The present invention provides a method for treating a disease in a patient comprising the step of transplanting dendritic cells prepared by the method of the present invention into the patient.
本発明の治療方法は、がん又は感染症を治療するための場合がある。 The treatment method of the present invention may be used to treat cancer or infection.
本発明の治療方法は、自己免疫疾患、アレルギー疾患又は１型糖尿病を治療するための場合がある。 The treatment methods of the present invention may be for treating autoimmune diseases, allergic diseases or type 1 diabetes.
本発明の治療方法は、本発明の調製方法によって調製される樹状細胞と異なるＨＬＡ遺伝子型を有する患者に移植するステップを含む場合がある。 The treatment method of the present invention may comprise the step of transplanting into a patient having an HLA genotype different from the dendritic cells prepared by the preparation method of the present invention.
本発明の技術的範囲は、添付する特許請求の範囲の記載に基づいて定められる。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。 The technical scope of the present invention is defined based on the description of the appended claims. Modifications of the present invention, for example, addition, deletion, and replacement of the configuration requirements of the present invention can be made on the condition that the gist of the present invention is not deviated.
本発明の樹状細胞又はその前駆細胞の出所は、末梢血と骨髄とリンパ節と臍帯血とを含むが、これらに限定されない組織の場合がある。本発明の樹状細胞又はその前駆細胞は末梢血から調製されることが好ましい。しかし、胚性幹細胞、成体幹細胞及び人工多能性幹（ｉＰＳ）細胞から生成される造血幹細胞から調製されてもかまわない。 The source of dendritic cells or progenitor cells thereof of the present invention may be tissues including but not limited to peripheral blood, bone marrow, lymph nodes and umbilical cord blood. It is preferable that the dendritic cell or its precursor cell of the present invention is prepared from peripheral blood. However, it may be prepared from hematopoietic stem cells generated from embryonic stem cells, adult stem cells and induced pluripotent stem (iPS) cells.
本発明の樹状細胞又はその前駆細胞は、樹状細胞移植療法の患者自身か、患者と同じ遺伝子型を有する人間かに由来する場合がある。代替的には、前記免疫療法の有効性を損なわないことを条件として、患者と異なる遺伝子型を有する人間に由来する場合がある。 The dendritic cells of the present invention or progenitor cells thereof may be derived from a patient who has undergone dendritic cell transplantation therapy or a human having the same genotype as the patient. Alternatively, it may be derived from a human having a genotype different from that of the patient, provided that the effectiveness of the immunotherapy is not impaired.
本発明の樹状細胞を用意するためには、本技術分野の専門家に知られたさまざまな手順を用いることができる。例えば末梢血から白血球を分離する際には、Ｆｉｃｏｌｌ比重遠心法を用いることができる。前記白血球からＴ細胞を分離除去する際には、Ｉｎｖｉｔｒｏｇｅｎ社から販売されるＤｙｎａｌ社製Ｄｙｎａｂｅａｄｓ（商標）や、ミルテニーバイオテック社のＣｌｉｎｉＭＡＣＳ（商標）を含むがこれらに限定されない免疫磁気ビーズを用いて、細胞表面抗原ＣＤ３を発現する細胞を分離除去することができる。 In order to prepare the dendritic cells of the present invention, various procedures known to those skilled in the art can be used. For example, when separating leukocytes from peripheral blood, Ficoll specific gravity centrifugation can be used. When separating and removing T cells from the leukocytes, immunomagnetic beads including, but not limited to, Dynabeads (trademark) manufactured by Dynal and CliniMACS (trademark) manufactured by Miltenyi Biotech are used. Thus, cells expressing the cell surface antigen CD3 can be separated and removed.
本発明の方法において、Ｔ細胞を除去するのは、単にリンパ球を除去して濃縮するのではない。本明細書の実施例に示すとおり、本発明のＧＭ−ＣＳＦ及びＳＣＦを含むサイトカインカクテルが添加された培地で単核球を培養すると、樹状細胞自体が増殖するよりもＴ細胞の増殖促進が著しく、樹状細胞が非常に希釈されてしまう。これは樹状細胞がＴ細胞と相互作用するが、樹状細胞自体の増殖及び／又は分化が妨げられるためと考えられる。そこで、樹状細胞が不必要にＴ細胞と相互作用しないように、Ｔ細胞を除去するのである。 In the method of the present invention, removing T cells is not merely removing and enriching lymphocytes. As shown in the Examples of the present specification, when mononuclear cells are cultured in a medium to which a cytokine cocktail containing GM-CSF and SCF of the present invention is added, the proliferation of T cells is promoted rather than the proliferation of dendritic cells themselves. Significantly, dendritic cells are very diluted. This is thought to be because dendritic cells interact with T cells, but the growth and / or differentiation of the dendritic cells themselves is hindered. Therefore, the T cells are removed so that the dendritic cells do not unnecessarily interact with the T cells.
本発明の方法において、Ｔ細胞を前記単核球から除去するステップは、Ｔ細胞に発現するが、樹状細胞又はその前駆細胞では発現しないいかなる細胞マーカーを用いて行われてもかまわない。好ましい細胞マーカーは、ＣＤ３か、ＣＤ４及びＣＤ８の組合せかである。また、Ｔ細胞を除去するには、前記細胞マーカーが細胞表面マーカーの場合には、磁気ビーズその他の固相に固定された、当該細胞表面マーカーに対する抗体その他の特異的結合パートナーを用いて、前記単核球の細胞集団から分離される場合がある。あるいは、フロー・サイトメトリー法その他の免疫学的分離手段が用いられる場合がある。また、Ｔ細胞に対する特異的結合パートナーを利用して、Ｔ細胞だけを選択的に傷害又は死滅させる場合がある。なお、前記Ｔ細胞を前記単核球から除去するステップは、他の単核球の細胞タイプ、例えば、Ｂ細胞及び／又はＮＫ細胞をＴ細胞とともに除去するステップであってもかまわない。 In the method of the present invention, the step of removing T cells from the mononuclear cells may be performed using any cell marker that is expressed in T cells but not in dendritic cells or their precursor cells. A preferred cell marker is CD3 or a combination of CD4 and CD8. Further, in order to remove T cells, when the cell marker is a cell surface marker, the antibody against the cell surface marker or other specific binding partner immobilized on a magnetic bead or other solid phase is used. May be isolated from a mononuclear cell population. Alternatively, flow cytometry methods or other immunological separation means may be used. In some cases, only T cells are selectively injured or killed using specific binding partners for T cells. The step of removing the T cells from the mononuclear cells may be a step of removing other mononuclear cell types such as B cells and / or NK cells together with T cells.
得られた細胞を培養するための培地としては、ＩＭＤＭ培地の他、ＭＥＭ、ＤＭＥＭ、ＲＰＭＩ−１６４０、Ｘ−ＶＩＶＯ１５培地を含むが、これらに限らない、血液系細胞の増殖に適する培地が用いられる。これらの培地には、ウシ胎仔血清が添加される場合がある。ウシ胎仔血清の濃度は５ないし２０％の場合があり、１０％がより好ましい。代替策として、ＢｉｏＷｈｉｔｔａｋｅｒ社その他から入手可能なヒトＡＢ型血清や、日本赤十字社から入手可能な献血ヒト血清アルブミンが添加される場合がある。ヒトＡＢ型血清は０ないし１５％の濃度で添加されることが好ましく、献血ヒト血清アルブミンは０ないし１０％の濃度で添加されることが好ましい。 As a medium for culturing the obtained cells, in addition to IMDM medium, MEM, DMEM, RPMI-1640, and X-VIVO15 medium are used, but not limited to these, a medium suitable for the growth of blood cells is used. . Fetal calf serum may be added to these media. The concentration of fetal calf serum may be 5-20%, more preferably 10%. As an alternative, human AB serum available from BioWhittaker et al. Or donated human serum albumin available from Japanese Red Cross may be added. Human AB serum is preferably added at a concentration of 0 to 15%, and blood donated human serum albumin is preferably added at a concentration of 0 to 10%.
前記培地には、ＧＭ−ＣＳＦ及びＳＣＦを含むが、これら以外のサイトカインを含まないサイトカインカクテルか、ＧＭ−ＣＳＦ及びＩＬ−４を含むが、ＳＣＦを含まないサイトカインカクテルかが添加される。これらのサイトカインは、ヒトのアミノ酸配列を有することが好ましく、安全上、組換えＤＮＡ技術で生産されることが好ましい。ＧＭ−ＣＳＦの濃度は、１ないし５００ｎｇ／ｍＬ、１ないし２００ｎｇ／ｍＬ又は１ないし１００ｍｇ／ｍＬの範囲で用いられる場合があり、より好ましくは、２ないし３００ｎｇ／ｍＬ、５ないし２００ｎｇ／ｍＬ、１０ないし１５０ｎｇ／ｍＬ、２０ないし１２０ｎｇ／ｍＬ又は３０ないし１００ｎｇ／ｍＬの範囲で用いられる。ＳＣＦ又はＩＬ−４の濃度は、０．５ないし５００ｎｇ／ｍＬ、０．５ないし１００ｎｇ／ｍＬ又は０．５ないし５０ｎｇ／ｍＬの範囲で用いられる場合があり、より好ましくは、１ないし３００ｎｇ／ｍＬ、２ないし２００ｎｇ／ｍＬ、５ないし１００ｎｇ／ｍＬ、１０ないし７０ｎｇ／ｍＬ、２０ないし６０ｎｇ／ｍＬ又は２５ないし５０ｎｇ／ｍＬの範囲で用いられる。 The medium contains GM-CSF and SCF, but a cytokine cocktail that does not contain any other cytokine or a cytokine cocktail that contains GM-CSF and IL-4 but does not contain SCF. These cytokines preferably have human amino acid sequences, and for safety, are preferably produced by recombinant DNA technology. The concentration of GM-CSF may be used in the range of 1 to 500 ng / mL, 1 to 200 ng / mL, or 1 to 100 mg / mL, more preferably 2 to 300 ng / mL, 5 to 200 ng / mL, 10 It is used in the range of from 150 to 150 ng / mL, from 20 to 120 ng / mL, or from 30 to 100 ng / mL. The concentration of SCF or IL-4 may be used in the range of 0.5 to 500 ng / mL, 0.5 to 100 ng / mL or 0.5 to 50 ng / mL, more preferably 1 to 300 ng / mL. Used in the range of 2 to 200 ng / mL, 5 to 100 ng / mL, 10 to 70 ng / mL, 20 to 60 ng / mL or 25 to 50 ng / mL.
本明細書において、あるサイトカインが含まれない培地とは、正常血清中の当該サイトカインの濃度を著しく超えない範囲、すなわち、正常血清中の濃度の３倍、２倍又は１倍以下であることが好ましく、１／２、１／３又は１／５以下であることがより好ましい。あるいは、当該サイトカインの濃度が０の場合の樹状細胞又はその前駆細胞の挙動と実質的に同じ挙動をすることを条件として、５０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、３０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、５ｎｇ／ｍＬ、３ｎｇ／ｍＬ又は１ｎｇ／ｍＬ以下であることを指す。 In the present specification, a medium not containing a certain cytokine is a range that does not significantly exceed the concentration of the cytokine in normal serum, that is, three times, two times, or less than the concentration in normal serum. Preferably, it is 1/2, 1/3, or 1/5 or less. Alternatively, 50 ng / mL, 40 ng / mL, 30 ng / mL, 20 ng / mL, 10 ng, on the condition that it behaves substantially the same as the behavior of dendritic cells or their progenitor cells when the concentration of the cytokine is 0 / ML, 5 ng / mL, 3 ng / mL or 1 ng / mL or less.
本発明の方法では、ＧＭ−ＣＳＦ及びＳＣＦを含む培地で増殖した樹状細胞の前駆細胞は、ＧＭ−ＣＳＦ及びＩＬ−４を含む培地に切り替えることによって樹状細胞のサブセットの変更が可能になる。ここで、ＧＭ−ＣＳＦ及びＳＣＦを含む培地で増殖する細胞は、いつでもＧＭ−ＣＳＦ及びＩＬ−４によって樹状細胞に分化するように決定された細胞である。したがって、ＧＭ−ＣＳＦ及びＳＣＦを含む培地からＧＭ−ＣＳＦ及びＩＬ−４を含む培地への切り替えは、培養開始後どの時点で行ってもかまわない。しかし、本発明の目的は樹状細胞を大量に調製することであるから、ＧＭ−ＣＳＦ及びＳＣＦを含む培地で培養する期間が長いほどより多くの樹状細胞が得られるので有利である。一方、培養期間が短いと、より多くの患者について樹状細胞移植療法を実施することができるので、有用である。実際には、実施例１に示すとおり、ＧＭ−ＣＳＦ及びＳＣＦを含む培地での培養開始から３週間後まではＣＤ１１ｃ陽性細胞はどんどん増加するが、４週間後になると、増殖速度が低下する。そこで、より大量の樹状細胞を得ることと、より短期間に樹状細胞を得ることとの折り合いをつけるのは、ＧＭ−ＣＳＦ及びＳＣＦを含む培地での増殖が、治療効果に必要な樹状細胞の細胞数（約１０９個）を達成できるとき、又は、増殖速度が低下して、これ以上ＧＭ−ＣＳＦ及びＳＣＦを含む培地で培養しても樹状細胞の前駆細胞があまり増えない状態となるときである。したがって、ＧＭ−ＣＳＦ及びＳＣＦを含む培地からＧＭ−ＣＳＦ及びＩＬ−４を含む培地への切り替えは、治療効果に必要な樹状細胞の細胞数まで増殖することを条件として、培養開始後いつ行ってもかまわない。しかし、ＧＭ−ＣＳＦ及びＳＣＦを含む培地での増殖速度が落ち始める３週間後以降であることが好ましく、ＧＭ−ＣＳＦ及びＳＣＦを含む培地での増殖がほぼ止まる４週間後であることがより好ましい。 In the method of the present invention, dendritic cell progenitor cells grown in a medium containing GM-CSF and SCF can be changed to a dendritic cell subset by switching to a medium containing GM-CSF and IL-4. . Here, cells that grow in a medium containing GM-CSF and SCF are cells that have been determined to differentiate into dendritic cells at any time by GM-CSF and IL-4. Therefore, switching from a medium containing GM-CSF and SCF to a medium containing GM-CSF and IL-4 may be performed at any time after the start of culture. However, since the object of the present invention is to prepare a large amount of dendritic cells, a longer period of culturing in a medium containing GM-CSF and SCF is advantageous because more dendritic cells can be obtained. On the other hand, a short culture period is useful because dendritic cell transplantation therapy can be performed for more patients. Actually, as shown in Example 1, the number of CD11c positive cells increases steadily until 3 weeks after the start of culture in a medium containing GM-CSF and SCF, but the growth rate decreases after 4 weeks. Therefore, a compromise between obtaining a larger amount of dendritic cells and obtaining dendritic cells in a shorter period of time is that the growth in a medium containing GM-CSF and SCF is necessary for the therapeutic effect. When the number of dendritic cells (about 10 9 ) can be achieved, or the proliferation rate decreases, the number of precursor cells of dendritic cells does not increase so much even when cultured in a medium containing GM-CSF and SCF. It is time to enter a state. Therefore, switching from a medium containing GM-CSF and SCF to a medium containing GM-CSF and IL-4 is performed at any time after the start of culture, provided that the cells grow to the number of dendritic cells necessary for the therapeutic effect. It doesn't matter. However, it is preferably after 3 weeks after the growth rate in the medium containing GM-CSF and SCF begins to drop, and more preferably after 4 weeks when the growth in the medium containing GM-CSF and SCF almost stops. .
増殖刺激された細胞は５ないし７％ＣＯ２存在下３７°Ｃで培養される場合がある。細胞は５×１０５個／ｍＬの濃度で播種され、２ないし４日後に培地交換され、新たな培地に２．５×１０５個／ｍＬないし１×１０６個／ｍＬの濃度で播種される場合がある。好ましい播種濃度は５×１０５個／ｍＬである。総数１×１０８個以上の大量培養の場合には、ＧＥ Ｈｅａｌｔｈｃａｒｅ社のＷＡＶＥ Ｂｉｏｒｅａｃｔｏｒ ２／１０（商標）のような装置が用いられる場合がある。 Growth-stimulated cells may be cultured at 37 ° C. in the presence of 5-7% CO 2 . Cells are seeded at a concentration of 5 × 10 5 cells / mL, medium changed after 2 to 4 days, and seeded at a concentration of 2.5 × 10 5 cells / mL to 1 × 10 6 cells / mL in fresh media. There is a case. A preferred seeding concentration is 5 × 10 5 cells / mL. In the case of mass culture of 1 × 10 8 or more in total, an apparatus such as WAVE Bioreactor 2/10 (trademark) manufactured by GE Healthcare may be used.
本発明の方法において、採取された末梢血から分離された単核球は凍結保存される場合がある。また、ＣＤ３発現細胞が除去された後で、残った単球細胞が凍結保存される場合がある。血球細胞の凍結及び解凍は当業者に周知のいかなる方法を用いてもかまわない。細胞の凍結には、株式会社リンフォテック製のバンバンカー（商標）その他の市販の細胞凍結保存液が用いられる場合がある。 In the method of the present invention, mononuclear cells isolated from collected peripheral blood may be cryopreserved. In addition, after the CD3-expressing cells are removed, the remaining monocyte cells may be cryopreserved. Any method known to those skilled in the art may be used for freezing and thawing blood cells. In order to freeze the cells, a commercially available cell cryopreservation solution such as bun bunker (trademark) manufactured by Lymphtec Corporation may be used.
本発明の方法で調製された樹状細胞は、ＯＫ−４３２、ＴＮＦ−α、ＩＬ−１β、ＩＬ−６、ＰＧＥ２等の向炎症性メディエーター等で刺激されて、Ｔ細胞刺激能を獲得した後に、患者に移植される場合がある。癌の治療のためには、それぞれのがんに特異的に発現する抗原で刺激される場合がある。例えば、前立腺癌、非小細胞肺がん及び高リスクメラノーマでは、それぞれ、前立腺酸性ホスファターゼ（ＰＡＰ）、腫瘍抗原ＭＡＧＥ−Ａ３及び癌精巣抗原ＮＹ−ＥＳＯ−１で刺激される場合がある。単一の癌に対する治療のために、複数の癌特異的抗原でそれぞれ単独に刺激された樹状細胞を組み合わせたり、あるいは、複数の癌特異的抗原で同時に刺激された樹状細胞を用いたりする場合もある。 Dendritic cells prepared by the method of the present invention were stimulated with pro-inflammatory mediators such as OK-432, TNF-α, IL-1β, IL-6, and PGE 2 to acquire T cell stimulating ability. Later, it may be transplanted into the patient. For the treatment of cancer, there are cases where stimulation is caused by an antigen specifically expressed in each cancer. For example, prostate cancer, non-small cell lung cancer, and high-risk melanoma may be stimulated with prostate acid phosphatase (PAP), tumor antigen MAGE-A3, and cancer testis antigen NY-ESO-1, respectively. For the treatment of a single cancer, combine dendritic cells that are individually stimulated with multiple cancer-specific antigens, or use dendritic cells that are simultaneously stimulated with multiple cancer-specific antigens. In some cases.
本発明の樹状細胞を含む医薬品組成物は、本発明の方法で調製された樹状細胞とともに、他の療法のための薬剤、例えば、化学療法剤、放射線療法剤が配合される場合がある。かかる薬剤には、ＣＤＤＰ、ＴＳ−１、Ｇｅｍｚａｒ等の癌細胞の増殖を抑制する薬剤や、ＩＬ−１２ｐ７０、ＰＧＥ２等の癌細胞を攻撃する細胞傷害性Ｔ細胞の活性を増強する因子の組換えタンパク質や、ＩＬ−１０等の前記細胞傷害性Ｔ細胞の活性を抑制する因子に対する阻害剤、例えば、抗体、短鎖干渉ＲＮＡ等が含まれるが、これらに限定されない。 The pharmaceutical composition containing the dendritic cell of the present invention may be mixed with a dendritic cell prepared by the method of the present invention together with another therapeutic agent such as a chemotherapeutic agent or a radiation therapeutic agent. . Such agents include recombination of agents that suppress the growth of cancer cells such as CDDP, TS-1, and Gemzar, and factors that enhance the activity of cytotoxic T cells that attack cancer cells such as IL-12p70 and PGE2. Examples include, but are not limited to, inhibitors for proteins and factors that suppress the activity of cytotoxic T cells such as IL-10, such as antibodies and short interfering RNAs.
本発明の医薬品組成物は、医薬品として許容される担体を含む場合がある。生細胞を懸濁することができるいずれかの溶液、例えば、生理食塩水、リン酸緩衝生理食塩水（ＰＢＳ）、培地、血清等が代表例である。また、樹状細胞をワクチンとして使用する場合には、免疫原性を増強するために、サイトカイン、細菌毒素、結核菌菌体成分等の免疫促進剤や、ミョウバン、フロイントアジュバントその他のアジュバント剤を含むが、これらに限定されない、免疫原性増強剤を本発明の医薬品組成物は含む場合がある。 The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Representative examples are any solution capable of suspending living cells, such as physiological saline, phosphate buffered saline (PBS), medium, serum, and the like. In addition, when dendritic cells are used as vaccines, in order to enhance immunogenicity, immunosuppressants such as cytokines, bacterial toxins, Mycobacterium tuberculosis, and other adjuvants such as alum, Freund's adjuvant, etc. However, the pharmaceutical composition of the present invention may contain an immunogenicity enhancer, which is not limited thereto.
以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。 The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims. Modifications of the present invention, for example, addition, deletion, and replacement of the configuration requirements of the present invention can be made on the condition that the gist of the present invention is not deviated.
末梢血からの採血
健康なボランティア被検者から末梢血が採取された。本実験は、九州大学医系地区部局臨床研究倫理審査委員会の承認（承認日：平成22年３月１２日）を得て実施され、前記ボランティア被検者からは書面による同意が得られている。採血には、２２〜２３Ｇ針を取り付けた真空採血管（ＴＥＲＵＭＯ ベノジェクトＩＩ ＥＤＴＡ−２Ｎａ）が用いられた。
Blood collection from peripheral blood Peripheral blood was collected from healthy volunteer subjects. This experiment was conducted with the approval of the Kyushu University Medical District Department Clinical Research Ethics Review Board (approval date: March 12, 2010), and written consent was obtained from the volunteer subjects. Yes. For blood collection, a vacuum blood collection tube (TERUMO Venoject II EDTA-2Na) equipped with a 22-23G needle was used.
末梢血からの単核球の分離
得られた血液は、常温に保たれた希釈液（１ｍＭ ＥＤＴＡ及び２％ウシ胎仔血清が添加されたＰＢＳ）で２倍希釈され、各遠心管に、希釈血２０ないし３５ｍＬが、１０ないし１５ｍＬのＦｉｃｏｌｌ Ｐａｑｕｅ（比重１．０７７）に重層された。遠心は、５８０×ｇ、室温で２０分間行われ、ブレーキをかけずに停止された。遠心上清（血漿部分）は数ｍＬを残して除去され、中間層が回収された。遠心管１ないし２本から回収された前記中間層が１本の新たな遠心管に集められ、前記希釈液により体積が５０ｍＬに調整された。２回目の遠心は、４２０×ｇ、室温、５分間又は１５分間の条件で行われた。上清は除去され、ペレットが、前記希釈液３０ｍＬに懸濁された。３回目の遠心は、２５０×ｇ、室温１０分間の条件で行われた。上清は除去され、ペレットは、細胞濃度が１×１０７個／ｍＬになるように、２ｍＭ ＥＤＴＡと、０．１％ＢＳＡとが添加されたＰＢＳに懸濁された（以下、「単核球懸濁液」という。）。
Separation of mononuclear cells from peripheral blood The obtained blood was diluted 2-fold with a diluent kept at room temperature (PBS supplemented with 1 mM EDTA and 2% fetal calf serum), and diluted blood was added to each centrifuge tube. 20 to 35 mL was overlaid on 10 to 15 mL of Ficoll Paque (specific gravity 1.077). Centrifugation was performed at 580 × g for 20 minutes at room temperature and stopped without braking. The centrifugal supernatant (plasma part) was removed leaving a few mL, and the intermediate layer was recovered. The intermediate layer collected from one or two centrifuge tubes was collected in one new centrifuge tube, and the volume was adjusted to 50 mL with the diluent. The second centrifugation was performed under the conditions of 420 × g, room temperature, 5 minutes or 15 minutes. The supernatant was removed and the pellet was suspended in 30 mL of the diluent. The third centrifugation was performed under conditions of 250 × g and room temperature for 10 minutes. The supernatant was removed, and the pellet was suspended in PBS supplemented with 2 mM EDTA and 0.1% BSA so that the cell concentration was 1 × 10 7 cells / mL (hereinafter referred to as “mononuclear”). Sphere suspension ”).
ＣＤ３発現細胞の除去
抗ＣＤ３抗体が固定化された磁気ビーズ（Ｄｙｎａｂｅａｄｓ ＣＤ３）は、０．１％ＢＳＡが添加されたＰＢＳで１回洗浄された後、前記単核球懸濁液に細胞１０７個あたり５０μＬが添加された。前記ビーズを含む単核球懸濁液は、４°Ｃで３０分間ローテータにて攪拌された。その後、前記磁気ビーズは磁石によって前記懸濁液から分離され、ＣＤ３を表面に発現する細胞が除去された。
CD3 magnetic beads removal of cells expressing the anti-CD3 antibody is immobilized (Dynabeads CD3), after being washed once with 0.1% BSA was added PBS, the cells 10 in the monocyte suspension 7 50 μL was added per piece. The mononuclear cell suspension containing the beads was stirred with a rotator at 4 ° C. for 30 minutes. Thereafter, the magnetic beads were separated from the suspension by a magnet, and cells expressing CD3 on the surface were removed.
ＣＤ３発現細胞が除去された細胞集団の培養
前記懸濁液に残った細胞（以下、「ＣＤ３−細胞」という。）は、１００ｎｇ／ｍＬの組換えヒトＧＭ−ＣＳＦと、５０ｎｇ／ｍＬの組換えヒトＳＣＦと、１０％ウシ胎仔血清と、抗生物質（ペニシリンＧ１００単位／ｍＬ、硫酸ストレプトマイシン１００μｇ／ｍＬ）とが添加されたＩＭＤＭ（以下、「ＧＭ／ＳＣＦ培地」という。）で５×１０５個／ｍＬになるように希釈され、２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）を含む生体適合性ポリマーでコーティングされた６ウェルプレート（リピジュアコートプレート（登録商標）、日油株式会社）で培養された。
Culture of cell population from which CD3-expressing cells have been removed Cells remaining in the suspension (hereinafter referred to as “CD3 - cells”) were recombinant with 100 ng / mL recombinant human GM-CSF and 50 ng / mL recombinant. 5 × 10 5 IMDM (hereinafter referred to as “GM / SCF medium”) supplemented with human SCF, 10% fetal bovine serum, and antibiotics (penicillin G100 units / mL, streptomycin sulfate 100 μg / mL). The cells were cultured in a 6-well plate (Lipidure Coated Plate (registered trademark), NOF Corporation) coated with a biocompatible polymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC).
ＣＤ３発現細胞を含む単核球集団の培養
対照実験として、ＣＤ３発現細胞を含む単核球集団が培養された。前記単核球懸濁液２００μＬ（２×１０６個）が４００ないし８００×ｇ、５分間の条件で遠心され、得られたペレットが前記ＧＭ／ＳＣＦ培地３ｍＬに懸濁され、ＣＤ３−細胞の培養に用いられるのと同じＭＰＣポリマーでコーティングされた６ウェルプレートで培養された。
Culture of mononuclear cell population containing CD3 expressing cells As a control experiment, a mononuclear cell population containing CD3 expressing cells was cultured. The mononuclear cell suspension 200μL (2 × 10 6 cells) are centrifuged at 400 to 800 × g, 5 min, the resulting pellet is suspended in the GM / SCF medium 3 mL, CD3 - cells Incubated in 6-well plates coated with the same MPC polymer used for culture.
培地交換
培地交換は３ないし４日ごとに行われた。細胞が回収され、４００×ｇ、５分間の遠心により培地が除去され、ペレットの細胞が、２．５ないし５×１０５個／ｍＬの濃度になるように前記ＧＭ／ＳＣＦ培地に懸濁され、ＭＰＣポリマーでコーティングされた新たな６ウェルプレートで培養された。
Medium change Medium change was performed every 3 to 4 days. Cells are collected, the medium is removed by centrifugation at 400 × g for 5 minutes, and the pellet cells are suspended in the GM / SCF medium to a concentration of 2.5 to 5 × 10 5 cells / mL. Incubated in new 6-well plates coated with MPC polymer.
培養中の細胞のモニタリング
培養開始から１週間ごとに、当業者に周知の試薬、装置及び手順に従って、細胞数の計測の他、細胞表面マーカーＣＤ３、ＣＤ１１ｃ及びＣＤ１４の発現がフロー・サイトメトリー法で解析された。
Monitoring of cells in culture Every week after the start of culture, the expression of cell surface markers CD3, CD11c and CD14 is determined by flow cytometry in addition to the measurement of cell number according to reagents, devices and procedures well known to those skilled in the art. Was analyzed.
結果
図１Ａ及び２Ａは、末梢血から分離された単核球をＧＭ／ＳＣＦ培地で、それぞれ１及び２週間培養後、ＣＤ３及びＣＤ１１ｃに対する抗体で２重染色しフロー・サイトメトリー法で解析した結果図である。図１Ｂ及び２Ｂは、末梢血から分離された単核球をＧＭ／ＩＬ−４培地で１及び２週間培養後、ＣＤ３及びＣＤ１１ｃに対する抗体で２重染色しフロー・サイトメトリー法で解析した結果図である。図１Ａの条件では、ＣＤ１１ｃ陽性かつＣＤ３陰性の細胞は２７．９％、ＣＤ１１陰性かつＣＤ３陽性の細胞は４４．２９％であったが、図１Ｂの条件では、それぞれ、１０．６６％及び５５．３１％であった。これに対し、図２Ａの条件では、ＣＤ１１ｃ陽性かつＣＤ３陰性の細胞は５．１６％、ＣＤ１１陰性かつＣＤ３陽性の細胞は７５．３１％であったが、図１Ｂの条件では、それぞれ、２．３２％及び７４．２９％であった。ＣＤ１１ｃ陽性かつＣＤ３陰性の細胞は、樹状細胞又はその前駆細胞である。これらの結果から明らかなとおり、ＧＭ／ＳＣＦ培地のほうがＧＭ／ＩＬ−４培地よりも樹状細胞又はその前駆細胞の割合が高くなった。しかし、いずれの培地でも、１週間より２週間のほうが樹状細胞又はその前駆細胞の割合が急激に下がり、逆にＣＤ１１陰性かつＣＤ３陽性の細胞の割合は非常に高くなった。ＣＤ３陽性の細胞はＴ細胞であり、試験管内でも樹状細胞はＴ細胞と相互作用してＴ細胞を刺激できることが知られている。そこで、これらの結果から、Ｔ細胞が共存すると、樹状細胞はＴ細胞を刺激するが、樹状細胞自体の増殖及び／又は分化が妨げられた可能性がある。実際に、図３Ａ及びＢの結果から、ＣＤ１１ｃ陰性の細胞のうち図３Ａでは８７．０７％、図３Ｂでは８８．２７％と、約９割がＣＤ３陽性、すなわち、Ｔ細胞系列の細胞であった。そこで、培養開始前に末梢血由来の単核球からＴ細胞を除去することで、Ｔ細胞との相互作用に煩わされることなく樹状細胞自体の増殖及び分化に専念させることができるのではないか、との発想が得られた。
Results FIG. 1A and FIG. 2A show the results obtained by analyzing mononuclear cells isolated from peripheral blood in GM / SCF medium for 1 and 2 weeks, respectively, and then double-staining with antibodies against CD3 and CD11c and analyzing by flow cytometry. FIG. FIGS. 1B and 2B show the results obtained by culturing mononuclear cells isolated from peripheral blood in GM / IL-4 medium for 1 and 2 weeks and then double-staining with antibodies against CD3 and CD11c and analyzing by flow cytometry. It is. In the condition of FIG. 1A, the CD11c positive and CD3 negative cells were 27.9%, and the CD11 negative and CD3 positive cells were 44.29%, but in the condition of FIG. 1B, 10.66% and 55 respectively. It was 31%. On the other hand, in the condition of FIG. 2A, the CD11c positive and CD3 negative cells were 5.16% and the CD11 negative and CD3 positive cells were 75.31%. 32% and 74.29%. CD11c positive and CD3 negative cells are dendritic cells or their progenitor cells. As is clear from these results, the ratio of dendritic cells or their precursor cells was higher in the GM / SCF medium than in the GM / IL-4 medium. However, in any medium, the proportion of dendritic cells or their progenitor cells decreased sharply for 2 weeks rather than 1 week, and conversely, the proportion of CD11-negative and CD3-positive cells became much higher. CD3-positive cells are T cells, and it is known that dendritic cells can interact with T cells and stimulate T cells even in vitro. Thus, from these results, when T cells coexist, dendritic cells stimulate T cells, but the proliferation and / or differentiation of dendritic cells themselves may be hindered. In fact, from the results of FIGS. 3A and B, among CD11c negative cells, 87.07% in FIG. 3A and 88.27% in FIG. 3B, about 90% were CD3 positive, that is, T cell lineage cells. It was. Therefore, by removing T cells from mononuclear cells derived from peripheral blood before the start of culture, it is not possible to concentrate on the proliferation and differentiation of dendritic cells themselves without being bothered by the interaction with T cells. I got the idea.
図４は、末梢血中の単核球からＣＤ３−細胞を培養した場合（実線）と、ＣＤ３発現細胞を除去しないで単核球集団をそのまま培養した場合（破線）とにおける、ＣＤ１１ｃ陽性細胞数の増殖曲線である。いずれの場合も、３人の被検者から採取された末梢血１ｍＬあたりのＣＤ１１ｃ陽性細胞の数が、培養開始時と、１週間後、２週間後、・・・、５週間後の時点での全細胞数と、それぞれの時点でのＣＤ１１ｃ陽性細胞の割合との積として算出された。末梢血中の単核球からＣＤ３発現細胞を除去した残りの細胞を培養した場合には、培養開始から４週間後までＣＤ１１ｃ陽性細胞の数が増加し続け、５週間後には極大に達する。この間、３週間後までは増加速度も上昇し続けるが、３週間後を過ぎると増加速度は低下し、４週間後から５週間後までの間は増加はあまりみられない。ＣＤ１１ｃ陽性細胞の割合も、培養開始時に約２０％であったのに、培養４週間後には９５％を超えていた。これに対し、ＣＤ３発現細胞を除去しないで単核球集団をそのまま培養した場合には、培養開始時からＣＤ１１ｃ陽性細胞の数の増加は全く認められず、図１Ａ及び２Ａで示されたのと同様に、２週間後には著しく減少してしまった。これらの結果から、ＣＤ３発現細胞、すなわち、Ｔ細胞を除去したうえで前記ＧＭ／ＳＣＦ培地で培養することにより、ほぼ樹状細胞だけを選択的に１０倍ないし１００倍に増幅できることが示された。 FIG. 4 shows the number of CD11c positive cells when CD3 − cells are cultured from mononuclear cells in peripheral blood (solid line) and when the mononuclear cell population is cultured as it is without removing CD3 expressing cells (broken line). It is a growth curve. In any case, the number of CD11c-positive cells per mL of peripheral blood collected from 3 subjects was measured at the start of culture, 1 week, 2 weeks, ..., 5 weeks later. Was calculated as the product of the total number of cells and the percentage of CD11c positive cells at each time point. When the remaining cells from which CD3-expressing cells are removed from mononuclear cells in peripheral blood are cultured, the number of CD11c positive cells continues to increase until 4 weeks after the start of culture, and reaches a maximum after 5 weeks. During this time, the rate of increase continues to increase until after 3 weeks, but the rate of increase decreases after 3 weeks, and there is little increase between 4 and 5 weeks. The proportion of CD11c positive cells was about 20% at the start of culture, but exceeded 95% after 4 weeks of culture. In contrast, when the mononuclear cell population was cultured as it was without removing CD3-expressing cells, no increase in the number of CD11c positive cells was observed from the beginning of the culture, as shown in FIGS. 1A and 2A. Similarly, it decreased significantly after 2 weeks. From these results, it was shown that by removing CD3-expressing cells, ie, T cells, and culturing in the GM / SCF medium, only dendritic cells can be selectively amplified 10 to 100 times. .
培地の切り替えによる樹状細胞の成熟
実施例１のプロトコールに従って、ＣＤ３−細胞が前記ＧＭ／ＳＣＦ培地で４週間培養された後、樹状細胞の前駆細胞を樹状細胞に分化させるために、培地が切り替えられた。新しい培地は、１００ｎｇ／ｍＬの組換えヒトＧＭ−ＣＳＦと、５０ｎｇ／ｍＬの組換えヒトＩＬ−４と、１０％ウシ胎仔血清と、抗生物質（ペニシリンＧ１００単位／ｍＬ、硫酸ストレプトマイシン１００μｇ／ｍＬ）とが添加されたＩＭＤＭ（以下、「ＧＭ／ＩＬ−４培地」という。）で、培養開始から４週間後に、ＧＭ／ＳＣＦ培地からＧＭ／ＩＬ−４培地に切り替えられ、さらに１週間培養された。対照実験では、ＣＤ３−細胞は、４週間後に培地を切り替えないで、５週間連続してＧＭ／ＳＣＦ培地で培養された。
Maturation of dendritic cells by switching of medium In order to differentiate the precursor cells of dendritic cells into dendritic cells after CD3 - cells were cultured for 4 weeks in the GM / SCF medium according to the protocol of Example 1, Has been switched. The new medium is 100 ng / mL recombinant human GM-CSF, 50 ng / mL recombinant human IL-4, 10% fetal bovine serum, and antibiotics (penicillin G100 units / mL, streptomycin sulfate 100 μg / mL) And IMDM (hereinafter referred to as “GM / IL-4 medium”) to which 4 and 4 were added, 4 weeks after the start of the culture, the GM / SCF medium was switched to the GM / IL-4 medium, and further cultured for 1 week. . In control experiments, CD3 - cells were cultured in GM / SCF media for 5 consecutive weeks without switching media after 4 weeks.
樹状細胞分化マーカーの検討
培養開始から５週間後に、それぞれの実験条件で培養された細胞が、常法に従うフロー・サイトメトリー法による解析に供された。用いられた細胞表面マーカーは、ＣＣＲ５、ＣＣＲ７、ＣＤ４０、ＣＤ５４、ＣＤ８０、ＣＤ８３、ＣＤ８６、ＨＬ−ＡＢＣ及びＨＬＡ−ＤＲであった。また、ＯＫ−４３２による刺激への応答を検討するために、前記５週間の培養後の細胞は、１×１０６個／ｍＬの濃度で、０．５ＫＥ／ｍＬのＯＫ−４３２が添加されたＩＭＤＭに懸濁され、さらに４８時間培養された。その後、ＩＬ−１ｂ、ＩＬ−６、ＩＬ−８、ＩＬ−１０、ＩＬ−１２ｐ７０及びＴＮＦ−αの各サイトカインの産生量が測定された。
Examination of Dendritic Cell Differentiation Marker Five weeks after the start of culture, cells cultured under each experimental condition were subjected to analysis by a flow cytometry method according to a conventional method. The cell surface markers used were CCR5, CCR7, CD40, CD54, CD80, CD83, CD86, HL-ABC and HLA-DR. In addition, in order to examine the response to stimulation by OK-432, the cells after 5 weeks of culture were supplemented with 0.5KE / mL of OK-432 at a concentration of 1 × 10 6 cells / mL. It was suspended in IMDM and further cultured for 48 hours. Thereafter, the production amounts of IL-1b, IL-6, IL-8, IL-10, IL-12p70, and TNF-α were measured.
図５Ａないし１３Ｂに、各細胞表面マーカーごとのフロー・サイトメトリー解析結果を比較したグラフを示す。また、図１４に、各細胞表面マーカーのフロー・サイトメトリー解析結果の平均蛍光強度（ＭＦＩ）の測定値を示す。これらの図において「ＧＭ／ＩＬ−４」は、培養開始から４週間後に、ＧＭ／ＳＣＦ培地からＧＭ／ＩＬ−４培地に切り替えられ、さらに１週間培養された、本発明の方法で調製された細胞での解析結果を表す。「ＧＭ／ＳＣＦ」は、５週間連続してＧＭ／ＳＣＦ培地で培養された、対照実験の細胞での解析結果を表す。図２から明らかなとおり、ＣＤ８３及びＣＤ８６については、本発明の方法で調製された細胞のほうが、対照実験の細胞より強く発現していた。ＣＤ８３及びＣＤ８６は、樹状細胞が分化すると発現が増大することが知られている。また、樹状細胞が抗原提示細胞として機能する際に、抗原の断片と複合体を形成するＨＬＡ分子も、本発明の方法で調製された細胞のほうが、対照実験の細胞より強く発現していた。つまり、これらの図から明らかなとおり、本発明の方法で調製された細胞は、樹状細胞としての分化マーカーを発現し、抗原提示細胞としての機能を備えていることが示された。 5A to 13B show graphs comparing the results of flow cytometry analysis for each cell surface marker. Moreover, the measured value of the mean fluorescence intensity (MFI) of the flow cytometry analysis result of each cell surface marker is shown in FIG. In these figures, “GM / IL-4” was prepared by the method of the present invention, which was switched from the GM / SCF medium to the GM / IL-4 medium after 4 weeks from the start of the culture, and further cultured for 1 week. Represents the analysis results in cells. “GM / SCF” represents the result of analysis in cells of a control experiment cultured in GM / SCF medium for 5 consecutive weeks. As is clear from FIG. 2, for CD83 and CD86, the cells prepared by the method of the present invention were expressed more strongly than the cells of the control experiment. CD83 and CD86 are known to increase in expression when dendritic cells are differentiated. In addition, when dendritic cells function as antigen-presenting cells, HLA molecules that form a complex with antigen fragments were also expressed more strongly in cells prepared by the method of the present invention than in control experiments. . That is, as is apparent from these figures, it was shown that the cells prepared by the method of the present invention expressed differentiation markers as dendritic cells and had a function as antigen-presenting cells.
図１５ＡないしＦは、本発明の方法で調製された樹状細胞のＴ細胞刺激能を示すグラフである。図１５ＡないしＦにおいて、「Ｇ４」は、培養開始から４週間後に、ＧＭ／ＳＣＦ培地からＧＭ／ＩＬ−４培地に切り替えられ、さらに１週間培養された、本発明の方法で調製された細胞での解析結果を表す。「ＧＳ」は、５週間連続してＧＭ／ＳＣＦ培地で培養された、対照実験の細胞での解析結果を表す。図１５ＡないしＦから明らかなとおり、ＩＬ−１２ｐ７０は本発明の方法で調製された細胞だけで発現し、逆に、ＩＬ−１ｂ、ＩＬ−６及びＩＬ−８は対照実験の細胞だけで発現していた。このことから、ＯＫ−４３２による刺激に対する応答能力の点からも、本発明の方法で調製された細胞は成熟した樹状細胞であることが証明された。 15A to 15F are graphs showing the T cell stimulating ability of dendritic cells prepared by the method of the present invention. 15A to 15F, “G4” is a cell prepared by the method of the present invention, which was switched from the GM / SCF medium to the GM / IL-4 medium after 4 weeks from the start of the culture, and further cultured for 1 week. Represents the analysis result. “GS” represents the result of analysis in cells of a control experiment cultured in GM / SCF medium for 5 consecutive weeks. As is apparent from FIGS. 15A to 15F, IL-12p70 is expressed only in cells prepared by the method of the present invention, and conversely, IL-1b, IL-6, and IL-8 are expressed only in control experimental cells. It was. From this, it was proved that the cells prepared by the method of the present invention were mature dendritic cells also from the viewpoint of the ability to respond to stimulation by OK-432.
末梢血中の単核球からＣＤ３発現細胞を除去した残りの細胞を培養した場合には、ＣＤ１１ｃ陽性細胞は５週間で１０ないし１００倍に増殖したが、ＣＤ３発現細胞を除去しなかった場合には、ＣＤ１１ｃ陽性細胞は全く増殖が認められず、むしろ、１週間後には数分の１、２週間後には１０分の１と減少していった。図１から明らかなとおり、末梢血由来の単核球からＣＤ３発現細胞を除去することによって、ＣＤ１１ｃ陽性細胞を大量に増殖させることができた。また本発明の方法で調製された細胞は、実施例２の実験結果で証明されたとおり、生体から回収されたばかりの樹状細胞と同じ能力を有する。現在採用されている樹状細胞の調製方法では、促成法で２日、通常法でも５ないし７日しか培養されないため、ＣＤ１１ｃ陽性細胞の数は試験管内では増加せず、むしろ５分の１程度に減少することが知られている。これに対し、本発明の方法によれば、１０倍ないし１００倍に増加させることができるので、本発明の方法は非常に有用である。 When the remaining cells from which CD3 expressing cells were removed from mononuclear cells in peripheral blood were cultured, CD11c positive cells grew 10 to 100 times in 5 weeks, but CD3 expressing cells were not removed. The CD11c positive cells did not grow at all, but rather decreased to 1/10 of a few minutes after 1 week and 1/10 after 1 week. As is clear from FIG. 1, CD11c positive cells could be proliferated in large quantities by removing CD3-expressing cells from peripheral blood-derived mononuclear cells. Moreover, the cell prepared by the method of the present invention has the same ability as the dendritic cell just recovered from the living body, as proved by the experimental result of Example 2. In the method of preparing dendritic cells currently employed, the number of CD11c positive cells does not increase in vitro because it is cultured only for 2 days in the forcing method and 5 to 7 days in the normal method. It is known to decrease. On the other hand, according to the method of the present invention, it can be increased 10 times to 100 times, so the method of the present invention is very useful.
Claims (6)
（ａ）被検者から採取された末梢血、臍帯血、骨髄及び／又はリンパ節由来の血球細胞から単核球を分離するステップ、
（ｂ）前記単核球からＴ細胞を選択的に除去するステップ、及び、
（ｃ）前記Ｔ細胞が除去された残りの細胞集団を、ＧＭ−ＣＳＦ及びＳＣＦを含む培地で培養するステップ
を含む方法であって、
前記ステップ（ｂ）におけるＴ細胞の除去は、細胞集団からＣＤ３を表面に発現する細胞を除去することによって達成されることを特徴とする、
方法。 A method for preparing a dendritic cell population comprising:
(A) separating mononuclear cells from peripheral blood, umbilical cord blood, bone marrow and / or lymph node-derived blood cells collected from a subject;
(B) selectively removing T cells from the mononuclear cells; and
(C) culturing the remaining cell population from which the T cells have been removed in a medium containing GM-CSF and SCF,
The removal of T cells in the step (b) is achieved by removing cells expressing CD3 on the surface from the cell population,
Method.
前記ステップ（ｂ）が、前記単核球から、Ｔ細胞に加えて、Ｂ細胞及び／又はＮＫ細胞をも選択的に除去するステップであることを特徴とする、
方法。 The method of claim 1, comprising:
The step (b) is a step of selectively removing B cells and / or NK cells in addition to T cells from the mononuclear cells,
Method.
前記ステップ（ｃ）において、前記Ｔ細胞が除去された残りの細胞集団に占めるＣＤ１１ｃ陽性細胞の割合が高められることを特徴とする、
方法。 The method according to claim 1 or 2, comprising:
In the step (c), the proportion of CD11c positive cells in the remaining cell population from which the T cells have been removed is increased.
Method.
前記ステップ（ｃ）は、少なくとも３週間実行されることを特徴とする、
方法。 The method according to any one of claims 1 to 3, wherein
Step (c) is performed for at least 3 weeks,
Method.
前記ステップ（ｃ）は、少なくとも４週間実行され、前記ステップ（ｃ）の後、
（ｄ）ＧＭ−ＣＳＦ及びＩＬ−４を含む培地に切り替えて培養するステップ
を含むことを特徴とする、
方法。 A method according to any one of claims 1-4,
Said step (c) is carried out for at least 4 weeks, and after said step (c),
(D) comprising a step of culturing by switching to a medium containing GM-CSF and IL-4,
Method.
前記血球細胞は末梢血からアフェレーシス法により採取されることを特徴とする、
方法。 A method according to any one of claims 1-5,
The blood cells are collected from peripheral blood by an apheresis method,
Method.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| WO2010055900A1 (en) * | 2008-11-14 | 2010-05-20 | ディナベック株式会社 | Method for producing dendritic cells |
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|---|---|---|---|---|
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Cited By (1)
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