JP2013532469A - ナチュラルキラー細胞を生成させる方法 - Google Patents
ナチュラルキラー細胞を生成させる方法 Download PDFInfo
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Abstract
【選択図】図1
Description
本出願は、その開示がその全体として引用により本明細書中に組み込まれている、2010年7月13日に出願された米国仮特許出願第61/363,981号、及び2011年6月16日に出願された米国仮特許出願第61/497,897号の恩典を主張する。
ナチュラルキラー細胞、例えば、胎盤に由来する、例えば、胎盤灌流液(例えば、ヒト胎盤灌流液)に由来するナチュラルキラー細胞、例えば、胎盤由来中間ナチュラルキラー細胞、又は他の組織、例えば、臍帯血もしくは末梢血に由来するナチュラルキラー細胞の集団を産生する方法が本明細書で提供される。本明細書で提示された方法によって産生される拡大されたナチュラルキラー細胞集団も本明細書で提供される。胎盤灌流液、及びそれに由来するナチュラルキラー細胞を用いて、腫瘍細胞の増殖を抑制する方法が本明細書で更に提供される。ある実施態様では、該ナチュラルキラー細胞を、1以上の免疫調節化合物、例えば、IMiDs(商標)と呼ばれる免疫調節化合物と併用するか、又は該免疫調節化合物で処理する。
ナチュラルキラー(NK)細胞は、自然免疫系の主要な構成要素となる細胞傷害性リンパ球である。NK細胞は、T細胞抗原受容体(TCR)も、CD3も、表面免疫グロブリン(Ig)B細胞受容体も発現していない。NK細胞は、ヒトでは通常、表面マーカーCD16(FcγRIII)及びCD56を発現するが、あるサブクラスのヒトNK細胞はCD16-である。NK細胞は細胞傷害性であり;その細胞質中の小顆粒は、パーフォリン、及びグランザイムとして知られるプロテアーゼなどの特殊なタンパク質を含有している。死滅させる標的となった細胞のすぐ近くに放出されると、パーフォリンは、標的細胞の細胞膜に孔を形成し、そこからグランザイム及び関連分子が進入可能になり、アポトーシスが誘導される。1つのグランザイム、グランザイムB(グランザイム2及び細胞傷害性Tリンパ球関連セリンエステラーゼ1としても知られている)は、細胞性免疫応答において標的細胞アポトーシスの迅速な誘導に重要なセリンプロテアーゼである。
細胞、例えば、造血細胞、例えば、造血幹細胞、例えば、CD34+造血幹細胞を拡大し、ナチュラルキラー細胞に分化させる方法が本明細書で提供される。一態様では、ナチュラルキラー(NK)細胞を産生する方法であって、造血幹細胞又は前駆細胞、例えば、CD34+幹細胞又は前駆細胞を第一の培地中で培養して、拡大され、分化した細胞を産生すること、及びその後、該拡大された細胞を、該細胞を更に拡大し、ナチュラルキラー細胞に分化させる第二の培地中で培養することを含む、方法が本明細書で提供される。第一の工程及び第二の工程は、該細胞を独特な組合せの細胞因子を含む培地中で培養することを含む。ある実施態様では、該細胞因子(例えば、サイトカイン)は、該培地の不確定成分(例えば、血清)に含まれず、例えば、該細胞因子(例えば、サイトカイン)は、該培地の不確定成分(例えば、血清)にとって外因性のものである。ある実施態様では、該方法は二工程方法である。ある実施態様では、該方法は、該細胞を接触させる第三の工程又は中間工程を含まない。具体的な実施態様では、活性化ナチュラルキラー(NK)細胞の集団を産生する方法であって:(a)造血幹細胞又は前駆細胞の集団が拡大し、かつ該拡大することの間に、該造血幹細胞又は前駆細胞の集団内の複数の造血幹細胞又は前駆細胞がNK細胞に分化するように、インターロイキン-15(IL-15)、並びに任意に幹細胞因子(SCF)及びインターロイキン-7(IL-7)のうちの1つ又は複数を含み、ここで、該IL-15並びに任意のSCF及びIL-7が該培地の不確定成分に含まれない、第一の培地中に、該集団を播種すること;並びに(b)第一の工程由来の細胞をインターロイキン-2(IL-2)を含む第二の培地中で拡大して、活性化NK細胞の集団を産生することを含む、方法が本明細書で提供される。本明細書で提供される方法(例えば、二工程方法)によって産生されるナチュラルキラー細胞は、本明細書では、TSNK細胞と呼ばれる。
本明細書で使用されるように、用語「免疫調節化合物」及び「IMiD(商標)」は、サリドマイドを包含しない。
造血細胞、例えば、造血幹細胞又は前駆細胞からNK細胞を産生し、それを拡大する新規の方法が本明細書で提供される。NK細胞を産生するために用いられる造血細胞を、任意の供給源、例えば、限定するものではないが、胎盤、臍帯血、胎盤血、末梢血、脾臓、又は肝臓から単離してもよい。ある実施態様では、NK細胞を、拡大された造血細胞、例えば、造血幹細胞及び/又は造血前駆細胞から産生する。一実施態様では、造血細胞を、そのような細胞の供給源、例えば、胎盤灌流液、臍帯血、胎盤血、末梢血、脾臓、肝臓、及び/又は骨髄から回収する。具体的な実施態様では、造血細胞を、フィーダー細胞を用いないで、第一の培地中で、連続的に拡大し、分化させる。その後、該細胞を、フィーダー細胞の存在下、第二の培地中で培養する。そのような単離、拡大、及び分化は、中心施設で実施することができ、それにより、使用地点、例えば、病院、軍事基地、軍部の前線などでの分散的な拡大及び分化のための輸送用の拡大された造血細胞が提供される。
本明細書に開示された方法において有用な造血細胞は、NK細胞に分化することができる任意の造血細胞、例えば、前駆細胞、造血前駆細胞、造血幹細胞などであることができる。造血細胞は、例えば、骨髄、臍帯血、胎盤血、末梢血、肝臓など、又はそれらの組合せなどの組織源から得ることができる。造血細胞は、胎盤から得ることができる。具体的な実施態様では、造血細胞は、胎盤灌流液から得られる。胎盤灌流液由来の造血細胞は、胎児造血細胞と母親造血細胞の混合物、例えば、母親細胞が造血細胞の総数の5%超を含む混合物を含むことができる。好ましくは、胎盤灌流液由来の造血細胞は、少なくとも約90%、95%、98%、99%、又は99.5%の胎児細胞を含む。
ある実施態様では、本明細書で提供される方法で用いられる造血細胞は胎盤造血細胞である。本明細書で使用されるように、「胎盤造血細胞」は、胎盤それ自体から得られる造血細胞であって、胎盤血からも臍帯血からも得られないものを意味する。一実施態様では、胎盤造血細胞はCD34+である。具体的な実施態様では、該胎盤造血細胞は、主に(例えば、少なくとも約50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、又は98%)CD34+CD38-細胞である。別の具体的な実施態様では、該胎盤造血細胞は、主に(例えば、少なくとも約50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、又は98%)CD34+CD38+細胞である。胎盤造血細胞は、当業者に公知の任意の手段によって、例えば、灌流によって、分娩後の哺乳動物(例えば、ヒト)胎盤から得ることができる。
本方法によるNK細胞の産生は、造血細胞の集団を拡大することを含む。細胞拡大の間に、該造血細胞集団内の複数の造血細胞はNK細胞に分化する。
ある実施態様では、本明細書で提供される方法は、造血細胞の集団を、該造血細胞集団内の複数の造血幹細胞又は前駆細胞がNK細胞に分化する、第一の培地中で培養し、拡大する第一の工程を含む。
R1は、H、(C1-C8)アルキル、(C3-C7)シクロアルキル、(C2-C8)アルケニル、(C2-C8)アルキニル、ベンジル、アリール、(C0-C4)アルキル-(C1-C6)ヘテロシクロアルキル、(C0-C4)アルキル-(C2-C5)ヘテロアリール、C(O)R3、C(S)R3、C(O)OR4、(C1-C8)アルキル-N(R6)2、(C1-C8)アルキル-OR5、(C1-C8)アルキル-C(O)OR5、C(O)NHR3、C(S)NHR3、C(O)NR3R3'、C(S)NR3R3'、又は(C1-C8)アルキル-O(CO)R5であり;
R2は、H、F、ベンジル、(C1-C8)アルキル、(C2-C8)アルケニル、又は(C2-C8)アルキニルであり;
R3及びR3'は、独立に、(C1-C8)アルキル、(C3-C7)シクロアルキル、(C2-C8)アルケニル、(C2-C8)アルキニル、ベンジル、アリール、(C0-C4)アルキル-(C1-C6)ヘテロシクロアルキル、(C0-C4)アルキル-(C2-C5)ヘテロアリール、(C0-C8)アルキル-N(R6)2、(C1-C8)アルキル-OR5、(C1-C8)アルキル-C(O)OR5、(C1-C8)アルキル-O(CO)R5、又はC(O)OR5であり;
R4は、(C1-C8)アルキル、(C2-C8)アルケニル、(C2-C8)アルキニル、(C1-C4)アルキル-OR5、ベンジル、アリール、(C0-C4)アルキル-(C1-C6)ヘテロシクロアルキル、又は(C0-C4)アルキル-(C2-C5)ヘテロアリールであり;
R5は、(C1-C8)アルキル、(C2-C8)アルケニル、(C2-C8)アルキニル、ベンジル、アリール、又は(C2-C5)ヘテロアリールであり;
R6の各々の出現は、独立に、H、(C1-C8)アルキル、(C2-C8)アルケニル、(C2-C8)アルキニル、ベンジル、アリール、(C2-C5)ヘテロアリール、もしくは(C0-C8)アルキル-C(O)O-R5であるか、又はR6基は結合して、ヘテロシクロアルキル基を形成することができ;
nは0又は1であり;かつ
*は、キラル炭素中心を表す。
別の実施態様では、該免疫調節化合物は、以下の構造を有する化合物、又は医薬として許容し得るその塩、水和物、溶媒和物、包摂化合物、エナンチオマー、ジアステレオマー、ラセミ化合物、もしくは立体異性体の混合物であり、
X及びYのうちの一方はC=Oであり、もう一方は、CH2又はC=Oであり;
Rは、H又はCH2OCOR'であり;
(i)R1、R2、R3、もしくはR4の各々は、他のものとは独立に、ハロ、炭素原子1〜4個のアルキル、もしくは炭素原子1〜4個のアルコキシであるか、又は(ii)R1、R2、R3、もしくはR4のうちの1つは、ニトロもしくは-NHR5であり、かつR1、R2、R3、もしくはR4のうちの残りは水素であり;
R5は、水素又は炭素1〜8個のアルキルであり
R6は、水素、炭素原子1〜8個のアルキル、ベンゾ、クロロ、又はフルオロであり;
R'はR7-CHR10-N(R8R9)であり;
R7は、m-フェニレン又はp-フェニレン又は-(CnH2n)-であり、ここで、nは0〜4の値を有し;
R8及びR9の各々は、他方とは独立に、水素もしくは炭素原子1〜8個のアルキルであるか、又はR8及びR9は一緒になって、テトラメチレン、ペンタメチレン、ヘキサメチレン、もしくは-CH2CH2X1CH2CH2-であり、ここで、X1は、-O-、-S-、もしくは-NH-であり;
R10は、水素、炭素原子〜8個のアルキル、又はフェニルであり;かつ
*は、キラル炭素中心を表す。
本明細書で提供される方法では、造血細胞、例えば、幹細胞又は前駆細胞、及び第一の工程から得られる、ナチュラルキラー細胞を、例えば、フィーダー層を用いないで、又はフィーダー細胞の存在下、第二の工程で更に拡大し、分化させる。本明細書で提供されているような細胞の培養は、第一の工程由来のNK細胞の連続的な拡大、分化、及び成熟をもたらす。第二の工程では、該NK細胞を、例えば、該第一の培地とは異なるサイトカイン及び/又は生体活性分子を含む第二の培養培地中で、連続的な様式で、拡大し、分化させ、かつ成熟させる。ある実施態様では、第二の培養培地は、動物成分不含培地である。例示的な動物成分不含細胞培養培地は、上の第6.2.1節に記載されている。
ナチュラルキラー細胞を単離する方法は当技術分野で公知であり、該方法を用いて、TSNK細胞を単離することができる。ナチュラルキラー細胞は、組織源、例えば、末梢血由来の細胞をCD56及びCD3に対する抗体で染色し、かつCD56+CD3-細胞を選択することによって単離又は濃縮することができる。TSNK細胞は、市販のキット、例えば、NK細胞単離キット(Miltenyi Biotec)を用いて単離することができる。TSNK細胞は、該TSNK細胞を含む細胞の集団内のNK細胞以外の細胞の除去によって単離又は濃縮することもできる。例えば、TSNK細胞は、例えば、CD3、CD4、CD14、CD19、CD20、CD36、CD66b、CD123、HLA DR、及び/又はCD235a(グリコフォリンA)のうちの1つ又は複数に対する抗体を用いた非NK細胞マーカーを示す細胞の除去によって単離又は濃縮することができる。陰性単離は、市販のキット、例えば、NK細胞陰性単離キット(Dynal Biotech)を用いて実行することができる。これらの方法によって単離される細胞を更に選別して、例えば、CD16+及びCD16-細胞を分離することができる。
TSNK細胞は、任意の供給源、例えば、胎盤組織、胎盤灌流液、臍帯血、胎盤血、末梢血、脾臓、肝臓などに由来する造血細胞、例えば、造血幹又は前駆細胞から産生することができる。ある実施態様では、該造血幹細胞は、胎盤灌流液由来及び該胎盤灌流液を生成させるために用いられる同じ胎盤に由来する臍帯血由来の組み合わされた造血幹細胞である。例えば、その開示がその全体として引用により本明細書中に組み込まれる、米国特許第7,045,148号及び第7,468,276号に開示された方法によって得ることができる胎盤灌流液細胞を含む胎盤灌流液。
例えば、本明細書で提供されるような、TSNK細胞と組み合わせて、造血幹もしくは前駆細胞をそこから単離し得るか、或いは腫瘍抑制、又は腫瘍細胞、癌、もしくはウイルス感染を有する個体の治療において有用な、胎盤灌流液及び灌流液細胞は、胎盤細胞回収組成物を用いる、哺乳動物、例えば、ヒトの分娩後胎盤の灌流によって回収することができる。灌流液は、任意の生理的に許容し得る溶液、例えば、生理食塩水溶液、培養培地、又はより複雑な細胞回収組成物を用いる胎盤の灌流によって、胎盤から回収することができる。胎盤の灌流に、並びに灌流液細胞の回収及び保存に好適な細胞回収組成物は、その全体が引用により本明細書中に組み込まれている、関連米国特許出願公開第2007/0190042号に詳細に記載されている。
通常、ヒトの胎盤を、出産後のその娩出後すぐに回収する。好ましい実施態様では、インフォームドコンセント後、及び患者の全病歴を取得して、該胎盤と関連付けた後、胎盤を患者から回収する。好ましくは、この病歴は分娩後も続く。
哺乳動物の胎盤を灌流する方法及び胎盤灌流液を得る方法は、例えば、その各々の開示が、その全体として引用により本明細書中に組み込まれている、Haririの米国特許第7,045,148号及び第7,255,879号、並びに米国特許出願公開第2007/0190042号及び第20070275362号に開示されている。
通常、単回の胎盤灌流由来の胎盤灌流液は、約1億〜約5億個の有核細胞を含み、これには、本明細書に開示された方法によってTSNK細胞を産生し得る造血細胞が含まれる。ある実施態様では、該胎盤灌流液又は灌流液細胞は、CD34+細胞、例えば、造血幹細胞又は前駆細胞を含む。そのような細胞は、より具体的な実施態様では、CD34+CD45-幹細胞もしくは前駆細胞、CD34+CD45+幹細胞もしくは前駆細胞、又は同様の細胞を含むことができる。ある実施態様では、該灌流液又は灌流液細胞は、それから造血細胞を単離する前に凍結保存される。ある他の実施態様では、該胎盤灌流液は、胎児細胞のみ、もしくは胎児細胞と母親細胞の組合せを含むか、又は該灌流液細胞は、胎児細胞のみ、もしくは胎児細胞と母親細胞の組合せを含む。
一態様では、TSNK細胞、本明細書に記載の方法(例えば、二工程方法)によって産生されるNK細胞が本明細書で提供される。本明細書に記載の方法(例えば、二工程方法)によって産生されるTSNK細胞を含む細胞の集団が本明細書で更に提供される。具体的な実施態様では、該NK細胞(例えば、TSNK細胞)はCD3-CD56+である。具体的な実施態様では、該NK細胞(例えば、TSNK細胞)はCD3-CD56+CD16-である。別の具体的な実施態様では、該NK細胞(例えば、TSNK細胞)は更にCD94+CD117+である。別の具体的な実施態様では、該NK細胞(例えば、TSNK細胞)は更にCD161-である。別の具体的な実施態様では、該NK細胞(例えば、TSNK細胞)は更にNKG2D+である。別の具体的な実施態様では、該NK細胞は更にNKp46+である。別の具体的な実施態様では、該該NK細胞は更にCD226+である。
例えば、腫瘍細胞又は複数の腫瘍細胞の増殖の抑制で用いるための、胎盤灌流液、胎盤灌流液細胞、及び/又は接着性胎盤細胞と組み合わせたTSNK細胞を含む組成物が本明細書で更に提供される。
TSNK細胞と胎盤灌流液及び/又は胎盤灌流液細胞の組合せを含む組成物が本明細書で更に提供される。一実施態様では、例えば、TSNK細胞が補充された、一定の容積の胎盤灌流液が本明細書で提供される。具体的な実施態様では、例えば、胎盤灌流液の各ミリリットルに、約1×104、5×104、1×105、5×105、1×106、5×106、1×107、5×107、1×108、5×108個、又はそれより多くのTSNK細胞を補充する。別の実施態様では、胎盤灌流液細胞にTSNK細胞を補充する。ある他の実施態様では、胎盤灌流液細胞をTSNK細胞と組み合わせる場合、該胎盤灌流液細胞は、通常、細胞の総数の約50%、45%、40%、35%、30%、25%、20%、15%、10%、8%、6%、4%、2%、もしくは1%超、又は細胞の総数の約50%、45%、40%、35%、30%、25%、20%、15%、10%、8%、6%、4%、2%、もしくは1%未満を含む。ある他の実施態様では、TSNK細胞を複数の胎盤灌流液細胞及び/又は組み合わされたナチュラルキラー細胞と組み合わせる場合、該NK細胞は、通常、細胞の総数の約50%、45%、40%、35%、30%、25%、20%、15%、10%、8%、6%、4%、2%、もしくは1%超、又は細胞の総数の約50%、45%、40%、35%、30%、25%、20%、15%、10%、8%、6%、4%、2%、もしくは1%未満を含む。ある他の実施態様では、TSNK細胞を用いて胎盤灌流液を補充する場合、細胞を懸濁する溶液(例えば、生理食塩水溶液又は培養培地など)の容積は、灌流液+細胞の全容積の約50%、45%、40%、35%、30%、25%、20%、15%、10%、8%、6%、4%、2%、もしくは1%超、又は灌流液+細胞の全容積の約50%、45%、40%、35%、30%、25%、20%、15%、10%、8%、6%、4%、2%、もしくは1%未満を含み、その場合、TSNK細胞は、補充前の1ミリリットル当たり約1×104、5×104、1×105、5×105、1×106、5×106、1×107、5×107、1×108、5×108個、又はそれより多くの細胞になるように懸濁される。
他の実施態様では、単独の又は胎盤灌流液もしくは胎盤灌流液細胞と組み合わせたTSNK細胞に、例えば、その開示がその全体として引用により本明細書中に組み込まれている、Haririらの米国特許第7,045,148号及び第7,255,879号、並びに米国特許出願公開第2007/0275362号に記載されているような、単離された接着性胎盤細胞、例えば、胎盤幹細胞及び胎盤寡能性細胞を補充する。「接着性胎盤細胞」は、該細胞が、組織培養物表面、例えば、組織培養プラスチックに接着することを意味する。本明細書に開示された組成物及び方法において有用な接着性胎盤細胞は、栄養芽細胞でも、胚性生殖細胞でも、胚性幹細胞でもない。ある実施態様では、接着性胎盤幹細胞を、上記のようなプロセス(例えば、二工程方法)でフィーダー細胞として用いる。
TSNK細胞及び更に馴化培地を含む組成物であって、腫瘍抑制性であるか、又は癌もしくはウイルス感染の治療において効果がある、組成物の使用も本明細書で提供される。上の第6.6.2節に記載されているような接着性胎盤細胞を用いて、腫瘍細胞抑制性、抗癌性、又は抗ウイルス性である馴化培地、すなわち、検出可能な腫瘍細胞抑制効果、抗癌効果、又は抗ウイルス効果を有する細胞によって分泌又は排出される1以上の生体分子を含む培地を産生することができる。様々な実施態様では、馴化培地は、該細胞が、少なくとも1、2、3、4、5、6、7、8、9、10、11、12、13、14日、又はそれより長い間、その中で増殖した(すなわち、培養された)培地を含む。他の実施態様では、馴化培地は、そのような細胞が、少なくとも30%、40%、50%、60%、70%、80%、90%コンフルエンス、又は100%コンフルエンスにまでその中で成長した培地を含む。そのような馴化培地を用いて、細胞、例えば、胎盤細胞、又は別の種類の細胞の別々の集団の培養を支援することができる。別の実施態様では、本明細書で提供される馴化培地は、単離された接着性胎盤細胞、例えば、単離された接着性胎盤幹細胞又は単離された接着性胎盤寡能性細胞、及び単離された接着性胎盤細胞以外の細胞、例えば、非胎盤幹細胞又は寡能性細胞がその中で培養された培地を含む。
細胞、例えば、TSNK細胞、又は造血幹細胞もしくは前駆細胞を含む胎盤灌流液細胞を保存する、すなわち、長期保存を可能にする条件下、又は例えば、アポトーシスもしくは壊死による細胞死を阻害する条件下に置くことができる。
本明細書で提供されるTSNK細胞は、癌を有する個体、例えば、固形腫瘍細胞及び/もしくは血液癌細胞を有する個体、又はウイルス感染を有する人を治療する方法で用いることができる。本明細書で提供されるTSNK細胞は、腫瘍細胞の増殖を抑制する方法で用いることもできる。
一実施態様では、癌、例えば、血液癌又は固形腫瘍を有する個体を治療する方法であって、該個体に、治療的有効量のTSNK細胞を投与することを含む、方法が本明細書で提供される。ある実施態様では、該個体は、ナチュラルキラー細胞の欠損、例えば、該個体の癌に対して活性があるNK細胞の欠損を有する。具体的な実施態様では、該方法は更に、該個体に、単離された胎盤灌流液又は単離された胎盤灌流液細胞、例えば、治療的有効量の胎盤灌流液又は単離された胎盤灌流液細胞を投与することを含む。別の具体的な実施態様では、該方法は更に、該個体に、有効量の免疫調節化合物、例えば、上の第6.2.1節に記載の免疫調節化合物、又はサリドマイドを投与することを含む。本明細書で使用されるように、「有効量」は、例えば、該個体が罹患している癌の1以上の症状の検出可能な改善、該症状の進行の軽減、又は該症状の消失をもたらす量である。
腫瘍細胞の増殖を抑制する方法であって、該腫瘍細胞をTSNK細胞と接触させることを含む、方法が本明細書で更に提供される。任意に、該腫瘍細胞及び/又はTSNK細胞を、単離された胎盤灌流液又は単離された胎盤灌流液細胞と接触させる。別の具体的な実施態様では、該腫瘍細胞及び/又はTSNK細胞を、該腫瘍細胞の増殖が、TSNK細胞と接触させていない同じタイプの腫瘍細胞と比較して、検出可能な程度に低下するように、免疫調節化合物、例えば、上の第6.2.1節に記載の免疫調節化合物、又はサリドマイドと更に接触させる。任意に、免疫調節化合物と接触させた腫瘍細胞及び/又はTSNK細胞を、単離された胎盤灌流液又は単離された胎盤灌流液細胞と接触させる。
本明細書に記載のTSNKを用いた、癌を有する個体の治療は、1以上の他の抗癌剤を含む抗癌治療レジメンの一部であることができる。そのような抗癌剤は当技術分野で周知である。TSNK細胞、及び任意に灌流液、灌流液細胞、TSNK細胞以外のナチュラルキラー細胞に加えて、癌を有する個体、例えば、腫瘍細胞を有する個体に投与し得る具体的な抗癌剤としては、限定するものではないが:アシビシン;アクラルビシン;塩酸アコダゾール;アクロニン;アドゼレシン;アドルシル;アルデスロイキン;アルトレタミン;アンボマイシン;酢酸アメタントロン;アムサクリン;アナストロゾール;アントラマイシン;アスパラギナーゼ;アスペルリン;アバスチン(ベバシズマブ);アザシチジン;アゼテパ;アゾトマイシン;バチマスタット;ベンゾデパ;ビカルタミド;塩酸ビサントレン;ジメシル酸ビスナフィド;ビゼレシン;硫酸ブレオマイシン;ブレキナルナトリウム;ブロピリミン;ブスルファン;カクチノマイシン;カルステロン;カラセミド;カルベチマー;カルボプラチン;カルムスチン;塩酸カルビシン;カルゼレシン;セデフィンゴール;セレコキシブ(COX-2阻害剤);クロラムブシル;シロレマイシン;シスプラチン;クラドリビン;メシル酸クリスナトール;シクロホスファミド;シタラビン;ダカルバジン;ダクチノマイシン;塩酸ダウノルビシン;デシタビン;デキソルマプラチン;デザグアニン;メシル酸デザグアニン;ジアジコン;ドセタキセル;ドキソルビシン;塩酸ドキソルビシン;ドロロキシフェン;クエン酸ドロロキシフェン;プロピオン酸ドロモスタノロン;デュアゾマイシン;エダトレキセート;塩酸エフロミチン;エルサミトルシン;エンロプラチン;エンプロメート;エピプロピジン;塩酸エピルビシン;エルブロゾール;塩酸エソルビシン;エストラムスチン;リン酸エストラムスチンナトリウム;エタニダゾール;エトポシド;リン酸エトポシド;エトプリン;塩酸ファドロゾール;ファザラビン;フェンレチニド;フロクスウリジン;リン酸フルダラビン;フルオロウラシル;フルロシタビン;ホスキドン;フォストリエシンナトリウム;ゲムシタビン;塩酸ゲムシタビン;ヒドロキシ尿素;塩酸イダルビシン;イホスファミド;イルモホシン;イプロプラチン;イリノテカン;塩酸イリノテカン;酢酸ランレオチド;レトロゾール;酢酸ロイプロリド;塩酸リアロゾール;ロメトレキソールナトリウム;ロムスチン;塩酸ロソキサントロン;マソプロコール;メイタンシン;塩酸メクロレタミン;酢酸メゲストロール;酢酸メレンゲストロール;メルファラン;メノガリル;メルカプトプリン;メトトレキセート;メトトレキセートナトリウム;メトプリン;メツレデパ;ミチンドミド;マイトカルシン;マイトクロミン;マイトギリン;マイトマルシン;マイトマイシン;マイトスペル;ミトタン;塩酸ミトキサントロン;ミコフェノール酸;ノコダゾール;ノガラマイシン;オルマプラチン;オキシスラン;パクリタキセル;ペガスパルガーゼ;ペリオマイシン;ペンタムスチン;硫酸ペプロマイシン;ペルホスファミド;ピポブロマン;ピポスルファン;塩酸ピロキサントロン;プリカマイシン;プロメスタン;ポルフィマーナトリウム;ポルフィロマイシン;プレドニムスチン;塩酸プロカルバジン;ピューロマイシン;塩酸ピューロマイシン;ピラゾフリン;リボプリン;サフィンゴール;塩酸サフィンゴール;セムスチン;シムトラゼン;スパルホセートナトリウム;スパルソマイシン;塩酸スピロゲルマニウム;スピロムスチン;スピロプラチン;ストレプトニグリン;ストレプトゾシン;スロフェヌル;タリソマイシン;テコガランナトリウム;タキソテール;テガフール;塩酸テロキサントロン;テモポルフィン;テニポシド;テロキシロン;テストラクトン;チアミプリン;チオグアニン;チオテパ;チアゾフリン;チラパザミン;クエン酸トレミフェン;酢酸トレストロン;リン酸トリシリビン;トリメトレキセート;グルクロン酸トリメトレキセート;トリプトレリン;塩酸ツブロゾール;ウラシルマスタード;ウレデパ;バプレオチド;ベルテポルフィン;硫酸ビンブラスチン;硫酸ビンクリスチン;ビンデシン;硫酸ビンデシン;硫酸ビネピジン;硫酸ビングリシネート;硫酸ビンロイロシン;酒石酸ビノレルビン;硫酸ビンロシジン;硫酸ビンゾリジン;ボロゾール;ゼニプラチン;ジノスタチン;及び塩酸ゾルビシンが含まれる。
別の実施態様では、ウイルス感染を有する個体を治療する方法であって、該個体に、治療的有効量のTSNK細胞を投与することを含む、方法が本明細書で提供される。ある実施態様では、該個体は、ナチュラルキラー細胞の欠損、例えば、該個体のウイルス感染に対して活性があるNK細胞の欠損を有する。ある具体的な実施態様では、該投与することは、該個体に、単離された胎盤灌流液、単離された胎盤灌流液細胞、単離されたナチュラルキラー細胞、例えば、胎盤ナチュラルキラー細胞、例えば、胎盤由来中間ナチュラルキラー細胞、単離され、組み合わされたナチュラルキラー細胞、及び/又はそれらの組合せのうちの1つ又は複数を投与することを更に含む。ある具体的な実施態様では、該投与することの前に、該TSNK細胞を、免疫調節化合物、例えば、上の第6.2.1節に記載の免疫調節化合物、又はサリドマイドと接触させる。ある他の具体的な実施態様では、該投与することは、該TSNK細胞に加えて、免疫調節化合物、例えば、上の第6.2.1節に記載の免疫調節化合物、又はサリドマイドを、該個体に投与することを含み、ここで、該量は、例えば、該ウイルス感染の1以上の症状の検出可能な改善、該症状の進行の軽減、又は該症状の消失をもたらす量である。具体的な実施態様では、ウイルス感染は、アデノウイルス科、ピコルナウイルス科、ヘルペスウイルス科、ヘパドナウイルス科、フラビウイルス科、レトロウイルス科、オルトミクソウイルス科、パラミクソウイルス科、パピローマウイルス科、ラブドウイルス科、又はトガウイルス科ファミリーのウイルスによる感染である。より具体的な実施態様では、該ウイルスは、ヒト免疫不全ウイルス(HIV)、コクサッキーウイルス、A型肝炎ウイルス(HAV)、ポリオウイルス、エプスタイン・バーウイルス(EBV)、単純ヘルペスウイルス1型(HSV1)、単純ヘルペスウイルス2型(HSV2)、ヒトサイトメガロウイルス(CMV)、ヒトヘルペスウイルス8型(HHV8)、帯状疱疹(herpes zoster)ウイルス(水痘帯状疱疹ウイルス(VZV)もしくは帯状疱疹(shingles)ウイルス)、B型肝炎ウイルス(HBV)、C型肝炎ウイルス(HCV)、D型肝炎ウイルス(HDV)、E型肝炎ウイルス(HEV)、インフルエンザウイルス(例えば、A型インフルエンザウイルス、B型インフルエンザウイルス、C型インフルエンザウイルス、もしくはトゴトウイルス)、麻疹ウイルス、流行性耳下腺炎ウイルス、パラインフルエンザウイルス、パピローマウイルス、狂犬病ウイルス、又は風疹ウイルスである。
細胞、例えば、胎盤灌流液細胞、例えば、胎盤灌流液由来の有核細胞、組み合わされたナチュラルキラー細胞、及び/又は単離されたナチュラルキラー細胞、例えば、TSNK細胞の数の決定、並びに免疫調節化合物、例えば、上の第6.2.1節の免疫調節化合物、又はサリドマイドの量の決定は、互いに独立に行なうことができる。
ある実施態様では、TSNK細胞を、ウイルス感染、癌、又は腫瘍細胞を有する個体、例えば、腫瘍細胞、固形腫瘍、又は血液癌を有する個体、例えば、癌患者に対して検出可能な治療的利益をもたらす任意の量又は数、例えば、有効量で、該個体に用いる、例えば、投与する。そのような細胞を、そのような個体に、細胞の絶対数で投与することができ、例えば、該個体に、約、少なくとも約、又は多くとも約1×105、5×105、1×106、5×106、1×107、5×107、1×108、5×108、1×109、5×109、1×1010、5×1010、又は1×1011個のTSNK細胞で投与することができる。他の実施態様では、TSNK細胞を、そのような個体に、細胞の相対数で投与することができ、例えば、該個体に、該個体のキログラム当たり約、少なくとも約、又は多くとも約1×105、5×105、1×106、5×106、1×107、5×107、1×108、5×108、1×109、5×109、1×1010、5×1010、又は1×1011個のTSNK細胞で投与することができる。TSNK細胞を、いくつかのTSNK細胞、並びに任意にTSNK細胞以外の胎盤灌流液細胞及び/又はナチュラルキラー細胞と、該個体内のいくつかの腫瘍細胞(例えば、推定数)との近似比率に従って、そのような個体に投与することができる。例えば、TSNK細胞を、個体内の腫瘍細胞の数に対して約、少なくとも約、又は多くとも約1:1、1:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1、15:1、20:1、25:1、30:1、35:1、40:1、45:1、50:1、55:1、60:1、65:1、70:1、75:1、80:1、85:1、90:1、95:1、又は100:1の比率で、該個体に投与することができる。そのような個体内の腫瘍細胞の数は、例えば、該個体由来の組織の試料、例えば、血液試料、生検などにおける腫瘍細胞の数を計数することによって推定することができる。例えば、固形腫瘍についての具体的な実施態様では、該計数することを、腫瘍(単数又は複数)のイメージングと組み合わせて行ない、おおよその腫瘍容積を得る。具体的な実施態様では、TSNK細胞、任意にTSNK細胞以外の胎盤灌流液細胞及び/又はナチュラルキラー細胞に加えて、免疫調節化合物又はサリドマイド、例えば、有効量の免疫調節化合物又はサリドマイドを個体に投与する。
(7.1.実施例1:ヒト胎盤灌流液及び臍帯血からの造血幹細胞の回収)
ヒト胎盤灌流液(HPP)及び臍帯血(UCB)細胞を、通常、Ficoll又は塩化アンモニウムのいずれかを用いて精製し、全有核細胞(TNC)を得た。その後、TNCを陽性選択手順で用い、製造業者のプロトコル(StemCell Technologies, Inc.)に従って抗CD34ビーズ及びRoboSepを用いて、CD34+細胞を単離した。この実験では、CD34+細胞を90%を超える純度で単離した。或いは、EasySep(登録商標)ヒト前駆細胞濃縮キット(StemCell Technologies, Inc.)を陰性選択手順で用い、以下のヒト細胞表面抗原:CD2、CD3、CD11b、CD11c、CD14、CD16、CD19、CD24、CD56、CD66b、及びグリコフォリンAに対するモノクローナル抗体を含むヒト前駆細胞濃縮カクテルを用いることによって、系譜が確定した(lineage committed)細胞を除去した。陰性選択を用いて、90%のCD34+細胞を原材料から回収した。回収されたHSCの細胞組成を表1にまとめた。
CD34+細胞を以下の培地製剤中で最大48日間培養し、細胞のアリコートを、細胞数、細胞生存率の評価(assessment)、ナチュラルキラー細胞分化の特徴付け、及び機能的評価(evaluation)のために採取した。
27日目(D27)に、NK2A培地中で培養されたCD34+細胞を、以下の培地のうちの1つの中で更に培養した:
・CNK培地及び維持培地を含む、二段階培地。CNK培地は、10%のFCS(Hyclone)、200IU/mLのIL-2(R&D Systems)、35μg/mLのトランスフェリン(Sigma-Aldrich)、5μg/mLのインスリン(Sigma-Aldrich)、2×10-5Mのエタノールアミン(Sigma-Aldrich)、1μg/mLのオレイン酸(Sigma-Aldrich)、1μg/mLのリノール酸(Sigma-Aldrich)、0.2μg/mLのパルミチン酸(Sigma-Aldrich)、2.5μg/mLのBSA(Sigma-Aldrich)、及び0.1μg/mLのフィトヘマグルチニン(PHA-P、Sigma-Aldrich)が補充されたIMDM(Invitrogen)である。NK2A培地中で培養されたCD56+CD3- NK細胞を、細胞培養液で処理した24ウェルプレートもしくはT字フラスコ中のCNK培地に2.5×105個の生細胞/mLで再懸濁した。マイトマイシンC処理した同種異系PBMC及びK562細胞(慢性骨髄性白血病細胞株)を両方とも、フィーダー細胞として、1mL当たり1×106個の最終濃度になるようCNK培地に添加した。NK細胞を、37℃、5%CO2で、5〜6日間培養した。5〜6日後、及びその後は3〜4日毎に、等容積の維持培地(10%FCS、2%ヒトAB血清、抗生物質、L-グルタミン、及び1mL当たり400ユニットのIL-2を含むIMDM)を該培養物に添加した。
・フィーダー細胞としてのマイトマイシンC処理したCD34-、CD10+、CD105+、CD200+組織培養プラスチック接着性胎盤幹細胞を含むNK2A(PDAC)培地;
・フィーダー細胞としてのマイトマイシンC処理した間葉系幹細胞(MSC)を含むNK2A(MSC)培地;又は
・対照としてのフィーダー不含NK2A(FF)培地。
二段階培地中で培養されたNK細胞を、Wollらの文献(Blood 113(4):6094-6101(2009))の方法によって産生される、胚性幹細胞(ESC)由来のNK細胞と比較した。具体的には、両方の該細胞型の培養のプロセスの間、CD94及びCD117の発現レベルの違いを観察した。図6は、CD117の発現が、7日(D7)から35日(D35)まで、二段階NK細胞で高いか、又は「+」であったのに対し、CD94の発現が徐々に増加したことを示している。35日目(D35)に、CD56+CD3-(二工程)細胞の約44%はCD94+CD117+細胞であり、CD56+CD3-細胞の37.6%はCD94-CD117+であり、該CD56+CD3-細胞の14.7%はCD94+CD117-であった。従って、二工程方法によって産生されるNK細胞は、ESC由来のNK細胞と区別可能であり、該ESC由来のNK細胞の78%は、培養14日〜35日までCD117low/-のままであった。下の実施例6に記載するように、CD117+ NK細胞は、様々な組織由来の腫瘍細胞株に対して細胞傷害性であるので、このCD117発現の違いは有用である。
造血幹細胞(HSC)のナチュラルキラー細胞への分化に対するCD10+、CD34-、CD105+、CD200+組織培養プラスチック接着性胎盤幹細胞(この実施例ではPDACと呼ぶ)の効果を評価するために、HSCを、0日目及び21日目に、10:1のPDAC/MSC:HSCの比率で、マイトマイシンC処理したPDAC又は骨髄由来間葉系幹細胞(MSC)で刺激し、一方、フィーダー不含培養物を対照として用いた。NK2A培地を培養培地として用いた。PDACは、培地のみと比較して、培養NK細胞の拡大倍率を高めることが分かった。しかしながら、フィーダー層ありで成長した細胞又はフィーダー層なしで成長した細胞の間で細胞傷害性の有意な差は見られなかった。図7に示すように、MSCで処理された細胞は、最も高い拡大倍率を示したが、最も低い細胞傷害性を示した。
この実施例は、上記の二段階プロセスを用いて拡大し、分化させたCD34+細胞から産生されるNK細胞が、腫瘍細胞株にとって細胞傷害性であることを示す。
この実施例は、プールされた臍帯血(UCB)及びヒト胎盤灌流液(HPP)(組合せ)からのCD34+細胞の単離を示す。UCB:HPPプール比率を評価するために、3つの異なるプール比率の並列比較を以下のように行なった:(1)最大プール:1×UCB(最大容積)+1×HPP(最大容積);(2)HPPの部分プール(33%):1×UCB(最大容積)+0.33×HPP(HPP容積の1/3);及び(3)HPPの部分プール(10%):1×UCB(最大容積)+0.10×HPP(HPP容積の1/10)。合計N=3の実験反復を実施した。初期のTNC及び容積を記録した。その後、プール試料をCD34+細胞について純化し、CD34+純度を解凍後に決定した。その後、最適プール比率を、解凍後のCD34+純度対容積又は細胞カウント(TNC)比率(組合せの%として)プロットからグラフによって決定した。図8に示すように、終点CD34+純度は、HPP容積含有量とはよく相関するが、HPP TNC含有量とはそれほど相関しない。全体としては、UCB 85%v/v、HPP 15%v/vが、80%以上の純度のCD34+細胞を得るための最適プール比率であることが分かった。
この実施例は、2つのGBGMベースの培地を用いるNK細胞培養プロセス(三段階プロセス及び二段階プロセス)の比較を示す。この2つのプロセスを表10にまとめる。両プロセスとも、胎盤起源のNK細胞及びCD34+細胞の分化のための基本培地としてGBGMを利用した。
(1)新鮮なUCB由来のCD34+細胞:N=6
(2)新鮮な「組合せ」由来のCD34+細胞:N=2
(3)凍結保存された「組合せ」由来のCD34+:N=8
マルチウェルディッシュからT-25、最大で複数のT-75フラスコまで、1〜80mLの培養容積
(1)二段階プロセス
(2)三段階プロセス
(1)フィーダーなし
(2)不活化したK562及びPBMCフィーダーを21日目に培養物に添加
20000〜50000細胞/mL
この研究は、異なる基本培地を用いてCD34+由来NK細胞の分化及び拡大を評価することを目的としている。
この実施例は、NK細胞を保存及び凍結保存する方法を示す。ヒト胎盤灌流液(HPP)及び臍帯血細胞から単離されたCD34+造血幹細胞を、先の実施例に記載されたプロトコルを用いて拡大し、NK細胞に分化させた。細胞を、HPP及び臍帯血から単離された直後(0日目)に、又はNK細胞の最初の成長期の間(単離9日、14日、21日、又は35日後)に凍結保存した。
この実施例は、NK細胞を保存及び凍結保存する別の方法を示す。ヒト胎盤灌流液(HPP)及び臍帯血細胞から単離されたCD34+造血幹細胞を、上記のプロトコルを用いて拡大し、NK細胞に分化させた。細胞を、HPP及び臍帯血から単離した直後(0日目)に、又はNK細胞の最初の成長期の間(単離9日、14日、21日、又は35日後)に凍結保存した。
生存率アッセイ。実施例10又は11に見られるような様々な製剤中で凍結保存されたNK細胞を解凍した。細胞を2×106〜3×107細胞/mLの密度で凍結させた。解凍したNK細胞を、新鮮な細胞又は凍結前の細胞と比較して、解凍0日、3日、及び18日後に、Countess(登録商標)自動細胞カウンター(Invitrogen)を用いて、細胞生存率について評価した。簡潔に述べると、10μlの細胞試料を10μlのトリパンブルーと混合した。該細胞混合物をピペッティングして、Countess(登録商標)チャンバースライドに入れた。該スライドを装置に挿入し、細胞をカウントした。凍結-解凍後細胞は、凍結保存製剤の違いによって、約80%〜90%超の生存率の細胞生存率を示した。
この実施例は、プロセス内凍結保存細胞バンキングの評価を示す。HS-AB又はFBSのいずれかを血清供給源として用い、Spanholtzらの文献(PLoS One. 5(2):e9221(2010))に記載されているような方法を用い、UCB CD34+細胞を用いて、細胞培養を開始した。細胞濃度を決定し、調整し、必要に応じて培地を補充した。7日、9日、10日、又は14日で、約106〜3×106個の細胞を細胞培養物から除去し、遠心分離し、凍結保存培地(5.5%v/vデキストラン-40、10%v/v HSA、5%v/v DMSO)に再懸濁した。該細胞を速度制御フリーザーで凍結させ、凍結保存用の液相窒素保存場所に移した。約1mLの細胞の各バイアルを、1mL当たり106〜107個の範囲の濃度で凍結保存した。残りの培養物は、終点(35日)まで持ち越され、「新鮮(プロセス内凍結保存なし)」と呼ばれた。表現型解析を細胞培養21日目、28日目、及び35日目に行なった。インビトロ機能性(K562細胞傷害性、10:1のE:T)を培養35日(終点)で評価した。
この実施例は、動物におけるNK細胞投与のための解凍後媒体の開発を示す。NK細胞の生存率、細胞傷害性、細胞回復、及び凝集塊形成に対する注射媒体及び細胞密度の効果を試験した。生存率はトリパンブルー染色により評価し;細胞傷害性はFACS(10:1の比のNK:K562)により評価し、凝集塊形成(clump formulation)は顕微鏡アッセイにより評価した。結果を、様々なタイプの注射媒体及び細胞密度について表9〜12に示す。
NK細胞の生存率、細胞傷害性、細胞回復、及び凝集塊形成に対する様々なHSA濃度の効果も試験した。実施例19から同じ方法を利用した。結果を、様々なタイプの注射媒体及び細胞密度について表14〜16に示す。
この実施例は、IL-2の非存在下でのNK細胞の培養を示す。細胞培養を実施例11に記載の二段階プロセスで行なった。第一の培地中の5つの異なる濃度:0、200、500、1000、2000U/mLのIL-2を試験した。
本発明は、本明細書に記載の具体的な実施態様によって範囲が限定されるべきではない。実際、記載された変更に加えて、本発明の様々な変更が、上の説明及び添付の図面から当業者に明白となるであろう。そのような変更は、添付の特許請求の範囲内に含まれることが意図される。
Claims (25)
- 活性化ナチュラルキラー(NK)細胞の集団を産生する方法であって:
(a)造血幹細胞又は前駆細胞の集団が拡大し、かつ該拡大することの間に、該造血幹細胞又は前駆細胞の集団内の複数の造血幹細胞又は前駆細胞がNK細胞に分化するように、インターロイキン-15(IL-15)、並びに任意に幹細胞因子(SCF)及びインターロイキン-7(IL-7)のうちの1つ又は複数を含み、ここで、該IL-15並びに任意のSCF及びIL-7が該培地の不確定成分に含まれない、第一の培地中に、該集団を播種すること;並びに
(b)工程(a)由来の細胞をインターロイキン-2(IL-2)を含む第二の培地中で拡大して、活性化NK細胞の集団を産生すること
を含む、前記方法。 - 活性化ナチュラルキラー(NK)細胞の集団を産生する二工程方法であって、該方法の第一の工程が、幹細胞因子(SCF)、インターロイキン-7(IL-7)、及びインターロイキン-15(IL-15)のうちの1つ又は複数を含む第一の培地中で、造血幹細胞又は前駆細胞の集団を拡大することを含み、ここで、該SCF、IL-7、及びIL-15が該培地の不確定成分に含まれず、かつここで、該拡大することの間に、該造血幹細胞又は前駆細胞の集団内の複数の造血幹細胞又は前駆細胞がNK細胞に分化し;かつ
該方法の第二の工程が、該第一の工程由来の細胞をインターロイキン-2(IL-2)を含む第二の培地中で拡大して、活性化NK細胞を産生すること
を含む、前記方法。 - 前記第一の培地が、Fms様チロシンキナーゼ3リガンド(Flt3-L)、トロンボポエチン(Tpo)、インターロイキン-2(IL-2)、又はヘパリンのうちの1つ又は複数を更に含む、請求項1記載の方法。
- 前記第一の培地が、胎仔ウシ血清又はヒト血清を更に含む、請求項3記載の方法。
- 前記SCFが、前記第一の培地中、約1〜約150ng/mLの濃度で存在する、請求項3記載の方法。
- 前記Flt3-Lが、前記第一の培地中、約1〜約150ng/mLの濃度で存在する、請求項3記載の方法。
- 前記IL-2が、前記第一の培地中、約50〜約1500IU/mLの濃度で存在する、請求項3記載の方法。
- 前記IL-7が、前記第一の培地中、約1〜約150ng/mLの濃度で存在する、請求項3記載の方法。
- 前記IL-15が、前記第一の培地中、1〜約150ng/mLの濃度で存在する、請求項3記載の方法。
- 前記Tpoが、前記第一の培地中、約1〜約150ng/mLの濃度で存在する、請求項3記載の方法。
- 前記ヘパリンが、前記第一の培地中、約0.1〜約30U/mLの濃度で存在する、請求項3記載の方法。
- 前記第二の工程における前記IL-2が、前記第二の培地中、50〜約1500IU/mLの濃度で存在する、請求項1記載の方法。
- 該第二の培地が、胎仔ウシ血清(FCS)、トランスフェリン、インスリン、エタノールアミン、オレイン酸、リノール酸、パルミチン酸、ウシ血清アルブミン(BSA)、及びフィトヘマグルチニンのうちの1つ又は複数を更に含む、請求項1記載の方法。
- 前記造血幹細胞又は前駆細胞がCD34+である、請求項1記載の方法。
- 前記造血幹細胞又は前駆細胞が、ヒト胎盤灌流液由来の造血幹細胞又は前駆細胞、及び臍帯由来の造血幹細胞又は前駆細胞を含み、ここで、該胎盤灌流液及び該臍帯血が同じ胎盤に由来するものである、請求項1記載の方法。
- 工程(b)のフィーダー細胞が、マイトマイシンC処理した末梢血単核細胞(PBMC)、K562細胞、又は組織培養物接着性幹細胞を含む、請求項1記載の方法。
- 前記NK細胞が、CD3-CD56+CD16-である、請求項1記載の方法。
- 前記NK細胞が更にCD94+CD117+である、請求項17記載の方法。
- 前記NK細胞が更にCD161-である、請求項17記載の方法。
- 前記NK細胞が更にNKG2D+である、請求項17記載の方法。
- 前記NK細胞が更にNKp46+である、請求項17記載の方法。
- 前記NK細胞が更にCD226+である、請求項17記載の方法。
- (a)造血幹細胞又は前駆細胞の集団が拡大し、かつ該拡大することの間に、該造血幹細胞又は前駆細胞の集団内の複数の造血幹細胞又は前駆細胞がNK細胞に分化するように、インターロイキン-15(IL-15)、並びに任意に幹細胞因子(SCF)及びインターロイキン-7(IL-7)のうちの1つ又は複数を含み、ここで、該IL-15並びに任意のSCF及びIL-7が該培地の不確定成分に含まれない、第一の培地中に、該集団を播種すること;並びに
(b)工程(a)由来の細胞をインターロイキン-2(IL-2)を含む第二の培地中で拡大して、活性化NK細胞の集団を産生すること:
を含む方法によって得られる活性化NK細胞の集団。 - 腫瘍細胞の増殖を抑制する方法であって、該腫瘍細胞を複数のCD94+CD117+ NK細胞と接触させることを含み、ここで、該NK細胞が、請求項1記載の方法によって産生されたものである、前記方法。
- 前記腫瘍細胞が、原発性腺管癌細胞、膠芽腫細胞、白血病細胞、急性T細胞白血病細胞、慢性骨髄リンパ腫(CML)細胞、急性骨髄性白血病細胞、慢性骨髄性白血病(CML)細胞、肺癌細胞、結腸腺癌細胞、組織球性リンパ腫細胞、多発性骨髄腫細胞、結腸直腸癌細胞、結腸直腸腺癌細胞、前立腺癌細胞、又は網膜芽腫細胞である、請求項24記載の方法。
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