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METHOD FOR PREPARING GENETICALLY-MODIFIED T CELLS WHICH EXPRESS CHIMERIC ANTIGEN RECEPTOR

Foreign code F180009365
File No. (S2016-0025-N0)
Posted date Apr 19, 2018
Country WIPO
International application number 2016JP079989
International publication number WO 2017061615
Date of international filing Oct 7, 2016
Date of international publication Apr 13, 2017
Priority data
  • P2015-200458 (Oct 8, 2015) JP
Title METHOD FOR PREPARING GENETICALLY-MODIFIED T CELLS WHICH EXPRESS CHIMERIC ANTIGEN RECEPTOR
Abstract In order to improve the efficiency of gene introduction in CAR therapy employing a transposon technique, provided is a method for preparing genetically-modified T cells which express a chimeric antigen receptor, comprising: (1) a step for preparing non-proliferative cells obtained by stimulating a group of cells including T cells using an anti-CD3 antibody and an anti-CD28 antibody, and thereafter, subjecting the cells to a treatment for causing the cells to lose their proliferation capability; (2) a step for obtaining genetically-modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced using a transposon technique; (3) mixing the non-proliferative cells prepared at step (1) with the genetically-modified T cells obtained at step (2), and co-culturing the mixed cells while stimulating the mixed cells using the anti-CD3 antibody and the anti-CD28 antibody; and (4) a step for collecting the cultured cells. Also, provided is a method for preparing genetically-modified T cells which express a chimeric antigen receptor, comprising: (i) a step for preparing non-proliferative cells holding a viral peptide antigen, which cells are obtained by stimulating a group of cells including T cells using an anti-CD3 antibody and an anti-CD28 antibody, and thereafter, subjecting the cells to culturing in the presence of the viral peptide antigen and a treatment for causing the cells to lose their proliferation capability; (ii) a step for obtaining genetically-modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced using a transposon technique; (iii) mixing the non-proliferative cells prepared at step (i) with the genetically-modified T cells obtained at step (ii), and co-culturing the mixed cells; and (iv) a step for collecting the cultured cells.
Outline of related art and contending technology BACKGROUND ART
Chimeric antigen receptors (Chimeric Antigen Receptor. Hereinafter, also referred to as' CAR ') cell therapy using genetically engineered T (CAR therapy) is being clinical application. CAR is, typically, single-chain variable region of the antibody and the extracellular domain, transmembrane region thereto, and co-stimulatory signaling that transmits a signal molecule CD3 ζ intracellular domain of the connecting structure. According to the specific properties of the antibody by binding to an antigen on CAR-T cell is activated and, damage to target cells (such as cancer cells). CAR therapy, the preparation of the cells is relatively easy, high cytotoxic activity, such as the sustained effect can be expected which has the advantage, in particular, refractory or resistant to conventional therapy as a new therapy for case expected. Actually, chemoresistance in acute lymphoblastic leukemia patient (acute lymphoblastic leukemia), CAR CD19 antigen expressed on the surface of the cells, peripheral blood T cells taken from the patient and the gene introduction, culturing Europe for infusion performed in clinical trials, the remission rate 80-90% of good results have been reported (non-patent document 1-3). CAR therapy, cancer in the United States refractory 1 as one of the most promising therapy has attracted attention.
Conventional, a cell used in the therapy CAR (CAR-T cells) is a viral vector has been prepared using. However, commonly used retrovirus, receptor-a high frequency of insertion mutation to the gene (hematopoietic stem cells were used for gene therapy in leukemia is frequently), safety becomes a problem. In addition, in the conventional culture cell lines and fetal bovine serum and, in particular pediatric patients in the long-term safety concern. Further, when using the viral vector is added to the cell culture equipment is needed for, and be very expensive due to the fact that the cost of the therapy, there is a problem of economy (non-patent document 4).
Scope of claims (In Japanese)[請求項1]
以下のステップ(1)~(4)を含む、キメラ抗原受容体を発現する遺伝子改変T細胞の調製方法:
(1)T細胞を含む細胞集団を抗CD3抗体及び抗CD28抗体で刺激した後、増殖能を喪失させる処理を行うことによって得られる非増殖性細胞を用意するステップ;
(2)トランスポゾン法によって、標的抗原特異的キメラ抗原受容体遺伝子が導入された遺伝子改変T細胞を得るステップ;
(3)ステップ(1)で用意した非増殖性細胞とステップ(2)で得た遺伝子改変T細胞を混合し、抗CD3抗体及び抗CD28抗体で刺激しつつ共培養するステップ;
(4)培養後の細胞を回収するステップ。
[請求項2]
ステップ(3)とステップ(4)の間に、共培養後の細胞をT細胞増殖因子の存在下で培養するステップを行う、請求項1に記載の調製方法。
[請求項3]
ステップ(3)の共培養の期間が1日~14日である、請求項1又は2に記載の調製方法。
[請求項4]
ステップ(3)を、T細胞増殖因子の存在下で行う、請求項1~3のいずれか一項に記載の調製方法。
[請求項5]
T細胞増殖因子がIL-15である、請求項4に記載の調製方法。
[請求項6]
T細胞増殖因子としてIL-15とIL-7を併用する、請求項4に記載の調製方法。
[請求項7]
以下のステップ(i)~(iv)を含む、キメラ抗原受容体を発現する遺伝子改変T細胞の調製方法:
(i)T細胞を含む細胞集団を抗CD3抗体及び抗CD28抗体で刺激した後、ウイルスペプチド抗原存在下での培養及び増殖能を喪失させる処理を行うことによって得られる、ウイルスペプチド抗原を保持した非増殖性細胞を用意するステップ;
(ii)トランスポゾン法によって、標的抗原特異的キメラ抗原受容体遺伝子が導入された遺伝子改変T細胞を得るステップ;
(iii)ステップ(i)で用意した非増殖性細胞とステップ(ii)で得た遺伝子改変T細胞を混合し、共培養するステップ;
(iv)培養後の細胞を回収するステップ。
[請求項8]
ステップ(iii)とステップ(iv)の間に、共培養後の細胞をT細胞増殖因子の存在下で培養するステップを行う、請求項7に記載の調製方法。
[請求項9]
ステップ(iii)の共培養の期間が1日~14日である、請求項7又は8に記載の調製方法。
[請求項10]
ステップ(iii)を、T細胞増殖因子の存在下で行う、請求項7~9のいずれか一項に記載の調製方法。
[請求項11]
T細胞増殖因子がIL-15である、請求項10に記載の調製方法。
[請求項12]
T細胞増殖因子としてIL-15とIL-7を併用する、請求項10に記載の調製方法。
[請求項13]
T細胞を含む細胞集団が末梢血単核細胞(PBMCs)である、請求項1~12のいずれか一項に記載の調製方法。
[請求項14]
増殖能を喪失させる処理が放射線照射である、請求項1~13のいずれか一項に記載の調製方法。
[請求項15]
トランスポゾン法がPiggyBacトランスポゾン法である、請求項1~14のいずれか一項に記載の調製方法。
[請求項16]
標的抗原がCD19、CD19、GD2、GMCSF受容体又はIGF受容体である、請求項1~15のいずれか一項に記載の調製方法。
[請求項17]
非増殖性細胞と、遺伝子改変T細胞が同一の個体に由来する、請求項1~16のいずれか一項に記載の調製方法。
[請求項18]
請求項1~17のいずれか一項に記載の調製方法で得られた、キメラ抗原受容体を発現する遺伝子改変T細胞。
[請求項19]
請求項18に記載の遺伝子改変T細胞を治療上有効量含む、細胞製剤。
[請求項20]
請求項18に記載の遺伝子改変T細胞を、治療上有効量、がん患者に投与するステップを含む、がんの治療法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY
  • SHINSHU UNIVERSITY
  • Inventor
  • NISHIO Nobuhiro
  • NAKAZAWA Yozo
  • TANAKA Miyuki
  • MORITA Daisuke
  • TAKAHASHI Yoshiyuki
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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