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METHOD FOR CLONING T CELL RECEPTOR

Foreign code F140007957
File No. S2012-0940-C0
Posted date Sep 26, 2014
Country WIPO
International application number 2013JP070028
International publication number WO 2014017533
Date of international filing Jul 24, 2013
Date of international publication Jan 30, 2014
Priority data
  • P2012-164442 (Jul 25, 2012) JP
Title METHOD FOR CLONING T CELL RECEPTOR
Abstract The present invention addresses the problem of providing a T cell receptor (TCR) cloning system which enables the analysis of non-biased TCR repertories, enables the collection of an antigen-specific (TCR α cDNA)/( TCR β cDNA) pair, and also enables the evaluation of the function of the pair. Provided is a method for producing a TCR gene specific to an antigen (A), comprising the steps of: (1) stimulating a group of T cells which contains antigen-(A)-specific T cells or a single antigen-(A)-specific T cell under the conditions effective for the amplification of the TCR gene; (2) identifying the antigen-(A)-specific T cells among from the group of T cells which contains the antigen-(A)-specific T cells and then sorting the identified antigen-(A)-specific T cells into a container one by one; and (3) subjecting one of the activated antigen-(A)-specific T cells in the container to PCR to amplify the TCR gene specific to the antigen (A). According to the present invention, it becomes possible to clone a TCR gene of interest in a shorter time compared with the conventional methods, e.g., within about 10 days. According to the present invention, it also becomes possible to clone a gene for TCR α-chain and a gene for TCR β-chain with high efficiency. Under the conditions employed in examples, a (TCR α-chain)-(TCR β-chain) pair was able to be obtained with an efficiency of 100% from the T cells which had been sorted one by one after stimulation.
Outline of related art and contending technology BACKGROUND ART
Mainly applied to a specific cancer has been studied T cell receptor (TCR) in gene therapy, lymphocytes of the cancer patient, cancer antigen-specific TCR gene is introduced. The gene transduced lymphocytes after when a large number, is returned to the cancer patient, recognizes a peptide tumor antigen was expressed on TCR since lymphocytes, cancer cells which present the tumor antigen which is recognized and specifically attacks, finally can be expected to abolish the cancer cells.
For use in gene therapy of antigen-specific TCR gene to obtain, collected from the patient in the peripheral blood lymphocytes (PBL) from the cancer antigen is expressed in cells T T cells can be identified, it is necessary to clone the gene TCR. Approach is typically performed for this purpose, including antigen-specific T cell clones established, this is typically, requires several months.
On the other hand, antigen-specific T cells for numerous studies in the TCR repertoire have been made. This is, analysis is typically done in, for example, the product of a gene family TCR β V (TRB) targeting monoclonal antibody (mAb) of panel (non-patent document 1) a method based on FACS TRBV PCR and specific primers based on the method of panel (non-patent document 2-4) has been performed by. However, conventional repertoire analysis, both TCR α TRBV V (TRA) analysis and not, as TCR repertoire analysis, is incomplete. In addition, established T cell clones TCR repertoire including these methods can cause deviations are presented the concern that the (non-patent document 5 and 6). That is, step T cell clones were established, and grow easily from T cell clones will grow and, for analysis using PCR, amplified by the primers can differ depending on the efficiency.
On the other hand, the present inventors and other groups by a group, human (non-patent document 7) and mouse (non-patent document 8) and of the transcription products of the simultaneous CDR3 α TCR CDR3 β single cell RT-PCR protocols that enable the identification have been reported. However, single cell RT-PCR protocol thereof complete protein translation region cloned TCR α / β cDNA pairs, by expressing in the cell it, in order to determine the antigen specificity has not yet been produced.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 1)抗原Aに特異的なT細胞を含むT細胞群を、または抗原Aに特異的な1個のT細胞を、T細胞受容体(TCR)遺伝子増幅上有効な条件で刺激する工程;
 2)抗原Aに特異的なT細胞を含むT細胞群から、抗原Aに特異的なT細胞を特定して1個ずつ容器へソートする工程;および
 3)容器内の1個の活性化された抗原A特異的T細胞をPCRに供して、抗原Aに特異的なTCR遺伝子を増幅する工程
を含む、抗原Aに特異的なTCR遺伝子の製造方法。

[請求項2]
 さらに、
 4)取得したTCRをTCRを発現していないT細胞株に導入し、発現させ、その抗原特異性を検証する工程
を含む、請求項1に記載の製造方法。

[請求項3]
 すべての工程を10日間以内に行う、請求項1または2に記載の製造方法。

[請求項4]
 工程1)のTCR遺伝子増幅上有効な条件が、少なくともインターロイキン-2(IL-2)、インターロイキン-7(IL-7)、フィトヘマグルチニン(PHA)、ホルボール12-ミリステート13-アセテート((phorbol 12-myristate 13-acetate、PMA)、シクロヘキシミド(CHX)、抗CD3抗体、抗CD28抗体、および抗原ペプチドから選択される少なくとも一つの存在下で、細胞群または細胞を8時間~3日間維持することである、請求項1~3のいずれか1項に記載の製造方法。

[請求項5]
 工程1)のTCR遺伝子増幅上有効な条件が、IL-2またはIL-7およびPHA;抗CD3抗体、抗CD28抗体およびIL-2;抗原ペプチド、抗CD28抗体およびIL-2;PMAおよびCHXの存在下で細胞群または細胞を維持することある、請求項4に記載の製造方法。

[請求項6]
 工程1)が、T細胞群を刺激するものであり、工程1)、2)および3)が、この順で実施される、請求項1~5のいずれか1項に記載の製造方法。

[請求項7]
 工程2)がフローサイトメトリーまたはチップイムノスポットアッセイ(Immunospot-array assay on a chip、ISAAC)法により実施される、請求項1~6のいずれか1項に記載の製造方法。

[請求項8]
 工程2)が、主要組織適合遺伝子複合体(MHC)分子と抗原A由来抗原ペプチド(p)との複合体を多量体化したもの(典型的には四量体化したもの(MHC/p四量体))と、抗CD4抗体または抗CD8抗体とを用いてフローサイトメトリーによりソートする工程である、請求項7に記載の製造方法。

[請求項9]
 工程2)が、抗インターフェロン-γ(IFN-γ)抗体と、抗CD4抗体または抗CD8抗体とを用いてフローサイトメトリーによりソートする工程である、請求項7に記載の製造方法。

[請求項10]
 請求項1~9のいずれか1項に定義された工程を含み、得られた抗原Aに特異的なTCR遺伝子を別のT細胞に導入して抗原Aに特異的な組換えT細胞を得る工程をさらに含む、組換えT細胞の製造方法。

[請求項11]
 抗原AがTCR遺伝子治療により処置可能な疾患または状態に関連する抗原であり、別のT細胞が、当該処置可能な疾患または状態にある対象由来である、請求項10に記載の製造方法。

[請求項12]
 抗原Aががん関連抗原であり、がん特異的TCR遺伝子、またはがん特異的組換えT細胞が製造される、請求項1~11のいずれか1項に記載の製造方法。

[請求項13]
 がんの処置に用いるための、請求項12に記載の製造方法。

[請求項14]
 がんまたは感染症にある対象におけるTCRレパートリーの解析のために行われる、請求項1~9のいずれか1項に記載の方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION UNIVERSITY OF TOYAMA
  • Inventor
  • KISHI, Hiroyuki
  • MURAGUCHI, Atsushi
  • KOBAYASHI, Eiji
  • OZAWA, Tatsuhiko
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 DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS KE KG KN KP KR KZ LA LC LK LR LS LT 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 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 TD TG
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