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Method for inducing T cells for cellular immunotherapy from pluripotent stem cells

一种多能性干细胞、细胞的技术，应用在诱导细胞免疫治疗用T细胞领域，能够解决TCR特异性损失等问题

Active Publication Date: 2022-01-28
KYOTO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This additional TCRα gene rearrangement not only results in a loss of TCR specificity, but may also generate autoreactive T cells due to mispairing with an undesired chain

Method used

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  • Method for inducing T cells for cellular immunotherapy from pluripotent stem cells
  • Method for inducing T cells for cellular immunotherapy from pluripotent stem cells
  • Method for inducing T cells for cellular immunotherapy from pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Establishment of iPS cells from T cells with a homozygous HLA haplotype and regeneration of T cells from iPS cells thus obtained.

[0112] Peripheral blood obtained from a donor homozygous for HLA-A*3303-B*4403-C*1403-ERB1*1302 was used.

[0113] 1) Establishment of Homo-T-iPS cells

[0114] The culture medium used is as follows:

[0115] 【Table 1】

[0116] Culture medium for T cells (T cell culture medium)

[0117] quantity final concentration RPMI 45ml human AB serum 5ml 10％ Total 50ml

[0118] A. Activation of CD8-positive T cells

[0119] 1. Use Ficoll to refine peripheral blood mononuclear cells obtained from healthy volunteers, and use MACS beads to enrich CD8-positive cells.

[0120] 2. Disperse the enriched cells in T cell culture medium and add IL-2 (final concentration: 30U / mL), IL-7 (final concentration: 5ng / mL) and IL-15 (final concentration: 1ng / mL ). Dynabeads Human T-Activator CD3 / CD28 was added to form a be...

Embodiment 2

[0169] 1. Induction of CD8 positive T cells from T-iPS cells generated in Example 1.

[0170] 2. Using the iPS cell clones obtained in Example 1 with homozygous HLA haplotypes. Donor A (whose HLA haplotype has at least one match with the HLA haplotype of the iPS cell) and donor B (whose HLA haplotype does not match the HLA haplotype of the iPS cell at all) are selected.

[0171] Donor A: HLA-A*31:01 / 33:03; B*44:03 / 48:01; C*04:01 / 14:03; DRB1*04:03 / 13:02

[0172] Donor B: HLA-A*24:02 / 24:02; B*07:02 / 52:01; C*07:02 / 12:02; DRB1*01:01 / 15:02

[0173] 3. Using Ficoll to purify monocytes from the peripheral blood of donor A and donor B, and enrich CD8 positive cells by CD8 microbeads.

[0174] 4. Perform mixed lymphocyte reaction (MLR) using CD8-positive T cells regenerated from T-iPS cells as a stimulating factor. CD8 cells obtained from donors were used as effectors. Effector cells were fluorescently labeled using the CellTrace Violet Cell Proliferation Kit. Cell Trace CFSF Cell...

Embodiment 3

[0180] The T-iPS cells having a homozygous HLA haplotype obtained in Example 1 were differentiated into CD8-positive T cells (CTL) by conventional methods. It was examined whether CTLs would activate NK cells derived from peripheral blood of subjects with heterozygous HLA haplotypes, one of which matched the HLA haplotype of iPS cells.

[0181] 1. The CD8-positive T cells differentiated from iPS cells with a homozygous HLA haplotype by conventional methods in Example 1 were used as stimulators.

[0182] 2. Establish iPS cells from cells of donor A with heterozygous HLA haplotypes (one of which matches the HLA haplotype of the stimulator cell) by conventional methods, and differentiate the resulting iPS cells into CD8 positive T cells. And the obtained CD8 positive T cells were also used as stimulatory factors.

[0183] 3. Obtain peripheral blood from donor A, and use Ficoll to separate peripheral blood mononuclear cells (PBMC). NK cells were enriched from PBMCs by negative ...

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Abstract

Provided is a method for inducing T cells for cellular immunotherapy, comprising the following steps: (1) providing human pluripotent stem cells having a gene encoding a desired antigen-specific T cell receptor and knocking out Rag1 and / or Rag2 genes, and (2) inducing T cells from the pluripotent stem cells of step (1). Also provided are a cellular immunotherapy method using T cells for cellular immunotherapy and an iPS cell bank for cellular immunotherapy.

Description

technical field [0001] The present application relates to a method for inducing T cells for cellular immunotherapy. In particular, a method for efficiently and reliably inducing T cells having a desired antigen-specific T cell receptor gene from pluripotent stem cells such as iPS cells is provided. The present application also relates to a method for forming a cell bank using iPS cells, which can be effectively used for cellular immunotherapy. Background technique [0002] Each T cell expresses T cell receptors (TCRs) with different specificities. When an infectious disease develops, a T cell with the appropriate specificity proliferates to form a T cell population (clone) that fights the pathogen. This is the basic idea of ​​acquired immunity. If it is possible to artificially expand T cells with the desired antigen-specific TCR, the expanded T cells can be used for adoptive immunotherapy. The expansion of specific T cells is called "cloning". In fact, autologous trans...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10A61K35/17A61P35/00
CPCA61K35/14A61P35/00A61K35/17C12N2506/45C12N2501/515C12N2510/00C12N5/0696C12N5/0638C12N5/0636C07K14/4705C12N5/10
Inventor 金子新河本宏增田乔子南川淳隆堀田秋津岛津裕一濑大志
Owner KYOTO UNIV
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