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Preparation method of T cell with cell surface markers of CD45RA < + > and CCR7 < + >

A cell surface and marker technology, applied in the field of T cells with cell surface markers, can solve problems such as difficulty in completely removing feeder cells

Pending Publication Date: 2022-06-07
DAIICHI SANKYO CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the iT SCM When the cell production method is applied to adoptive T cell therapy, there are problems such as difficulty in completely removing the co-cultured feeder cells

Method used

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  • Preparation method of T cell with cell surface markers of CD45RA &lt; + &gt; and CCR7 &lt; + &gt;
  • Preparation method of T cell with cell surface markers of CD45RA &lt; + &gt; and CCR7 &lt; + &gt;
  • Preparation method of T cell with cell surface markers of CD45RA &lt; + &gt; and CCR7 &lt; + &gt;

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1: Preparation of memory stem cell-like T cells in the presence of OP9-hDLL1 cell culture supernatant

[0136] 1-1. Preparation of OP9-hDLL1 cell culture supernatant

[0137] A cell culture supernatant (OP9-hDLL1 CM) of OP9-hDLL1 cells (obtained from RIKEN) was prepared. will be 1.5 x 10 5 Each OP9-hDLL1 cell was seeded in a 10 cm dish for cell culture (353003, manufactured by FALCON), and added with fetal bovine serum (20%, FBS, manufactured by Sigma), penicillin-streptomycin solution (1%, Thermo The cells were grown in αMEM medium (manufactured by Gibco, 10 mL) of Fisher Corporation. Cell proliferation at 37 °C and 5 vol% CO 2 conditions. The culture medium was collected when the cells reached 100% confluence, filtered through a sterile membrane (pore size: 0.45 μm) to obtain the culture supernatant, and stored at 4°C.

[0138] 1-2. Activated T cells (activated CD8α) + T cells) preparation

[0139] [Screening step of raw T cells] Human peripheral bloo...

Embodiment 2

[0147] Example 2: Preparation of memory stem cell-like T cells in the presence of cell culture supernatants of OP9 and TSt-4 cells do

[0148] 2-1. Preparation of OP9 and TSt-4 cell culture supernatants (OP9 CM, TSt-4 CM)

[0149] Cell culture supernatants (OP9 CM, TSt-4 CM) of OP9 cells (obtained from RIKEN) and TSt-4 cells (obtained from RIKEN) were prepared. will be 1.5 x 10 5 OP9 cells or 1.5 x 10 5 Each TSt-4 cell was seeded in a 10-cm dish for cell culture (353003, manufactured by FALCON), in a solution containing FBS (20%, manufactured by Sigma) and penicillin-streptomycin solution (1%, Thermo Fisher). The cells were grown in αMEM medium (manufactured by Gibco, 10 mL). Cell proliferation at 37 °C and 5 vol% CO 2 conditions. The medium was collected when the cells reached 100% confluence, filtered through a sterile membrane (pore size: 0.45 μm) to obtain the culture supernatant (OP9 CM or TSt-4CM), and stored at 4°C.

[0150] 2-2. Activated T cells (activated C...

Embodiment 3

[0158] Example 3: Memory using OP9-hDLL1 cell culture supernatant / Notch signaling activation (NICD forced expression) Production of sex stem-like T cells

[0159] 3-1. Activated T cells forcibly expressing NICD (activated NICD forcibly expressing CD8α) + T cells) preparation

[0160] according to figure 1 The indicated experimental design for overexpression of the Notch intracellular domain (NICD, SEQ ID NO: 1) in activated T cells was made figure 2 The indicated NICD overexpressed retroviral vector (pMEI-5 DNA, manufactured by Takara Bio). Activated NICD-expressing T cells were produced using the produced retroviral vector.

[0161] Specifically, by the same method as the initiation step described in Example 1, human CD8α was + T cell activation. Next, 24 hours after activation, retroviral forced expression was performed using NICD. Screening of NICD-enforced T cells as Venus after 6 days of T cell activation + cells were reactivated using CD3 / 28 microbeads.

[0...

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Abstract

The present invention addresses the problem of providing a technique for eliminating the depletion of T cells, which is a problem in T cell transfer therapy and the like, and improving the activity of T cells. T cells having cell surface markers of CD45RA + and CCR7 + can be prepared by culturing activated T cells in the presence of (a) a cell culture supernatant of stromal cells or (b) CXCL12.

Description

technical field [0001] The present invention relates to the preparation of CD45RA + and CCR7 + A method of cell surface markers for T cells. In more detail, according to the present invention, by culturing T cells using the cell culture supernatant of stromal cells or CXCL12, it is possible to prepare cells with CD45RA + and CCR7 + cell surface markers of T cells. Background technique [0002] Adoptive T cell therapy is considered promising in cancer treatment (Non-Patent Document 1). For example, it has been reported that tumor infiltrating lymphocyte (TIL) therapy has a remarkable effect in patients with malignant melanoma (Non-Patent Documents 2 and 3). In this therapy, isolated TILs are restimulated, expanded and cultured using antigen-presenting cells that present tumor associated antigens (TAAs), and the TILs are administered to a patient. [0003] On the other hand, new adoptive T cell therapy using gene transfer technology has also shown great therapeutic effec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P31/00A61P37/02A61P37/08A61P35/00
CPCC12N5/0636A61P31/00A61P37/02A61P37/08A61P35/00C12N2502/1394C12N2501/42C12N2501/21C12N2501/105A61K39/4631A61K39/4611A61K39/464412A61K2239/38A61K2239/48A61K2239/31A61P37/06C12N2510/00C12N2502/1352C12N2501/2307C12N2501/51C12N2501/515C07K16/2803C07K2317/622A61P35/02C12N2501/72C12N2501/125C12N2501/26C07K2319/03C07K14/70589C07K14/7158
Inventor 富里亘吉村昭彦近藤泰介安藤真
Owner DAIICHI SANKYO CO LTD
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