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CD3 negative cell populations expressing chemokine receptors and cell adhesion molecules and uses and methods of making same

A cell population and cell technology, applied in chemical instruments and methods, biochemical equipment and methods, biological preparations for removing undesirable cells, etc., can solve the lack of chemokine receptors, CAR-T cells do not reach solid tumors, tumors Problems such as decreased cell chemotaxis

Pending Publication Date: 2020-11-10
GAIA BIOMEDICINE INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CAR-T cell therapy may not be very effective in treating solid tumors. One of the reasons for this is that CAR-T cells do not reach solid tumors (Non-Patent Document 1)
Regarding the targeting of CAR-T cells to tumors, it has been reported that chemokine receptors may be deleted during genetic modification, resulting in decreased chemotaxis to tumor cells (Non-Patent Document 2)

Method used

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  • CD3 negative cell populations expressing chemokine receptors and cell adhesion molecules and uses and methods of making same
  • CD3 negative cell populations expressing chemokine receptors and cell adhesion molecules and uses and methods of making same
  • CD3 negative cell populations expressing chemokine receptors and cell adhesion molecules and uses and methods of making same

Examples

Experimental program
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Effect test

preparation example Construction

[0098] [Preparation method of NK-like cell population]

[0099] The present invention provides a method for preparing CCR5-positive, CCR6-positive, CXCR3-positive and CD3-negative cell populations, including the steps of preparing primary mononuclear cell populations; removing CD3-positive cells from primary mononuclear cell populations; A step in which any one selected from the group consisting of monocytes and B cells is removed from the mononuclear cell population; after removing the CD3 positive cells and any one selected from the group consisting of monocytes and B cells A step in which the remaining cell population is cultured in medium comprising IL-2.

[0100] (primary mononuclear cell population)

[0101] In the preparation method of the present invention, the primary mononuclear cell population can be obtained through the step of isolating mononuclear cells from blood cells collected from a subject. Blood cells may be collected from any one selected from the group ...

Embodiment 1

[0145]

[0146] Peripheral blood of healthy volunteers was separated by density centrifugation to obtain peripheral blood mononuclear cells (hereinafter, PBMC). to each 1 x 10 7 Cells Using 5 μL of CliniMACS CD3 (Miltenyi Biotech, Cat. No. 130-017-601), CD3-positive cells were removed from PBMCs (cells recovered here were referred to as "primary NK"), and the remaining cells were cultured. In culture, the cells were divided into 5×10 5 The density of cells / mL was suspended in KBM-501 (Kohjin-bio, added with 5% AB serum), and cultured for 14 days using 6-well plates (Thermo Fisher Scientific, 140675) or T-75 flasks (Thermo Fisher Scientific, 156499) Medium was added on day 9). The cells of this cell population are referred to as "14-day cultured cells" and "cells of the present invention".

[0147] For the analysis of chemokine receptors, the cells of the present invention after 14 days of culture and "primary NK" of cells recovered before culture were used as a control. ...

Embodiment 2

[0160]

[0161] To analyze the content of CD3-positive cells and CD19-positive cells, cells of the present invention cultured for 14 days were used in the same manner as in Example 1, and PBMC were used as controls. The antibodies used for fluorescent labeling were PE-labeled anti-human CD3 antibody (Biolegend, 300408) and PerCP-Cy 5.5-labeled anti-human CD19 antibody (Biolegend, 302230). Fluorescent labeling was carried out in the same manner as in Example 1.

[0162] show the result in figure 2 . The cells of the present invention cultured for 14 days contained 0.025% of CD3 positive cells and 0.28% of CD19 positive cells. On the other hand, 64.7% of CD3-positive cells and 8.57% of CD19-positive cells were contained in PBMCs that were not cultured.

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Abstract

The present invention relates to immune cells with higher cytotoxic activity and pharmaceutical compositions that are expected to be efficiently used in NK cell therapy. The present invention providesa population of cells comprising CCR5 positive, CCR6 positive, and CXCR3 positive and CD3 negative cells. The present invention provides a population of cells in which CCR5-positive, CCR6-positive, and CXCR3-positive and CD3-negative cells further highly express CD11c. The present invention provides CCR5 positive, CCR6 positive and CXCR3 positive and CD3 negative cells that are infiltrated with solid tumors. The invention also provides pharmaceutical compositions comprising such populations of cells and pharmaceutically acceptable additives. The invention further provides a preparation methodof the cell population.

Description

technical field [0001] The present invention relates to CD3-negative cell populations expressing chemokine receptors and cell adhesion molecules and uses thereof. [0002] Cross References to Related Applications [0003] This application claims the priority of Japanese Patent Application No. 2018-59624 filed on Mar. 27, 2018, and the entire description thereof is hereby incorporated by reference as disclosure. Background technique [0004] Natural killer cells (NK cells) are cytotoxic lymphocytes that function as the main factor of natural immunity, and are responsible for the initial protection mechanism against tumor cells and virus infection. [0005] NK cell therapy, in which NK cells collected from the patient are cultured in vitro to proliferate hundreds to thousands of times and then infused back into the patient, has attracted attention as a treatment method with relatively few side effects. Usually, about 1×10 10 Mononuclear cells (mononuclear cells), if the com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61P35/00C07K14/705C07K14/7158C07K14/70503C07K14/7051C12N5/0081C12N5/0646C12N2501/2302C12N2501/21A61K2239/59A61K39/4644A61K39/4613A61K2239/31A61K2239/38
Inventor 米满吉和原田结
Owner GAIA BIOMEDICINE INC
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