Culture expansion method of CD8 T cells, and K3EC cells

A culture method and cell technology, applied in the field of biomedicine, can solve problems such as difficulty and unacceptable for patients, and achieve the effect of efficient preparation

Active Publication Date: 2020-01-21
SHANGHAI ICELL BIOTECH +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Obviously, such a large amount of blood collection is clinically difficult, and it is not easy for patients to accept it.

Method used

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  • Culture expansion method of CD8 T cells, and K3EC cells
  • Culture expansion method of CD8 T cells, and K3EC cells
  • Culture expansion method of CD8 T cells, and K3EC cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Antigen-specific CD8 T cell preparation process of the present invention ( figure 2 )

[0067] 1. MLPC-plus activates and expands antigen (CMV-pp65)-specific CD8 T cells

[0068] 1) Collect 20ml of peripheral blood and anticoagulate with heparin

[0069] 2) Ficoll gradient centrifugation to collect PBMC, PBS centrifugal washing 3 times

[0070] 3) Resting culture overnight in 10% FCS-RPMI

[0071] 4) After adding 20% ​​K3EC, centrifuge and discard the supernatant, adjust PBMC to 1x 10 with 3% SG-AIM-V 7 / ml

[0072] 5) Add 1 μg / ml CMV-pp65 antigen peptide (Miltenyi, Cat. No. 130-093-435), and incubate at 37°C for 2 hours

[0073] 6) Supplement 3% SG-AIM-V to 4ml, add 4ml 2x CM, culture and passage in 6-well plate

[0074] 7) Collect the cells when culturing for 7 days, adjust the cells to 1x 10 7 / ml

[0075] 8) Add 1 μg / ml CMV-pp65 antigen peptide and incubate at 37°C for 2hr

[0076] 9) Paraformaldehyde fixed K3EC cells

[0077] 10) Adjust cells t...

Embodiment 2

[0086] Embodiment 2 constructs K562-aAPC (K3EC)

[0087] 1. The basis for the selection of co-stimulatory molecules

[0088] The main purpose of constructing K3EC is to provide co-stimulatory signal for MLPC to activate antigen-specific CD8 T cells. It is currently known that the costimulatory signals required to activate such T cells can be provided by multiple receptors, among which CD28 is the most powerful costimulatory receptor. CD28 belongs to the Ig superfamily (Ig superfamily, IgSF), and its cytoplasmic tail contains three motifs: YMNM, TPRRP and PYAP. When CD28 binds to B7 ligand (CD80 / 86), YMNM activates AP1 (activator protein 1), NF-kB (κB nuclear factor) and NFAT (activated T cell nuclear factor) through PI3K-AKT pathway and Grb2-Vav-Sos pathway ) leads to the expression of Bcl-xL anti-apoptotic protein and IL2 cytokine. The PYAP motif can also activate AP1, NF-kB, and NFAT transcription factors through the Grb2-Vav-Sos pathway, while the TPRRP motif mainly acti...

Embodiment 3

[0107] Example 3 Polyclonal T cell proliferation test

[0108] aAPC usually needs to be inactivated (that is, to eliminate the ability to proliferate) before it can be used in T cell co-culture. This inactivation method includes irradiation, mitomycin treatment, and paraformaldehyde fixation. In order to evaluate whether K3EC still has co-stimulatory activity after fixation, CD3 antibody was used to provide polyclonal TCR signal, and 4% paraformaldehyde-fixed K3EC provided co-stimulatory signal. The proliferative activity of T cells was detected by CFSE dilution method, that is, PBMCs were labeled with CFSE (20 μM), and after washing, PBMCs and K3EC were mixed and cultured in 96 wells in different ratios (PBMC:K3EC=5:1, 10:1 and 20:1) In the plate, a CD3 antibody control group was set up at the same time, and 1 μg / ml CD3 antibody was added to each group. CD3 detection after 2 days and 5 days of culture + cells and CD3 + CFSE Low proliferating cells. CD3 + Proliferating c...

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Abstract

The invention provides an expansion culture method of CD8 T cells. The CD8 T cells are subjected to induced expansion culture in the presence of the following substances: K3EC cells and a peptide-fragment overlapping library of a target antigen; and the K3EC cells are K562 engineering cells expressing CD2 ligands CD58, NKG2D ligands MICA/B and CD137 ligands CD137L and are used for providing costimulatory receptor second signals activating the CD8 T cells, and the peptide-fragment overlapping library of the target antigen is used for providing T cell receptor (TCR) first signals activating theCD8 T cells. According to the method, quick and efficient expansion of the antigen (CMV-pp65) specific CD8 T cells; and the CD8 T cells obtained through expansion have high clinical application valuein prevention and treatment of CMV infection and treatment of CMV-related cancers.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for culturing and expanding T cells. Background technique [0002] CD8 T cells, also known as cytotoxic T lymphocytes (CTL), are an important line of defense for the adaptive immune system to construct antigen-specific immune protection mechanisms. CD8 T cells specifically recognize antigens through T cell receptors (TCR), which are expressed in the form of immunogenic peptide epitopes embedded in MHC-I molecules (pMHCI for short) on the cell surface. [0003] Enrichment and expansion of antigen-specific CD8 T cells from body fluids (such as blood) or tissues (such as tumor lesions) using modern cell engineering techniques, and then infuse them into patients in need of treatment. The treatment option is called adoptive T cell therapy or T cell therapy. Clinical data reveal that this T cell therapy can control refractory viral infections (such as CMV an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/10
CPCC12N5/0638C07K14/70535C07K14/70503C12N2501/515C12N2501/2302C12N2501/2307C12N2501/2315C12N2502/30
Inventor 董强刚张艳周瑾王春慧赵雅宁严小敏张光辉戴果鲜邵小燕
Owner SHANGHAI ICELL BIOTECH
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