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Non-activated t cells expressing exogenous virus-specific t cell receptor (TCR)

A cell receptor and specific technology, applied in the field of T cells, can solve problems such as remodeling virus immunity and hindering application

Pending Publication Date: 2019-03-15
LION TCR PTE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Concerns about toxicity have hampered the use of adoptive transfer therapy of such specific T cells and the possibility of remodeling the body's immunity against these viruses that infect vital organs

Method used

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  • Non-activated t cells expressing exogenous virus-specific t cell receptor (TCR)
  • Non-activated t cells expressing exogenous virus-specific t cell receptor (TCR)
  • Non-activated t cells expressing exogenous virus-specific t cell receptor (TCR)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0243] Example 1: Cell Lines

[0244]HepG2-Env and HepG2-Core cell lines expressing luciferase were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 20 mM HEPES, 0.5 mM sodium pyruvate, 100 U / ml penicillin, 100 μg / ml streptomycin , MeM amino acids, Glutamax, MeM non-essential amino acids (Invitrogen, Carlsbad, CA) in RPMI 1640 and supplemented with 2 μg / ml puromycin (BD Biosciences, San Jose, CA) to maintain selection. T2 cells were cultured in RPMI1640 supplemented as described above. HepG2 cells (HepG2.2.15) stably expressing the entire HBV genome and producing infectious virus were supplemented with 10% heat-inactivated FBS, 20mM HEPES, 0.5mM sodium pyruvate and 150μg / ml G418 (Sigma-Aldrich, St.Louis , MO) in DMEM. HepG2 cells stably expressing human sodium taurocholate cotransporting polypeptide (hNTCP) were cultured in DMEM supplemented with 10% heat-inactivated FBS, Glutamax, 100 U / ml penicillin, 100 μg / ml streptomycin, and 5 μg / ml Puromycin to maint...

Embodiment 2

[0245] Example 2: T cells

[0246] Peripheral blood mononuclear cells (PBMC) were collected from healthy donors with formal informed consent. To produce activated T cells, PBMC were treated with 600 IU / ml interleukin-2 (rlL-2; R&D Systems) and 50 ng / ml anti-CD3 antibody (OKT-3; eBioscience, San Diego, CA) in AIM-V+2% human Stimulate in AB serum for 8 days, and raise rlL-2 to 1000IU / ml one day before electroporation. Non-activated (resting) T cells were isolated using a Whole T Cell Isolation Kit (Miltenyi Biotec, GmbH, Germany) and cultured overnight in 100 IU / ml rlL-2 before electroporation.

Embodiment 3

[0247] Example 3: Flow Cytometry

[0248] Antibodies used for cell surface staining were purchased from BD Biosciences (anti-human CD8-PE-Cy7, CD8-V500, CD45RA-APC, CD62L-PECy7 antibody), eBioscience (anti-human CD45RO-eFluor650 antibody), Immudexand Proimmune (HLA-A201- HBs183-91-PE dextramer or pentamer) and R&D Systems (human LTβ receptor-Fc chimera) were obtained. Antibodies for intracellular cytokine staining were obtained from BD Biosciences (anti-human IFN-γ-APC, TNF-α-Alexa488, IL-2-PE, granzyme-APC antibody) and Diaclone (anti-human perforin-FITC antibody) get. Intracellular cytokine staining was performed by fixing and permeabilizing cells with cytofix / cytoperm (BD Biosciences). Flow cytometry was performed using a FACS Canto flow cytometer or LSRII (BD Biosciences), and data was analyzed using the FACS Diva program (BD Biosciences).

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Abstract

The present invention relates to T cells, in particular a non-activated T cell, comprising an exogenous nucleic acid encoding a T cell Receptor (TCR) specific for a virus. An embodiment of the invention is directed to a non-activated (resting) T cell expressing Hepatitis B virus (HBV) envelope s183-191 TCR capable of inhibiting viral replication and which shows reduced expression of perforins and / or granzymes in response to stimulation as compared to an activated T cell expressing the said TCR. Also encompassed are methods for producing such cells, compositions, pharmaceutical compositions andkits comprising such cells and medical uses thereof.

Description

technical field [0001] The present invention relates to T cells, in particular antiviral T cells, methods for producing these cells, compositions comprising these cells and their medical use. Background technique [0002] Current antiviral therapies can control HBV replication but cannot clear HBV cccDNA from infected hepatocytes. Standard therapeutic strategies are expensive and require lifelong therapy as in the case of nucleoside / nucleotide analogue therapy, or are associated with severe adverse side effects as with IFN-α therapy. HBV-specific T cells are often absent or functionally depleted in chronically infected patients several years after exposure to HBV infection. Clinical therapeutic strategies to remobilize such virus-specific T cell responses could treat chronic infection or prevent death associated with severe acute infection. [0003] T lymphocytes engineered by implanting T cell receptor (TCR) or chimeric antigen receptor (CAR) can specifically recognize tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17
CPCC12N5/0636C12N15/86C07K14/005C07K14/7051A61K48/005A61K48/0008A61K38/1774A61K35/17A61P31/14A61P31/20A61P31/22C12N2510/00C12N2740/10043C12N2740/15043C12N2730/10122C12N5/0638A61K39/464838A61K39/4632A61K39/4611Y02A50/30A61K39/292C07K14/02
Inventor 安东尼奥·贝托莱蒂萨琳·科
Owner LION TCR PTE LTD