Engineering nucleic acid, T cell, and applications and production method thereof

An engineering and nucleic acid technology, applied in the field of biomedicine, can solve problems such as failure to achieve results, achieve strong anti-tumor effects and reduce T cell exhaustion.

Active Publication Date: 2019-11-08
GUANGDONG TCRCURE BIOPHARMA TECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CAR-T cell therapy has been successful in patients with hematological malignancies, it has not achieved the desired effect in most solid tumors

Method used

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  • Engineering nucleic acid, T cell, and applications and production method thereof
  • Engineering nucleic acid, T cell, and applications and production method thereof
  • Engineering nucleic acid, T cell, and applications and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: preparation method

[0054] 1. Vector construction design: The MP71 reverse transcription vector construct comprising three coding regions constructed using standard molecular biology techniques, wherein the three coding regions are: (A) the human anti-E6 TCRα chain fused with the TCRα chain constant region Variable region (designated aE6_Va-Ca)); (B) β chain variable region of the same human anti-E6 TCR fused to TCR β chain constant region (designated aE6_Vb-Cb); (C) linked to TCR with GS linker The variable regions of the heavy chain (referred to as PD1_VH) and light chain (referred to as PD1_VL) of a novel anti-PD-1 antibody. The resulting retroviral vector is commonly designated E6.αPD1_m11. A schematic diagram of the retroviral vector used in this study is shown in figure 1 shown.

[0055] 2. Cell lines and media. HEK-293T and Ca Ski cells were purchased from ATCC. Peripheral blood mononuclear cells (PBMC) from anonymous donors were purchased fro...

Embodiment 2

[0061] Example 2: Expression of anti-PD-1 scFv in vitro.

[0062] 293T cells were transfected with retroviral vectors encoding E6, E6.αPD1_m11 or E6.αPD1_5C4 TCR, and cell culture supernatants were collected 48 hours after transfection. The expression of anti-PD-1 scFv in 20 μl supernatant was detected by ELISA.

[0063] image 3 Shows the expression level of anti-PD-1 scFv in the culture supernatant of expressing cells. E6 indicates the E6 TCR without anti-PD-1 scFV; E6.aPD1_m11 indicates the E6 TCR of the novel anti-PD-1 scFv; E6.αPD1_5C4 indicates the E6 TCR with the published anti-PD-1 scFv.

Embodiment 3

[0064] Example 3: E6 TCR-T expression in vitro.

[0065] transduction of T cells Figure 4 vector shown. After 72 hours of culture, the expression of recombinant TCR was detected by staining with anti-TCRβ antibody. TCRβ flow cytometry is based on a CD3-positive lymphocyte gate.

[0066] result: Figure 4 demonstrated that E6 TCR was highly expressed in E6.αPD1_m11-transduced T cells.

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Abstract

The invention discloses an engineering nucleic acid, which comprises nucleic acid of an antigen acceptor modified by gene engineering and can specifically binds an antigen from HPV; a nucleic acid ofan immune checkpoint acceptor for preventing tumor expression, wherein the nucleic acid can code one block protein; an engineering T cell, which comprises the above mentioned nucleic acid sequence; anapplication of the engineering T cell in the preparation of drugs for treating cancers; and a method for producing the engineering T cell. According to the method, a carrier is introduced into a T cell to obtain a TCR-T cell, then amplification is carried out to obtain the engineering T cell, and the carrier comprises the nucleic acid. The invention provides an engineering nucleic acid, a T cell,and an application and production method thereof. The obtained T cell can be used to recognize HPV E6 tumor antigen and can secrete a single chain antibody that blocks programmed cell death of protein PD-1. The engineering T cell has a strong antitumor function, the failure of T cell is reduced, and the nucleic acid and the T cell can be applied to immunological therapy of cancers of HPV E6 expression.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an engineered nucleic acid, T cells and their application and production method. Background technique [0002] Some cancers, such as cervical cancer, are primarily caused by human papillomavirus (HPV) infection. Despite the effectiveness of treatments such as chemotherapy, many cancers, including HPV-related cancers, can have a poor prognosis. Thus, there is a medical need for cancer, especially HPV-related cancers. [0003] HPV16 is a subtype of HPV that often causes malignant tumors. HPV16 can work through its E6 protein, which can affect the cell cycle and cause cancer. The E6 protein of HPV16 can be expressed in various cancers, including but not limited to cervical cancer, oropharyngeal cancer, anal cancer, anal canal cancer, anorectal cancer, vaginal cancer, vulvar cancer and penile cancer. [0004] T cells can specifically recognize HPV16 E6 antigen by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/705C12N5/10A61K35/17A61P35/00C12N15/867
CPCC07K14/705C07K14/70521C12N5/0636A61K35/17A61P35/00C12N2740/10043C12N2740/15043C12N2510/00C07K16/2818C07K2317/622C07K2317/565
Inventor 王品李思
Owner GUANGDONG TCRCURE BIOPHARMA TECHNOLOGY CO LTD
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