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Bispecific T-cell adapter, recombinant oncolytic virus thereof and application of bispecific T-cell adapter and recombinant oncolytic virus

A bispecific, linker technology

Pending Publication Date: 2022-03-29
SHANGHAI SINOBAY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing designs that use vaccinia virus as an oncolytic virus are relatively safe, and the clinical effectiveness needs to be further observed, but further optimization is needed in terms of immune regulation and precise tumor targeting
[0013] Based on the above, there is no bispecific T cell engager that is particularly suitable for recombinant oncolytic viruses in the prior art. Currently, safer and more targeted bispecific T cell engagers and oncolytic viruses containing them There is a need

Method used

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  • Bispecific T-cell adapter, recombinant oncolytic virus thereof and application of bispecific T-cell adapter and recombinant oncolytic virus
  • Bispecific T-cell adapter, recombinant oncolytic virus thereof and application of bispecific T-cell adapter and recombinant oncolytic virus
  • Bispecific T-cell adapter, recombinant oncolytic virus thereof and application of bispecific T-cell adapter and recombinant oncolytic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1: Construction and expression verification of the 293T recombinant expression plasmid of αCD47-αCD3-BITE

[0119] 1.1 Construction of hIgG1 WT vector with αCD47-αCD3-BITE target gene

[0120] First, screen five high-affinity anti-human CD47 antibodies, select their heavy chain variable regions and light chain variable regions, and construct fusion proteins according to the following formula: VH αCD47 -L-VH αCD3 -L-VL αCD3 -L-VL αCD47 ;

[0121] Among them, the VH αCD47 As shown in SEQ ID NO:19, the VL αCD47 As shown in any one of SEQ ID NO: 20-24, the VH αCD3 As shown in SEQ ID NO:25, VL αCD3 As shown in SEQ ID NO: 26, and the L is GGGGSGGGGSGGGGS.

[0122] And, according to VL αCD47 Different, Hu004-67 indicated to comprise the VL shown in SEQ ID NO:20 αCD47 αCD47-αCD3 BiTE; Hu004-68 indicates that it contains the VL shown in SEQ ID NO:21 αCD47 αCD47-αCD3 BiTE; Hu004-73 indicates that it contains the VL shown in SEQ ID NO:22 αCD47 αCD47-αCD3 BiTE...

Embodiment 2

[0131] Example 2: αCD47-αCD3-BITE mediates the in vitro anti-human ovarian adenocarcinoma effect of T cells

[0132] 1. SK-OV3 cell plating: SK-OV3 (stored in Shanghai Xinwan Biotechnology Co., Ltd.) is a Luciferase-expressing cell line. cells at 1 x 10 4 Cells / well were spread in 96-well plates and incubated overnight at 37°C until they adhered to the wall.

[0133]2. Remove the culture medium after 24 hours, set up 7 groups, add T cells respectively, B6H12-αCD3-BITE (the heavy chain variable region and light chain variable region of B6H12 are respectively shown in SEQ ID NO: 29 and 30, and the rest of the structure is the same as The BITE of Example 1 of the present invention is the same) and T cells, Hu004-67-αCD3-BITE and T cells, Hu004-68-αCD3-BITE and T cells, Hu004-73-αCD3-BITE and T cells, Hu004-100 - αCD3-BITE and T cells, Hu004-101-αCD3-BITE and T cells.

[0134] 3. Incubate overnight at 37°C for killing. After 24 hours, remove the supernatant, add 50 μL cell l...

Embodiment 3

[0136] Example 3: αCD47-αCD3-BITE mediates the in vitro anti-human lung cancer cell effect of T cells

[0137] 1. NCI-H292 cell plating: NCI-H292 (stored in Shanghai Xinwan Biotechnology Co., Ltd.) is a Luciferase-expressing cell line. cells at 1 x 10 4 The density of cells / well was spread in 96-well plates, and incubated overnight at 37°C until they adhered to the wall.

[0138] 2. Remove the medium after 24 hours, set up 7 groups, respectively add T cells, B6H12-αCD3-BITE and T cells, Hu004-67-αCD3-BITE and T cells, Hu004-68-αCD3-BITE and T cells, Hu004-73 - αCD3-BITE and T cells, Hu004-100-αCD3-BITE and T cells, Hu004-101-αCD3-BITE and T cells.

[0139] 3. Incubate overnight at 37°C for killing. After 24 hours, remove the supernatant, add 50 μL of cell lysate (Promega Company, product number E1531) to each well, incubate with shaking at room temperature for 30 minutes, and add 30 μL luciferase substrate (Promega Company, product number E151A) to each well. On-machine ...

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Abstract

The invention provides a bispecific T cell adapter, a recombinant oncolytic virus thereof and application of the bispecific T cell adapter and the recombinant oncolytic virus. The invention provides an alphaCD47 (alpha CD47) and alphaCD3 (alpha CD3) bispecific T cell adapter. The invention also provides an isolated nucleic acid molecule which encodes the bispecific T cell adapter. The invention also provides an expression framework of the bispecific T cell adapter BiTE. The invention also provides a recombinant oncolytic virus, and the oncolytic virus is operably inserted into or comprises the expression framework of the bispecific T cell adapter BiTE. According to the present invention, the bispecific T cell adapter is combined with the oncolytic virus, and the oncolytic virus significantly enhances the ability to inhibit malignant tumors compared to a pure gene therapy or viral therapy.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a novel bispecific T cell engager (BiTE) comprising human CD47 and human CD3 specific binding fragments. The present invention also relates to a recombinant oncolytic virus comprising the bispecific T cell engager (BiTE), and the present invention also provides the preparation method of the bispecific T cell engager and the oncolytic virus and their use in Antitumor applications. Background technique [0002] Cancer is the second leading cause of death worldwide with an estimated 9.6 million deaths in 2018. Globally, cancer is responsible for about one in six deaths. Related cancer treatment methods, especially tumor immunotherapy with the help of the body's own immune system, have increasingly become the focus of attention. [0003] Human CD47 is an integrin-related protein, which is a cell membrane surface glycoprotein and belongs to the immunoglobulin superfamily. CD47 is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N7/01C12N15/863A61K35/761A61K35/763A61K35/765A61K35/766A61K35/768A61P35/00C12R1/93
CPCC07K16/2809C07K16/2803C12N7/00C12N15/86A61K35/768A61K35/763A61K35/765A61K35/761A61K35/766A61P35/00C07K2317/56C07K2317/565C07K2319/02C12N2710/24021C12N2710/24043C07K2317/31Y02A50/30
Inventor 徐建青张晓燕丁相卿廖启彬王诗语周俊花
Owner SHANGHAI SINOBAY BIOTECH CO LTD
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