Framework for encoding double-specificity molecules of mediated secretion effector cells and killing target cells as well as virus

A bispecific, mediated technology, applied in the direction of viruses/phages, medical preparations containing active ingredients, microorganisms, etc., can solve the problems of high preparation cost, short half-life, complex fusion protein expression and purification, etc. High, low toxicity, and the effect of reducing the number of treatments

Active Publication Date: 2015-04-29
SHENZHEN BEIKE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most bispecific recombinant proteins are expressed in mammalian cells, the expression and purification of fusion proteins are relatively complicated, and the preparation cost is high
The limitation of using bispecific recombinant protein to treat tumors is that its half-life is relatively short, especially for some small molecular proteins, the half-life is only a few hours

Method used

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  • Framework for encoding double-specificity molecules of mediated secretion effector cells and killing target cells as well as virus
  • Framework for encoding double-specificity molecules of mediated secretion effector cells and killing target cells as well as virus
  • Framework for encoding double-specificity molecules of mediated secretion effector cells and killing target cells as well as virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, construction and identification of recombinant adenovirus

[0036] 1. Preparation of recombinant adenovirus

[0037] 1. Insert the double-stranded DNA molecule shown in Sequence 1 of the sequence listing between the EcoRI and SalI restriction sites of plasmid pDC316 to obtain a recombinant plasmid.

[0038] The double-stranded DNA molecule shown in sequence 1 of the sequence listing encodes the protein shown in sequence 2 of the sequence listing (in sequence 1, nucleotides 1-51 encode a signal peptide, which will be excised eventually). The protein shown in Sequence 2 of the sequence listing is named F3 protein, also known as Fcab-anti-CD3 protein. In sequence 2, the 1-243rd is an anti-CD3 fragment (the anti-CD3 fragment targets T cells), the 246-483rd is an Fcab fragment (the Fcab fragment targets the HER2 antigen on the surface of tumor cells), and the 484-493 bit for the myc tag.

[0039] 2. Spread 293A cells in a six-well plate (about 6×10 per well 5 ...

Embodiment 2

[0079] Embodiment 2, the construction of recombinant adenovirus

[0080] 1. Insert the double-stranded DNA molecule shown in sequence 3 in the sequence listing between the EcoRI and SalI restriction sites of plasmid pDC316 to obtain a recombinant plasmid.

[0081] The double-stranded DNA molecule shown in sequence 3 of the sequence listing encodes the protein shown in sequence 4 of the sequence listing (in sequence 3, nucleotides 1-51 encode a signal peptide, which will be excised eventually). The protein shown in sequence 4 in the sequence listing is named H3 protein, also known as HER2-anti-CD3 protein. In Sequence 4, the 1-242nd is the HER2 fragment (HER2 fragment targets the HER2 antigen on the surface of tumor cells), the 260-502 is the anti-CD3 fragment (the anti-CD3 fragment targets T cells), and the 505-514 The first bit is the myc tag, and the 516th-520th bit is the His tag.

[0082] Other steps are the same as in Example 1.

Embodiment 3

[0083] Embodiment 3, the construction of recombinant adenovirus

[0084] 1. Insert the double-stranded DNA molecule shown in Sequence 5 of the sequence listing between the EcoRI and SalI restriction sites of plasmid pDC316 to obtain a recombinant plasmid.

[0085] The double-stranded DNA molecule shown in sequence 5 of the sequence listing encodes the protein shown in sequence 6 of the sequence listing (in sequence 5, nucleotides 1-51 encode a signal peptide, which will be excised eventually). The protein shown in sequence 6 in the sequence listing is named M3 protein, also known as Mart1-anti-CD3 protein. In Sequence 6, positions 1-117 are Mart1 fragments (Mart1 fragments target the HLA-A2-MART1 polypeptide complex on the surface of tumor cells), and positions 137-379 are anti-CD3 fragments (anti-CD3 fragments target T cells) , the 382-391th position is the myc tag, and the 393-397th position is the His tag.

[0086] Other steps are the same as in Example 1.

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Abstract

The invention discloses a framework for encoding double-specificity molecules of mediated secretion effector cells and killing target cells as well as a virus. Recombinant protein provided by the invention comprises a functional area I and a functional area II, wherein the functional area I is specifically combined with tumor-associated antigens or HLA (human leukocyte antigen) polypeptide compounds on tumor cells; and the functional area II is specifically combined with surface markers of T cells or surface markers of NK cells. According to the invention, after the provided recombinant adenovirus infects the tumor cells, the tumor cells become places for double-specificity recombinant protein expression; compared with direct supply of fusion protein, treatment times can be reduced, the adenovirus is guided into a tumor in an orthotopic injection mode, and the expressed fusion protein can be combined with the tumor cells immediately without in-vivo metabolism; and compared with direct injection of the fusion protein, the efficiency is higher, and the tumor cells can be killed once the effector cells appear around the tumor. The framework and the virus have great significance for oncotherapy.

Description

technical field [0001] The invention relates to a frame and a virus encoding bispecific molecules secreted and mediated by effector cells to kill target cells. Background technique [0002] Tumor (neoplasm) refers to the gene change of cells under the action of tumorigenic factors, which loses the normal regulation of its growth and leads to abnormal proliferation. It can be divided into two categories: benign tumors and malignant tumors. Benign tumors grow slowly, have clear boundaries with surrounding tissues, do not metastasize, and do little harm to human health. Malignant tumors grow rapidly and can be transferred to other parts of the body, making the body dysfunctional and life-threatening. [0003] Malignant tumor, also called cancer, is the most serious disease that endangers human health. In the United States, the mortality rate of malignant tumors is second only to cardiovascular diseases. According to the 2000 Statistical Bulletin on the Development of Health ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K45/00A61P35/00
Inventor 高斌王磊何嫣然陶华
Owner SHENZHEN BEIKE BIOTECH
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