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Frameworks and viruses encoding bispecific molecules secreted to mediate effector cell killing of target cells

A bispecific, mediated technology, applied to viruses/bacteriophages, medical preparations containing active ingredients, microorganisms, etc., can solve problems such as high preparation costs, short half-life, complex expression and purification of fusion proteins, and achieve titer High, low toxicity, and the effect of reducing the number of treatments

Active Publication Date: 2017-07-14
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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  • Frameworks and viruses encoding bispecific molecules secreted to mediate effector cell killing of target cells
  • Frameworks and viruses encoding bispecific molecules secreted to mediate effector cell killing of target cells
  • Frameworks and viruses encoding bispecific molecules secreted to mediate effector cell killing of target cells

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 table 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 to 51 encode a signal peptide, which will eventually be excised). The protein shown in Sequence 2 of the sequence listing is named F3 protein, also known as Fcab-anti-CD3 protein. In sequence 2, positions 1-243 are anti-CD3 fragments (anti-CD3 fragments target T cells), positions 246-483 are Fcab fragments (Fcab fragments target HER2 antigen on the surface of tumor cells), and positions 484-493 Bits are myc tags.

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

Embodiment 2

[0079] Example 2. Construction of recombinant adenovirus

[0080] 1. Insert the double-stranded DNA molecule shown in sequence 3 of the sequence table 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, the 1-51 nucleotides encode a signal peptide, which will eventually be excised). The protein shown in SEQ ID NO: 4 of the sequence listing is named H3 protein, also known as HER2-anti-CD3 protein. In sequence 4, positions 1-242 are HER2 fragments (HER2 fragments target the HER2 antigen on the surface of tumor cells), positions 260-502 are anti-CD3 fragments (anti-CD3 fragments target T cells), and positions 505-514 Bits are myc tags, bits 516-520 are His tags.

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

Embodiment 3

[0083] Example 3. Construction of recombinant adenovirus

[0084] 1. Insert the double-stranded DNA molecule shown in Sequence 5 of the sequence table 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 to 51 encode a signal peptide, which will eventually be excised). The protein shown in SEQ ID NO: 6 of 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) , positions 382-391 are myc tags, and positions 393-397 are His tags.

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

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Abstract

The invention discloses a frame and a virus encoding bispecific molecules that secrete and mediate effector cells killing target cells. The recombinant protein provided by the present invention includes functional domain A and functional domain B; said functional domain A specifically binds to tumor-associated antigens or HLA polypeptide complexes on tumor cells; said functional domain B specifically binds to T cell surface markers or NK Cell surface markers. After the recombinant adenovirus provided by the present invention infects tumor cells, the tumor cells become factories for the expression of bispecific recombinant proteins, which can reduce the number of treatments compared with direct administration of fusion proteins. Introduce adenovirus into tumors by in situ injection , the expressed fusion protein can immediately bind to tumor cells without undergoing in vivo metabolism. Compared with direct injection of fusion protein, it is more efficient, and once there are effector cells around the tumor, it can be killed immediately. The invention has great value for tumor therapy.

Description

technical field [0001] The present invention relates to a framework and a virus encoding a bispecific molecule secreted to mediate effector cell killing of target cells. Background technique [0002] Tumors (neoplasms) refer to the changes in genes of cells under the action of tumorigenic factors, loss of normal regulation of their growth, resulting in abnormal proliferation, which 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 metastasize to other parts of the body, making the body dysfunctional and life-threatening. [0003] Malignant tumors, also known as cancers, are the most serious diseases that endanger human health. In the United States, the mortality rate of malignant tumors ranks second only to cardiovascular diseases. According to the Statistical Bulletin of my country's...

Claims

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Application Information

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Patent Type & Authority Patents(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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