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
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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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