Chimeric antigen receptors targeting cd19 and uses thereof
A technology of antigen and single-chain antibody, applied in the direction of antibody medical components, antibody mimics/stents, medical preparations containing active ingredients, etc., can solve the problems of apoptosis, persistence (poor persistence, unsatisfactory curative effect, etc.)
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Embodiment 1
[0100] Example 1: Determination of CD8 Leader Sequence-mCD19scFv-CD8α-CD28-CD3ζ Gene Sequence and Construction of Retroviral Vector
[0101] The gene sequence information of human CD8α hinge region, human CD28 transmembrane region, human CD28 intracellular region and human CD3ζ intracellular region was searched from the NCBI website database. The anti-CD19 single-chain antibody clone number is FMC63. These sequences are available on the website Codon optimization is performed on http: / / sg.idtdna.com / site to ensure that it is more suitable for human cell expression without changing the encoded amino acid sequence.
[0102] Using overlapping PCR, the above sequences were sequentially connected according to anti-CD19 scFv, human CD8α hinge region gene, human CD28 transmembrane region gene, human CD28 intracellular region gene, and human CD3ζ intracellular region gene sequence, and different restriction enzymes were introduced at the junction of each sequence. sites to form a comp...
Embodiment 2
[0109] Example 2: Retroviral Packaging
[0110] The retroviral vector purified and obtained in Example 1 was transfected into 293T cells by the calcium phosphate method to carry out the retroviral packaging experiment, and the specific steps were as follows:
[0111] Day 1: Select 293T cells less than 20 passages and not overly congested, and use 0.6×10 6 Cells / ml were plated, 10ml DMEM medium was added to a 10cm dish, the cells were thoroughly mixed, and cultured overnight at 37 degrees.
[0112] Day 2: 293T cell confluency reaches about 90% for transfection (usually about 14-18h after plating); prepare the plasmid complex, the amount of various plasmids is: the MSCV backbone vector prepared in Example 1 12.5ug, Gag -pol 10ug, VSVg6.25ug, CaCl 2 250ul,H 2 O 1ml, the total volume is 1.25ml; add HBSS (Hank’s Balanced Salt Buffer) equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture to...
Embodiment 3
[0114] Example 3: Retrovirus infection of human T cells
[0115] 1. Use Ficcol separation medium (Tianjin Haoyang) to separate and obtain relatively pure CD3+ T cells, and use X-VIVO (LONZA) medium containing 5% AB serum to adjust the cell density to 1×10 6 / mL. Inoculate the cells at 1ml / well on a cell culture plate coated with anti-human 50ng / ml CD3 antibody (Beijing Tongli Haiyuan) and 50ng / ml CD28 antibody (Beijing Tongli Haiyuan), and then add 100IU / ml Interleukin 2 (Beijing Shuanglu), stimulated and cultured for 48 hours before virus infection.
[0116] 2. The next day after T cell activation culture, PBS was diluted to a final concentration of 15 μg / ml, and Retronectin (Takara) was used to coat a non-tissue-treated culture plate, 250 μl per well of a 24-well plate. Protected from light, overnight at 4°C for later use.
[0117] 3. After T cell activation and culture for two days, two coated 24-well plates were taken out, the coating solution was discarded, and HBSS co...
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