Anti-CD19 fully human antibody and immune effector cells targeting CD19
A fully human antibody and immune cell technology, applied in the field of tumor immunotherapy or diagnosis, can solve problems affecting clinical use
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Embodiment 1
[0110] Example 1. Preparation of a specific single-chain antibody (scFv) that binds to human CD19
[0111] 1.1 Screening of human CD19-specific binding antibodies based on phage display
[0112] Using phage display technology, human CD19-specific antibodies were screened from a fully human natural antibody library. For this purpose, 400ml of 2×YT / ampicillin culture medium was inoculated with glycerol bacteria (purchased from Shanghai Ruijin Biotechnology Co., Ltd.) displaying a natural library of fully human single-chain antibodies on phage, so that the cell density reached OD600=0.1, at 37 Under the condition of ℃ and 200rpm, shake culture until the cell density reaches OD600=0.5. use 10 12 pfu of M13KO7 helper phage (purchased from Invitrogen) was infected and incubated at 30° C. and 50 rpm for 30 minutes. After adding 50mg / l kanamycin, shake and culture at 37°C and 200rpm for 30 minutes, separate the precipitate by centrifugation (15 minutes, 1600×g, 4°C), resuspend in 4...
Embodiment 2
[0129] Example 2, Expression and Purification of Anti-CD19 Single Chain Antibody
[0130] 2.1 Preparation of human CD19 single chain antibody
[0131] Use the primer pair V5-1F1-F (SEQ ID NO:17) and V5-1F1-R (SEQ ID NO:18) to amplify the scFv-1F1 fragment from the clone pCantab-1F1 obtained in Example 1; use the primer pair V5-1H11-F (SEQ ID NO: 19) and V5-1H11-R (SEQ ID NO: 20) amplified scFv-1H11 fragments from the obtained clone pCantab-1H11, and passed NheI / BamHI (purchased from NEB) double Digestion with T4 DNA ligase in the same double digestion with NheI / BamHI vector plasmid pCMV-V5-Fc (this vector is fused to express the Fc fragment of human IgG1 antibody downstream of the multiple cloning site, hereinafter referred to as V5-Fc, purchased from Shanghai Ruijin Biotechnology Co., Ltd.) was ligated and transformed into the host strain TOP10, and the positive clones were picked and identified by PCR and confirmed by sequencing, and the eukaryotic expression plasmids of V5...
Embodiment 3
[0137] Example 3. Antigen-binding activity analysis of single-chain antibody against human CD19
[0138] The binding activity of the screened antibodies to the antigen human CD19 was determined by concentration gradient ELISA experiments. For this purpose, use 0.1M NaHCO 3 (pH 9.6) Antigen human CD19 was diluted with coating solution, 100ng per well, 50μl / well, overnight at 4°C, and coated with 2% (w / v) BSA and 0.01% (v / v) Tween-20 Block with PBST blocking solution for 2 hours at room temperature. Plates were then rinsed three times with PBST and removed. Subsequently, 100 μl of PBST solution containing a series of concentrations (initial concentration 10 nM, 3-fold serial dilution, until diluted to 1:729) of each antibody protein was added to each well plate, and the determination of each sample was performed using parallel triplicate analysis. . After incubation at 37°C for 2 hours, rinse with PBST three times, then add 100 μl / well of horseradish peroxidase-labeled goat ...
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