Specificity assay for novel target antigen binding moieties
A technology for antigen-binding molecules and target antigens, applied in specific peptides, receptors/cell surface antigens/cell surface determinants, polypeptides containing positioning/targeting motifs, etc., can solve problems such as not providing pictures
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Embodiment 1
[0442] Anti-CD20 antibody GA101 was digoxigenylated and incorporation of digoxigenin (DIG) molecules was verified by Western blot analysis. For the conjugation reaction of antibody and digoxigenin, Zeba TM Antibody dissolved in 20 mM His, 140 mM NaCl, pH 6 was desalted and buffer exchanged to 0.1 M sodium bicarbonate (pH 8) buffer with a spin desalting column (ThermoFisher, cat# 89889). Mix equimolar or higher (1:3 ratio) amounts of antibody and digoxigenin-3-O-methylcarbonyl-e-aminocaproic acid-N-hydroxysuccinimide ester (Sigma Aldrich, catalog number 11333054001) Incubate on a shaker at 300 rpm for 1 hour at room temperature. The antibody-digoxigenin conjugate was again desalted and buffer exchanged to 20 mM His, 140 mM NaCl, pH6. Unconjugated Dig-NHS (7 kDa cut-off) was removed in the same step.
[0443] Digoxigenylation was detected by anti-digoxigenin-AP Fab fragment (Sigma Aldrich, catalog number 11093274910) in Western blots. 1 μg of the corresponding (un)conjugated...
Embodiment 2
[0445] The expression of anti-digoxigenin-ds-scFv-CD28ATD-CD28CSDCD3zSSD in Jurkat NFAT reporter CAR-T cells and the binding of digoxigenin-Cy5 to CAR were confirmed by FACS. The Jurkat NFAT reporter CAR-T cells transduced with anti-digoxigenin-ds-scFv-CD28ATD-CD28CSDCD3zSSD were pelleted at 300g for 3 minutes at room temperature and in an appropriate volume of fresh RPMI-1640+10%FCS+1% Resuspend in Glutamax (growth medium). Then place the 3x10 5 Cells were added to each well of a 96-well plate, spun down once at 300 g for 5 minutes and resuspended in 100 μl in PBS containing 2% FCS. Dig-Cy5 was added to a final concentration of 20 nM and incubated on ice for 45 minutes. Cells were then pelleted and resuspended in ice-cold PBS. The wash step was repeated two more times. Cells were then analyzed for Cy5 signal (APC channel) via flow cytometry ( Figure 7 ). As a negative control, untransduced Jurkat NFAT cells were similarly treated and analyzed.
Embodiment 3
[0447]Described here is a reporter CAR-T cell assay using CD20-expressing SUDHDL4 tumor cells as target cells and a sorted collection of anti-digoxigenin-ds-scFv-CD28ATD-CD28CSDCD3zSSD expressing Jurkat NFAT reporter CAR-T cells as a reporter cell ( Figure 9 ). Digoxigenylated GA101 IgG (antibody:Dig-NHS ratio 1:10) was used as IgG, which on the one hand recognizes tumor antigens and on the other hand is recognized by transduced Jurkat NFAT reporter CAR-T cells. As a positive control, a 96-well plate (Cellstar Greiner-bio-one, catalog number 655185) was treated with 10 μg / ml CD3 antibody (from ) either overnight at 4°C or at least 1 hour at 37°C. Wash the CD3-coated wells twice with PBS, removing the PBS completely after the final wash step. Reporter cells or Jurkat NFAT wild-type cells were counted and checked for viability using Cedex HiRes. Adjust cell number to 1x10 6 viable cells / ml. Therefore, an appropriate aliquot of the cell suspension was pelleted at 210 g fo...
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