Adcc reporter bioassay

Example 73

ADCC assay of three chimeric antibodies ch5C9, ch15G11, and ch9A 1 was executed by utilizing ADCC Reporter Bioassay (Promega). 2.5×10⁴/well CLDN 18.2 transiently expressed cells were seeded in a 96 well culture plate overnight. The culture medium was removed at second day and replaced with 50 μL 4% FBS RPMI medium with a series diluted chimeric anti-CLDN 18.2 antibody-ch5C9, ch15G 11 and ch9A 1. The engineered Jurkat cells 25 μL was added to each well at a ratio E:T=6:1, the cell mixture was incubated for 6 hours at 37° C., 5% CO₂, 90% humidity. The luminescence signal resulting from NFAF (neuclear factor of activated T-cells) response element driving expression of luciferase was recorded by Bio-tek synergy 4. EC50 was calculated through Prism Graphpad 8.0.1, nonlinear regression agonist vs response variable slope (4 parameters). The results are shown in FIG. 37.

Example 79

ADCC assay was executed by utilizing ADCC Reporter Bioassay (Promega). 1×10⁴/well CLDN18.2 stably expressed cells HEK293_CLDN 18.2 were seeded in a 96 well culture plate overnight. The culture medium was removed at second day and replaced with 50 μL 4% FBS RPMI medium with 1:3 series diluted anti-CLDN 18.2 antibodies h5C9o Fc, h5C9oao, h5C9oaq, h5C9ob, h5C9oap, h5C9oae. The engineered Jurkat cells 25 μL was added to each well at a ratio E:T=10:1, the cell mixture was incubated for 6 hours at 37° C., 5% CO₂, 90% humidity. The luminescence signal resulting from NFAF (neuclear factor of activated T-cells) response element driving expression of luciferase was recorded by Bio-tek synergy 4. EC50 is calculated through Prism Graphpad 8.0.1, nonlinear regression agonist vs response variable slope (4 parameters). The results are shown in FIG. 45.

Example 13

The capability of a few human antibodies of inducing antibody dependent cytotoxicity (ADCC) was tested using an ADCC Reporter Bioassay (Promega, Madison, Wis.). BT549 or SW48 cells were plated at 1×10⁴ cells per well 24 hours in advance of treatment in sterile 96-well black cell culture microplate (GrenierBio, Kremsmünster, Austria). Culture medium was aspirated in advance of treatment and 25u1 of RPMI 1640 supplemented with 4% ultra-low IgG FBS was added to cells. Human monoclonal antibodies were diluted in assay medium and added to cells in 25 ul/well in duplicate to achieve final antibody concentrations of 0, 0.0001, 0.001, 0.01, 0.1, and 1.0 ug/ml. Engineered Jurkat effector cells were thawed at 37° C. for 2 minutes into assay medium and added to the cells at an effector/target ratio of 7.5:1. The mixed cell culture was incubated at 37° C., 5% CO₂ for 6 hours. Cell viability was analyzed using Bio-Glo luciferase assay reagent as directed in technical manual. Relative Light Units (RLU) readout was graphed using Prism® software.

As shown in FIGS. 6A-6C, among tested antibodies, 41A9 was identified as the antibody with highest ADCC activity in both TNBC cell lines, BT549 and MDA231, as well as CRC cells SW48. 38E11 also exhibited ADCC activity in these cell lines.

Example 77

ADCC assay was executed by utilizing ADCC Reporter Bioassay (Promega). 1×10⁴/well CLDN 18.2 stably expressed cells HEK293_CLDN18.2 were seeded in a 96 well culture plate overnight. The culture medium was removed at second day and replaced with 50 μL 4% FBS RPMI medium with 1:3 series diluted anti-CLDN 18.2 antibodies h5C9oab, h5C9oaf, h5C9oag, h5C9oaj, h5C9oak, h5C9oam, h5C9oan and h5C9oh. The engineered Jurkat cells 25 μL was added to each well at a ratio E:T=10:1, the cell mixture was incubated for 6 hours at 37° C., 5% CO₂, 90% humidity. The luminescence signal resulting from NFAF (neuclear factor of activated T-cells) response element driving expression of luciferase was recorded by Bio-tek synergy 4. EC50 was calculated through Prism Graphpad 8.0.1, nonlinear regression agonist vs response variable slope (4 parameters). The results are shown in FIG. 42 and FIG. 43.