ADC Cell Killing Assay with MMAE conjugated GPRC5D Antibodies (Ab1 and Ab2)
Specific Cell Killing by Anti-GPRC5D Antibodies Conjugated with MMAE
In this experiment, three cell types were tested: K562 (negative control, left), K562-GPRC5D (positive cells, center), and H929 (GPRC5D-expressing natural cell line, right). The black and red curves represent MMAE-conjugated anti-GPRC5D antibodies (Ab1/2), showing specific killing of GPRC5D-positive cells. The blue curve represents MMAE-conjugated anti-GPRC5D positive control antibodies, and the green curve represents MMAE-conjugated IgG negative control. The data demonstrates the specific cytotoxicity of anti-GPRC5D antibodies against GPRC5D-positive cells following MMAE conjugation.
Cyno and mouse cross-reactivity tests
Cross-Reactivity Analysis of GPRC5D Candidate Antibodies with Cyno and Mouse GPRC5D Positive Cells Using FACS
In this study, we assessed four distinct cell types: 293T-hu GPRC5D, 293-cyno GPRC5D, 293-mouse GPRC5D, and 293T-negative control. The negative control, 293-PTT5 cells, lacked overexpressed GPRC5D proteins, while the other cells contained overexpressed GPRC5D proteins from human, cyno, and mouse species. We employed candidate antibodies, including G01~G09 (humanized antibodies developed by DIMA Bio) and G00 (a humanized antibody molecule developed by Eureka Therapeutics), to interact with these four cell types using FACS. Our data clearly reveals that G00, G05, G07, and G09 antibodies exhibit cross-reactivity with Cynomolgus Monkey GPRC5D, while the G00 antibody displays cross-reactivity with Mouse GPRC5D.
Antibody Internailzaiton Assay
Specific Internalization of GPRC5D ADC BMK-AME100001 Conjugate in GPRC5D-Positive Cells (K562-GPRC5D Stable Expression Cell Line, Right), with No Detection in K562 Cells Lacking GPRC5D Overexpression (Left).
Specific Internalization of GPRC5D ADC BMK-AME100002 Conjugate in GPRC5D-Positive Cells (K562-GPRC5D Stable Expression Cell Line, Right), with No Detection in K562 Cells Lacking GPRC5D Overexpression (Left).
ADC payload detection
Figure1. Elisa plates were pre-coated with IgG-MMAE (0.2μg/per well). Serial diluted anti-MMAE monoclonal antibody (DME101004) solutions were added, washed, and incubated with secondary antibody before Elisa reading. From above data, the EC50 for anti-MMAE monoclonal antibody binding with IgG-MMAE is 1.356ng/ml.
