Table 3. Activity of a dual antigen targeting bridging protein.
| Bridging protein binding affinity in flow cytometry assays (EC50) | ||||
| cell line | antigens expressed | anti-Her2-CD19-anti-EGFR (#460) | CD19-anti-Her2 (#311) | CD19-anti-EGFR (#416) |
| BT474 | Her2 only | 2.5 nM | 3.8 +/- 1.1 nM | - |
| n = 1 | n = 2 | |||
| K562-EGFR | EGFR only | 0.6 nM | 0 | 0.52 nM |
| n = 1 | n = 2 | n = 1 | ||
| SKOV3 | Her2/EGFR | 0.44 nM | 2.5 +/- 0.6 nM | 0.37 nM |
| n = 1 | n = 3 | n = 1 | ||
| Bridging protein mediated cytotoxicity by CAR-CD19 T cells (IC50) | ||||
| cell line | antigens expressed | anti-Her2-CD19-anti-EGFR (#460) | CD19-anti-Her2 (#311) | CD19-anti-EGFR (#416) |
| BT474 | Her2 only | 5.1 pM | 24.4 +/- 3.7 pM | 0 |
| n = 1 | n = 4 | n = 2 | ||
| K562-EGFR | EGFR only | 19.1 pM | - | 20.9 pM |
| n = 1 | n = 1 | |||
| SKOV3 | Her2/EGFR | 1.4 pM | 11.7 pM | 13 pM |
| n = 1 | n = 1 | n = 1 | ||
Data are presented as mean +/- standard error (if n ≥ 2). Top: Binding of single antigen and dual antigen bridging proteins to single and dual-antigen positive cell lines. Binding was detected with anti-CD19 antibody FMC63 staining and the EC50 values were derived from dose response flow cytometric analyses. Bottom: Bridging protein-mediated CAR-CD19 cytotoxicity against Her2-positive cell lines. CAR-CD19 T cells were used at an E:T ratio of 10:1. IC50 values were derived from a titration of the bridging protein concentration in the assays. A 0 indicates that binding or activity were not detectable above background levels; a ‘-‘ indicates that the experiment was not performed.