Table 3.
Summary of the validation assays for compounds targeting CTLA-4.
| Variable | D11 | A9 | B10 | D7 | A6 | Target Molecules |
|---|---|---|---|---|---|---|
| AlphaLISA (μM) | 4.1 | 6 | 5.2 | 8.9 | 3.1 | Human CTLA-4/CD80 (B7-1) |
| DSF | Y | Y | Y | Y | Y | Abatacept alone |
| SPR (μM) | 22.5 | 20.8 | 106.5 | 240 | No binding | Abatacept alone |
| INF-γ stimulation ex vivo (MC38) | Y | Y | Y | Y | Y | Mouse CTLA-4/CD80 |
| Tconv: Treg decrease (human cells) | Y | Y | NA | NA | NA | Human CTLA-4/CD80 |
| MC38 models | Y | Y | NA | NA | NA | Mouse or human CTLA-4/mouse CD80 |
| RO5 violations | 0 | 0 | 0 | 0 | 0 | |
| RO3 violations | 2 | 4 | 5 | 3 | 5 |
The 5 top compounds were tested with in vitro and in vivo assays. The Lipinski rule of 5 was also summarized.
AlphaLISA Amplified Luminescent Proximity Homogeneous LISA, CTLA-4 cytotoxic T-lymphocyte–associated protein 4, DSF differential scanning fluorimetry, IFN-γ interferon γ, NA not applicable, RO3 rule of 3, RO5 Lipinski Rule of 5, SPR surface plasmon resonance, Y yes.