Method Profile
Compare EGFR binding, growth effects, Vδ2 T-cell expansion and immune killing for two experimental conjugates.
Assess separate targeting and immune-effector properties of aminobisphosphonate antibody conjugates.
Five CRC cell lines; Caco-2/HCT-116 spheroids; two PDOs in T-cell expansion experiments; one separately identified PDO in cytotoxicity experiments; CRC tissue suspensions.
Cet-DC315 and Cet-DC310 were compared with native cetuximab, typically at 2 μg/mL. Growth was monitored through 120 hours for Cet-DC315 and 72 hours for Cet-DC310; immune killing lasted 48 hours and expansion 14–25 days with IL2 support.
Conjugates
MALDI estimated drug-to-antibody ratios of approximately 23 for Cet-DC315 and 6.8 for Cet-DC310. Binding curves measured apparent dissociation constants.
Immune models
Peripheral T cells from healthy donors and expanded Vδ2 cells. PDO-derived target cells in the killing assay are distinguished from intact three-dimensional spheroids.
Keep direct cell-growth experiments distinct from immune expansion and killing. Compare each conjugate with native cetuximab and untreated controls.
Target binding, two- and three-dimensional growth and immune co-culture comparisons.
LS-180 expansion: 6 healthy donors. PDO expansion: 2 donors and 2 PDOs in four experiments. Cytotoxicity: Vδ2 cells from 3 healthy donors and 3–6 cell replicates. Reported tissue-suspension expansion: 4 patients.
Native cetuximab; Cet-DC310; untreated target cells; immune cells without conjugate.
Cet-DC315 inhibited Caco-2 but not HCT-116 growth, expanded Vδ2 cells and lowered surviving CRC signal. Apparent dissociation constants rose to 24–189 μg/mL versus 0.01–0.02 for native cetuximab. Cet-DC310 failed the principal functional assays.
An experimental cetuximab–aminobisphosphonate conjugate inhibited selected colorectal models and increased Vδ2 T-cell expansion and killing. Its high drug loading reduced EGFR affinity, while a second conjugate failed the principal functional tests.
