Kinase experiment:

Cell pellets are collected 24 hours after ADC treatment. Cell count, diameter, and circularity are determined on Vi-Cell Counter. MMAE extraction and quantification method is performed. Briefly, tumors or cell pellets are homogenized with methanol and acetonitrile containing internal standard (d8-MMAE for MMAE detection and 13C-MMAF for MMAF detection). The homogenates are centrifuged at 10,000 rpm to precipitate protein and protein-bound payloads. The supernatant is then subjected to solid phase extraction, and signals of MMAE and MMAF are detected by LC-MS[1].

D8-MMAE is a deuterated labeled MMAE, a potent mitotic inhibitor and a tubulin inhibitor.

Antibody-drug conjugates (ADC) comprise targeting antibodies armed with potent small-molecule payloads. ADCs are generated to target different receptors on the anaplastic large cell lymphoma line L-82, but delivered the same cytotoxic payload (monomethyl auristatin E, MMAE), and the intracellular concentration of released MMAE correlated with in vitro ADC-mediated cytotoxicity independent of target expression or drug:antibody ratios. LC-MS is used to measure the concentration of MMAE in a parallel cohort of L-82 tumors with an identical treatment regimen. Although tumor volume is not different among treatment groups 3 days after dose, the intratumoral MMAE measurement reveals two patterns. First, intratumoral MMAE concentration increases proportionally to the ADC dose, which correspondes to stronger antitumor activity. Second, the intratumoral MMAE concentration obtained from treatment with both cOKT9-vcMMAE and cAC10-vcMMAE is similar at each dose, consistent with the observation that tumor responded similarly to these two ADCs[1].

Intratumoral MMAE concentrations consistently correlates with the extent of tumor growth inhibition in tumor xenograft models. IHC analysis reveals that nonbinding control-treated tumors consist of both CD30+ and CD30-cells, presumably because they do not kill either CD30+ or CD30- Karpas 299 cells. Only CD30- cells are found in cAC10-vcMMAF-treated tumors, illustrating that cAC10-vcMMAF eliminates most CD30+ cells. Interestingly, the two tumors that relapses from cAC10-vcMMAE treatment are also found to be CD30- by the end of study, indicating a small fraction of CD30- cells might have escaped from bystander killing in these two remaining tumors[1].

References:
[1]. Li F, et al. Intracellular Released Payload Influences Potency and Bystander-Killing Effects of Antibody-Drug Conjugates in Preclinical Models. Cancer Res. 2016 May 1;76(9):2710-9.