Panoply™ Human TNFRSF8 Over-expressing Stable Cell Line

The recent success of antibody-drug conjugates (ADCs), with seven new drugs approved by the FDA in three years, has attracted greater attention to antibody-based targeted therapies and fueled efforts to develop novel drug-linker technologies to improve next-generation ADCs. Here, researchers present a highly efficient phosphonamidate-based conjugation handle that combines a discrete hydrophilic PEG substituent, an established linker-payload, and a cysteine-selective electrophile in a compact building block. This reactive entity enables homogeneous ADCs with drug-to-antibody ratios (DARs) as high as 8 from non-engineered antibodies via a one-pot reduction and alkylation protocol. The compact branched PEG architecture introduces hydrophilicity without increasing the distance between the antibody and payload, allowing the generation of the first homogeneous DAR 8 ADC from VC-PAB-MMAE without increasing in vivo clearance. Compared with the FDA-approved VC-PAB-MMAE ADC Adcetris, this high-DAR ADC exhibited superior in vivo stability and higher antitumor activity in tumor xenograft models, clearly demonstrating the advantages of phosphonamidate-based building blocks as a versatile tool for efficient and stable antibody-based delivery of highly hydrophobic linker-payload systems.

Here, the researchers set out to evaluate DAR 8 brentuximab-7 by measuring its in vitro potency using three CD30 (also known as TNFRSF8) overexpressing cell lines in a cell-based viability assay and comparing its cell killing activity to that of DAR 4 brentuximab-7 and Adcetris (Figure 1a-c). The cell killing behavior of Adcetris and DAR 4 brentuximab-7 was nearly identical in all antigen-positive cell lines tested. These results suggest that modification of brentuximab with PEG12 phosphamide 7 had little effect on its recognition, binding, and subsequent internalization. In addition, both ADCs had no effect on the HL60 control cell line (Figure 1d). When the IC50 values of the DAR 4 ADC were further compared to the high-load DAR 8 ADC, a clear increase in potency was observed, consistent with the higher payload loading of DAR 8 brentuximab-7. To further evaluate the cleavage of cathepsin B after ADC internalization and the traceless release of MMAE from the VC-PAB linker, the researchers established an indirect bystander assay in which CD30-positive L-540 cells were incubated with Adcetris, brentuximab-7 DAR4, or DAR8. In this case, the ADC was internalized and exposed to intracellular cathepsin B activity. If the VC peptide is recognized and cleaved by cathepsin B, free MMAE can passively diffuse across the cell membrane into the cell culture medium, while the uncleaved cysteine adducts are trapped inside the cells. The supernatant of L-540 cells was then transferred to CD30-negative HL-60 cells and their viability was measured. As shown in Figure 1e, the potency trends of the three ADCs were the same, indicating that the PEG12-substituted phosphonamidate VC-PAB-MMAE does not inhibit cathepsin B-mediated payload release under in vitro potency conditions.

Figure 1. In vitro and in vivo efficacy evaluation of brentuximab-7. (Ochtrop P, et al., 2023)