Unlocking a Toxic Payload Inside Cancer Cells Without Antibodies: Immunoproteasome-Activated Prodrug Shrinks Lung Tumors in Mice

Antibody-drug conjugates deliver potent cytotoxic molecules to tumors, but this requires cancer cells to carry recognizable antigens on their surface. Researchers are now attempting to move the source of selectivity inside the cell: instead of seeking surface markers, they are using the greater immunoproteasome activity in cancer cells to unlock an otherwise sealed cytotoxic drug on site.

The study, published in *Signal Transduction and Targeted Therapy*, linked monomethyl auristatin E (MMAE) to the ATMW tetrapeptide and a self-immolative linker. MMAE is a microtubule inhibitor used in several antibody-drug conjugates and is highly toxic in its free form. Only after ATMW is recognized and cleaved by the immunoproteasome does the linker break down, releasing active MMAE. Purified enzyme assays showed that the standard proteasome's capacity to release MMAE was approximately fourfold lower.

Cell experiments provided further support for this mechanism. The prodrug retained cytotoxic activity at low-nanomolar or even lower concentrations in lymphoma and small cell lung cancer cells with higher immunoproteasome activity, while its toxicity was markedly reduced in cells with lower immunoproteasome activity. When the researchers blocked the immunoproteasome with inhibitors or reduced the β5i subunit in NCI-H446 small cell lung cancer cells, the prodrug's toxicity declined accordingly. After interferon-γ was used to increase β5i activity in another type of lung cancer cell, the prodrug's potency increased nearly tenfold.

The animal studies were small but provided preliminary in vivo evidence. In the first experiment, which included only three mice per group, the free-MMAE group rapidly developed severe weight loss, and all of the animals died. Mice receiving the prodrug at an equivalent MMAE dose completed nine days of dosing without significant weight loss. In another NCI-H446 human small cell lung cancer xenograft experiment with five mice per group, both the 0.83 and 1.66 mg/kg doses shrank tumors, with the high-dose group approaching complete regression. After the tumors were removed, free MMAE was detected only in the groups that received the prodrug.

The immunoproteasome is not exclusive to cancer cells. It is also present in immune cells and increases particularly in response to inflammatory signals, so unintended damage to the immune system is an issue this strategy must address. In tonsil organoids from 11 donors, the research team observed that the prodrug preserved populations such as plasmablasts and regulatory T cells better than free MMAE. However, such short-term ex vivo experiments still cannot replace comprehensive assessments of immunotoxicity and cytokines.

The evidence currently remains limited to cells, organoids, and mice, with small sample sizes, and the principal tumor experiment did not directly compare the prodrug with antibody-drug conjugates or standard treatment. It remains unclear whether the prodrug can circulate stably in the human body and enter tumors effectively, or whether immunoproteasome activity can serve as a reliable patient-selection marker. The maximum tolerated dose, long-term toxicity, and full pharmacokinetic profile also remain to be established. This work demonstrates a new principle for intracellular activation, not a therapy that is already capable of replacing antibody-targeted treatment.