Linker Remnants, Lysosomal Trapping and Why Clean Payload Release Matters

Oftentimes antibody drug conjugates have an enzymatically cleavable linker region that, upon cleavage, will release the drug within the targeted cell.

Post-cleavage, the payload needs to be released ‘cleanly’ as a free drug, without any linker remnants attached.  If the free drug is not released, then it has a higher likelihood of being trapped inside the lysosome and being degraded.  Escaping the lysosome is based on the free drugs hydrophobicity, lipophilicity and size of the payload, as well as reactive groups that become available post-cleavage.  Linker remnants post-enzymatic cleavage may inactivate the drugs properties, depending on the mechanism of action, therefore creating a vastly different potency than the free drug.

Lysosomal escape is necessary for the payload to have an effect at it’s desired location. About 1% of ADC’s end up having an effect at the desired location, so it is important to consider if there might be a linker remnant that may lead to lysosomal trapping or that may be deflating this number even further. 

Permeability is also important when considering payload selection.  The example of MMAE and MMAF comes to mind, where MMAE can freely cross into a cell membrane and is very cytotoxic at low concentrations.  MMAF was designed to have the additional phenylalanine group ('F' of MMAF), rendering it less cell permeable, but once inside the cell it is highly potent and cytotoxic.  In this way, the cytotoxicity is gated by the antibody binding kinetics and entry into the cell, and without it, it’s potency is highly reduced.

There is also a known effect in ADC biology called “bystander killing” which is useful for heterogeneous cancers where the target antigen may not be expressed homogeneously throughout the cancer. In the case of bystander killing, permability matters for getting out of the initially entered cell. The same cellular modifications that allow for lysosomal escape are the same modifications that allow for the permeability out of the initial cell and into the neighboring cell. The overlap between lysosomal escape (necessary for drug effect) and bystander killing is high.