Linker-Unstable Payload Connection
Connector-payload instability can cause the payload to be released prematurely into the bloodstream and lead to ADC miss toxicity. The choice of connectors is one of the main drivers behind the stability of ADC. The first generation of ADCs has acid cleavage bonds (such as hydrazine), which are stable in plasma with a neutral pH, and ADCs are released in lysosomes with a lower pH after internalization. Poor plasma stability is a common issue with these early ADCs, and free payloads are observable in systemic circulation.
The introduction of unbreakable connectors alleviates the cleavage problem of connectors in some cases and improves preclinical safety. The decrease in toxicity of uncleavable connectors is thought to be due to the reduced release of free cytotoxic payloads.
However, not all targets are suitable for non-cleavage ADC, as complete catabolism of McAbs is required to release the connector–payload. ADC with cleavable connectors may also improve efficacy through the bystander effect, which is the first choice for low copy numbers, heterogeneous tumor expression, or a low internalization rate of antigen. It should also be noted that in addition to the cleavability of the junction, the membrane permeability of the released payload may also affect the potential off-target cytotoxicity in normal cells, which can be used to affect TI.
The coupling sites may affect the stability and pharmacokinetic characteristics of ADC. Traditional nonspecific coupling methods use exposed amino acids, such as lysine or cysteine, to produce highly heterogeneous ADC (drug antibody ratio, 0 to 8), increased aggregation, and decreased plasma stability. Therefore, non-specifically coupled ADC may also help to increase target independent uptake and toxicity in normal cells.
Neutropenia is an important target-independent DLT in ADC that is related to the systemic release of membrane permeability free payload due to the instability of divisible connectors in plasma. Neutropenia is a common toxicity of many adenokinases. Valine-citrulline connectors can be cleaved by proteases, such as Brentuximab vedotin (ADCetris, Seattle Genetics), ASG-5ME (Agensys), Glembatumumab vedotin (Celldex Therapeutics), Indusatumab vedotin (Millennium Pharmaceuticals), Polatuzumab vedotin (Genentech), and PSMA ADC (Progenics Pharmaceuticals).
According to the chemical properties of connectors, valine-citrulline connectors are cleaved in lysosomes by intracellular cysteine proteases. Serine proteases secreted by local neutrophil differentiation in the bone marrow microenvironment contribute to the division of extracellular VC connectors and release membrane permeability MMAE, which leads to the cytotoxicity of neutrophil differentiation in the bone marrow.
Similarly, peripheral neuropathy (PN) is another important target-independent toxicity associated with microtubule inhibitor ADC (regardless of the target antigen). PN is thought to be driven by connector-payload instability and is associated with the premature release of membrane permeability free payloads (microtubule inhibitors) in systemic circulation. Microtubule inhibitors destroy interphase microtubule function, which is essential for the active transport of key essential proteins from neuronal bodies to distal synapses, resulting in peripheral neuropathy. PN is a common adverse event for almost all ADCs binding to cleavable linkers in permeable membranes (DM-1 and DM-4).
It is worth noting that PN observed clinically is not always predicted in preclinical animal models. For example, preclinical toxicological studies of VC-MMAE-based ADC did not detect PN. Other non-MMAE ADCs containing microtubule inhibitors, such as DM1 or DM4, PN, have been observed in preclinical species and have good clinical predictability.