Premature release of cytotoxin into systemic circulation is the main safety concern for ADCs. A safety margin of free-circulating
cytotoxin is established during drug development that accounts for freely circulated cytotoxin, but does not account for the
possibility that the circulating free cytotoxin levels may change from lot to lot due to inherent variation during ADC production.
Understanding the toxic potential of a particular ADC lot requires identifying all in vivo sources that could generate free cytotoxin, including intact ADCs, linker-bound cytotoxin, and partial linker-bound cytotoxin
fragments or degraded peptide fragments containing intact linker and cytotoxin (see Figures 4 and 5). A comparison of the
rate of cytotoxin released from each source with its respective PK elimination rate would enable assessment of the safety
impact of each particular source of free cytotoxin. If the rate of elimination from a particular free cytotoxin source were
slower than the release rate from that source, method development should include a measure of area under the curve (AUC) of
that particular free-cytotoxin source. Safety studies also should compare the AUC of free cytotoxin and fast-release sources,
as well as the safety margin for the free cytotoxin from toxicology studies. In addition to free cytotoxin analysis, the linker-bound
cytotoxin also should be evaluated for toxicity to ensure that it is inactive. Obviously, any major, active metabolites of
the free cytotoxin should be monitored in accordance with the Metabolites in Safety Testing guidelines (20).
The major determinants of efficacy are sufficient amounts of intact ADC to achieve the desired effect, as well as sufficiently
low levels of free antibody so that there is no competition with the intact ADC to compromise its efficacy. If the ADC and
free antibody have comparable affinity for the target, the intact ADC will compete equally with free antibody for the receptor.
However, if the ADC has a lower affinity, then even small amounts of free antibody will compromise efficacy. If ADC affinity
is higher than the free antibody, free-antibody levels will generally not significantly influence efficacy.
ADC assessment using an average cytotoxin load per antibody does not provide information on free-antibody levels that can
compete with the ADC. Similarly, after administration of an ADC there is significant cleavage of the cytotoxin that not only
poses a safety concern, but potentially generates a free-circulating antibody that may compete with the ADC for binding, thus
compromising its efficacy. Efficacy can be further complicated by the generation of ADC fragments in vivo (see Figures 4 and 5), whose binding and efficacy are unknown. Cleavage at the hinge region of the ADC could generate fragments
that bind to the receptor and evade ELISA detection, yet compromise ADC efficacy. The glyco microheterogeneity in normal lot-to-lot
variation can cause different degradation fragments between lots.
ADC technologies are rapidly expanding the use of these therapeutic modalities that combine the targeting potential of mAbs
with the pharmacology of small molecules. However, these ADC combinations also present significant challenges in meeting the
regulatory requirements for analytical method development. For example, establishing ADC equivalence requires characterization
of the metabolism and catabolism and excretion of all parts of the ADC. Because antibody fragments and the in vivo generation of free antibody can compromise efficacy, the rate at which these metabolites and catabolites are formed in vivo, as well as their potential effects on safety and efficacy, must be assessed. Any metabolite or catabolite that induces cytotoxicity
or the ability to bind and compete with the intact ADC for the receptor must be measured. These challenges require a thoughtful
and thorough development plan that combines expertise from both small-molecule and biologics development.
Alan Breau*, PhD, is vice-president of bioanalytical and analytical services at MPI Research, 54943 North Main St. Mattawan, MI 49071, tel.
269.668.3336, ext. 3230; firstname.lastname@example.org
. Monica Lee Whitmire, MS, BS, MT (ASCP), is study director at MPI Research.
*To whom all correspondence should be addressed.