Objective
Antibody-drug
conjugates (ADCs) are a promising class of biotherapeutics comprised of three parts, a
monoclonal antibody (mAb), a cytotoxic drug and a covalent linker. ADCs use the
high specificity and long circulating half-life of a mAb to selectively deliver
highly potent cytotoxic drugs to tumour cells, widening the therapeutic index
of cytotoxic agents.
Currently,
there are four ADCs on the market for the treatment of cancer and these are
generated by conjugation to either endogenous cysteine or lysine residues.
Cysteine conjugation is achieved by reduction and alkylation of interchain
disulfide bonds, of which there are four in a human IgG1. In comparison, lysine
conjugation uses any of the 80-100 lysine residues typically available in an IgG1.
Therefore, compared to lysine conjugation, cysteine conjugation generates a
less heterogeneous ADC, as a maximum of eight conjugation sites are available.
However, the reduction of disulfide bonds and conjugation to a hydrophobic
small molecule has the potential to negatively impact the stability of the
antibody.
In
this study, cysteine conjugation was investigated on solid and solution phase,
using partial and full reduction methodologies. Conditions were optimised for human
IgG1 conjugation allowing DARs 1 to 8 to be reproducibly generated. ADC stability
was then assessed to determine the impact of both the conjugation methodology
and the small molecule loading. The drug-to-antibody ratio (DAR), aggregation
and binding were investigated using HRMS, SEC-MALS and ELISA respectively. The ADCs
were assessed immediately after conjugation and under stress conditions
including freeze-thaw and heat stress up to 50 °C.
The results of these stability studies revealed a
preferential conjugation phase and reduction methodology, providing key insight
towards generating more stable cysteine ADCs. Furthermore, conditions
appropriate for long-term ADC storage were determined.
