A new wave of antibody-targeted anticancer therapies is showing great clinical promise, with the potential to transform cancer treatment. These antibody-drug conjugates (ADCs) are multicomponent systems that combine recombinant and chemical technologies to deliver a highly potent cytotoxic drug to a tumor thereby minimizing off-target toxicities and increasing the therapeutic window of the drug.
While there are multiple chemical strategies to conjugate a payload to an antibody, one of the more established methods utilizes the cysteine thiols derived from a reduced interchain disulfide bond. The linkers that are currently used conjugate only to a single cysteine thiol, which results in the loss of covalency between the cysteines that composed the original disulfide.
Reduction of the four interchain disulfides of a standard IgG1 antibody gives rise to up to eight thiols, so when a drug is conjugated a range of positional isomers with drug to antibody ratios (DAR) ranging from 0 to 8 are produced. Each drug positional isomer may have its own distinct biological profile, and it is difficult to control the conjugation reaction to achieve the required DAR.
If the disulfide can be re-bridged following conjugation, there will be fewer possible drug positional isomers than with the single thiol approach, with the DAR distribution limited to between 0 and 4 and a DAR between 2 and 4 achievable at greater than 94%.
In this article, a conjugation technology called ThioBridge™ from PolyTherics is described that allows site-specific targeting of disulfide bonds in a way that retains the covalent integrity of a disulfide bridge and does not require re-engineering of the antibody to obtain a less heterogeneous and stable ADC.
While great progress has been made in the selection and optimization of the antibody and cytotoxic payload, many of the ADCs in development are typically heterogeneous mixtures possessing populations with varying DARs. Producing a more homogeneous ADC with the optimal DAR distribution for safety and efficacy is challenging with current conjugation methodologies, and it is widely recognized that further improvements are needed if next-generation ADCs are to really fulfill their potential.
The development of a successful ADC is predicated on appropriate target selection, as well as optimization of the antibody, the linker, and the cytotoxic agent. While much attention is given to the linker in terms of its ability to release the payload after it reaches the target cell, less attention is given to the fact that the linker also has to serve to conjugate the payload as efficiently and as selectively as possible to the antibody.
Although high DARs, i.e., above 4, are observed to be more potent in vitro, higher loaded species are more hydrophobic due to the nature of the drug payload, with increased clearance rates and in vivo toxicity reported for these species. There is also increasing concern about the in vivo instability of ADCs due to the deconjugation of maleimide-based linkers from the antibody while in circulation. Such instability can cause the cytotoxic drug to be released before it reaches the tumor, potentially leading to off-target toxicity and reduced efficacy.
Once released, the cytotoxic payload can potentially conjugate to serum proteins and thus remain in the circulation rather than reaching the tumor.
Utilizing the latent reactivity of a reduced disulfide for conjugation avoids the need to re-engineer the antibody specifically for site-specific conjugation and is therefore more generally applicable to a wider range of antibodies. While antibodies possess many disulfide bonds, only the heavy to heavy and heavy to light interchain disulfides are both reducible and accessible for conjugation under mild conditions. These interchain disulfides are easily reduced either fully or partially using standard reductants.