Antibody+ PROTAC: Degradation agent-antibody conjugate (DAC)

Proteolysis-targeting chimeras (PROTACs), are heterobifunctional molecules that are rapidly revolutionizing the market landscape in biopharmaceuticals. These heterobifunctional compounds can degrade target proteins intracellularly, providing the possibility for better and extended biological activity compared to small molecule inhibitors targeting the same targets.

PROTAC is made up of three parts: an E3 ubiquitin ligase ligand, a target protein ligand, and a specifically engineered "linker" that connects the two, resulting in the triplet "PROTAC" active form. These heterobifunctional degraders, however, are frequently linked with poor DMPK features, such as low oral bioavailability and difficulties with rapid in vivo clearance.

Building on the ongoing clinical and commercial success of ADCs with cytotoxic payloads, a number of studies have explored coupling PROTAC degraders with monoclonal antibodies in order to improve their DMPK properties and enable more efficient delivery of PROTAC degraders in vivo.

The new molecules produced by the antibody and PROTAC degradants are called "degradant-antibody conjugates" (DACs), which have several potential advantages over PROTAC molecules: (1) they allow in vivo delivery of degradants with poor physical and chemical properties or DMPK characteristics; (2) they avoid complex formulations that are often necessary to make PROTAC active during in vivo exposure; and (3) targeting the PROTAC molecule of interest to a specific tumor or tissue.

While some of the strategies employed by ADCs can be used to prepare biologically active DACs, the construction of DACs must sometimes overcome a number of other challenges. Unlike the broad cytotoxicity of ADC payloads, the degradant payloads of DACs typically exhibit more targeted biological activity with specific cancers. Therefore, the antigen selected for DAC must not only meet ADC internalization and transport criteria, but should also be highly expressed on tumors, tissues, or other cells sensitive to the biological pathway targeted by the degradant.

Also since degradant payloads are typically relatively less toxic than ADC payloads, higher loadings may be required to produce functional antibody conjugates, often with DAR values greater than 4. And since DAC PROTACs typically have larger or more lipophilic payloads than ADCs. These differences can amplify aggregation and pharmacokinetic issues when PROTACs are attached to antibodies, so DACs may require the design of new linkers and attachment methods.

Many studies have reported that PROTAC does not contain chemical groups that can be used for the covalent attachment of cleavable linkers, e.g. primary amines and secondary amines. In such cases, careful consideration must be given to whether the necessary chemical groups are purposefully incorporated into the degradant structure or new linkage methods are used to link the degradant groups, e.g. hydroxyl/phenol groups. The use of non-cleavable linkers requires similar considerations, in which case the linker fragment attached to the degradant structure after antibody catabolism in the lysosome should not interfere with its biological properties, and the DAC payload should have good stability in the lysosomal environment as well as the ability to efficiently escape the lysosomal compartment.

One of the earliest examples of DAC published in the peer-reviewed literature appeared in 2020, which described highly potent, VHL-linked degradants (GNE-987) targeting bromodomain protein 4 (BRD4) attached to targeting CLL1 antibodies via new disulfide-containing cleavable linkers. Note that the carbonate fraction with uncertain biostability was used to attach the linker to the degradant via a hydroxyproline fragment present in the VHL binding region with a DAR value of 6.

There are currently many other reported use of novel linkers as well as linker methods and mAbs against other targets used to construct DACs, most of which use a higher payload (DAR value of 6) compared to most known cytotoxic ADCs (DAR = 2 to 4).

Although the field of DAC is still at an early stage of research, many DAC molecular entities have been successfully prepared using various PROTAC payloads, and subsequent experiments thereof have shown good in vitro and in vivo biological activity, demonstrating the potential of DAC to deliver PROTAC payloads to specific tumors or cells of interest.

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