There exist chemical moieties that are essentially invisible to the machinery in the mammalian cell; that inhabit the same physical space as the biological components, but a very different - orthogonal - chemical space.
The name given to these traffic-dodging chemical groups is “bioorthogonal” (BO). Coined by Carolyn R. Bertozzi in 2003, the development of BO chemistry was and is driven by a desire to study biomolecules in situ and on a molecular level.
Important early work from Bertozzi and colleagues details the use of a modified Staudinger reaction to produce stable cell-surface adducts. The reaction makes use of a BO reaction pair, two chemical moieties that will selectively react with one another in a biochemical environment and not interact with anything else to any significant degree. By attaching a tag to one reaction partner and installing the other reaction partner into a biochemical target by metabolism, the target (in this case a glycan) can be selectively tagged1.
Since this early work, BO chemistry has been used extensively to study biological processes by leveraging exquisite selectivity and biological silence2. BO chemistry builds upon foundations of so called “click” chemistry laid by Barry Sharpless and Morten Meldal. Click chemistry is the name given to reactions that are selective, fast and high yielding; a feat that is achieved by choosing reaction partners that feel a strong thermodynamic drive to react.
The contributions of these three scientists have earned them the 2022 Nobel prize in chemistry for, and the press release puts it well, “[taking] chemistry into the era of functionalism”. Now, BO chemistry represents a tantalising prospect for highly targeted therapy, and it is beginning to deliver the goods.
Click-to-Release
There is a clear need for targeted cancer therapies that reduce the damage to healthy cells, allow for greater dose tolerance and ultimately improve the prognoses for patients.
Shasqi’s Click Activated Protodrugs Against Cancer (CAPACTM) platform is an example of BO chemistry being utilised in a treatment at the human trial stage and demonstrates the potential of “click-to-release” treatments.
Shasqi3
The CAPAC platform provides targeted treatment for injectable tumours. First, sodium hyaluronate polymer functionalized with a tetrazine moiety (BO partner 1) is injected at the tumour. The polymer is inert and remains at the injection site for long periods of time: when injected in mice, approximately 50% of the polymer persisted at the injection site after 14 days.
Doxorubicin (Dox), the anti-cancer agent being delivered, is conjugated to a trans-cyclooctene (TCO) moiety (BO partner 2) and then administered systemically, meaning it circulates in the body. In its free form Dox is cardiotoxic, but the prodrug form of Dox is chemically attenuated by the modification to include a TCO moiety: the prodrug is 83-fold less toxic than free Dox in vitro and demonstrates improved tolerability in vivo.
At this stage the BO reaction pair are both in the body, one waiting at the tumour site, the other patrolling harmlessly. The magic happens when the two reaction partners meet at the tumour.
The azide on the polymer captures the circulating prodrug form of Dox through a fast and selective inverse electron-demand Diels-Alder reaction, removing the Dox prodrug from circulation. A subsequent spontaneous reaction then releases the active form of Dox at the tumour site – killing tumour cells. Studies in mice show good anti-tumour efficacy, increasing median survival from 31 days to 47.
The CAPAC mechanism is notably different from other common targeted approaches. The method does not rely on tumour biomarker expression or factors such as oxygen levels or pH. As an example, known methods have attached Dox to proteins that are cleaved by proteases known to be over-expressed at a tumour. The natural variability in biomarkers between patients and pathologies poses a problem that the CAPAC regime side-steps.
Other
The Shasqi phase 1 study has an estimated completion date in July 20264 . There are of course limitations to the treatment, notably that it can only be used on injectable tumours and is not universally applicable to any cancer drug. In the meantime, there are other approaches to pay attention to.
Photochemical BO reactions are trying to use light irradiation to achieve unprecedented control over when and where a reaction occurs. A challenge when developing these methods is maintaining bioorthogonality when creating excited electronic states. Often, photo-induced reactions proceed via high energy excited states that exchange electrons, creating highly reactive radicals that are not known for being very discerning when choosing what they react with5.
Mark Robillard (CEO of TagWorks) has proposed another click-to-release treatment using an anti-cancer drug that is caged in a diabody that targets a protein found in many solid tumours. Caging the drug like this reduces toxicity and leads to the uptake of the drug by tumour sites. A BO reaction with a tetrazine then releases the drug6.
Floris van Delft and the team at Synaffix have developed from using bicyclononymes for bioorthognal labelling of living cells7 to a commercialised conjugation approach, termed GlycoConnectTM, where toxic payloads are conjugated to monocolonal antibodies using similar chemical moieties8. This technology is in the clinic in antibody drug conjugates from companies such as ADC Therapeutics and Mersana.
Patent Considerations
These new synthetic approaches have the potential to be protected using the patent system both as platform technologies and when used to solve particular issues in the manner in which they are deployed. As with all recently developed approaches as their use becomes a routine tool in the toolbox chemists employ, it will likely become harder to obtain patent protection, but that day seems somewhat far off at the moment.
Conclusions
Two decades of development in the BO chemistry field now has the potential to create a raft of entirely new therapeutic platforms upon which new treatments can be built. The chemist in me is excited by the level of control this technology could bring to therapy; in this case tempering our drugs and realising their potential at the same time. The meta-chemist in me is excited by the fact that this technology makes use of an inherent asymmetry between the chemistry mammals have evolved to use, and that they have not.
As with any new technology, there is no shortage of things that could go wrong for a therapy making use of BO chemistry: maybe the BO reaction partners are not tolerated as well as hoped in humans; or maybe the efficacy is less impressive and only present when treating a handful of cancers. Regardless, the BO ball is rolling faster and faster each year.
References
- James Mitchell Crow, “The bioorthogonal revolution”, Chemistry World, 30 May 2022.
- Smeenk et al., “Recent developments in biorthogonal chemistry and the orthogonality within”, Current Opinion in Chemical Biology, Vol 60, 2021, Pages 79-88.
- Wu et al., “Click activated protodrugs against cancer increase the therapeutic potential of chemotherapy through local capture and activation”, Chem. Sci., 2021, 12, 1259.
- Clinical Trial NCT04106492
- Li et al., “Photo-controllable biorthogonal chemistry for spatiotemporal control of bio-targets in loving systems”, Chem. Sci. 2020, 11, 3390-3396.
- Bird et al., “Bioorthogonal Chemistry and Its Applications”, Bioconjugate Chem. 2021, 32, 12, 2457-2479.
- Dommerholt et al., “Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells”, Angew. Chem. Int. Ed. 2010, 49, 9422–9425.
- van Geel et al., “Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N‑Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates”, Bioconjugate Chem. 2015, 26, 11, 2233-2242
