NEXTGENERATION ADCs,

antibody. Through method development and optimisation, this process can be robust, reproducible, and high yielding. The limited number of steps and reagents keeps the costof-goods low and makes the process readily scalable and highly reproducible.

SMARTag tandem-cleavage linkers improve ADC stability

Common approaches to making a cleavable linker include incorporating substrates for protease cleavage, disulfide reduction, or acid-mediated hydrolysis into the design. Regardless of the cleavage mechanism, most cleavable linkers share a common design element: Site-specific conjugation of ADCs

Better Oncology Treatments Through Innovation

There are numerous technologies available to engineer ADCs with sitespecific conjugation. One such method uses an aldehyde tag, which is a six amino acid sequence that is genetically encoded into the desired location of antibody constant regions. The sequence is a substrate for the naturally occurring human enzyme, formylglycine-generating enzyme (FGE), which converts a cysteine residue in the tag sequence to a formylglycine residue, which contains an aldehyde functionality. The aldehyde chemical reactivity is bioorthogonal to other reactive groups within the antibody, and thus serves as the handle for site-specific bioconjugation.

There are a variety of locations on the antibody that can be modified without affecting its biophysical properties or negatively impacting product titres. This affords flexibility in payload placement, which provides opportunities for conjugate optimisation and design innovation. The enzymatic transformation of cysteine to formylglycine occurs co-translationally as the protein is being expressed in the cell, and to ensure full conversion, cell lines that overexpress the FGE enzyme can be used. The antibody is secreted into the cell culture medium with aldehydes installed and is purified using standard techniques. At this point, the antibody can be conjugated by adding the linker-payload to the modified only one cleavage event is required for payload release. This reduces the stability of the ADC, as only one “lock” protects conjugate integrity, which increases the potential for loss of payload as the drug circulates in vivo, leading to reduced efficacy, greater toxicity and side effects.

To increase stability, a cleavable linker system has been developed to increase stability of ADCs in circulation, thereby improving the therapeutic index.

The principle behind the design was that by adding a second “lock” to the linker— namely, a second enzymatic cleavage event that would be dependent on ADC internalisation— in vivo stability would be improved.

The first “lock” in the tandem-cleavage system is a standard valinealanine dipeptide, which is a substrate for cathepsins and other proteases. For the second “lock”, a glucuronic acid was placed very close to the dipeptide to sterically prevent access by proteases to the dipeptide substrate. In order to release the payload, the tandem-cleavage linker requires two orthogonal enzymatic activities to occur in sequence. First, glucuronidase—which is only active in low pH environments, such as lysosomes—removes the monosaccharide, liberating the dipeptide from protection. Then, a protease can cleave the dipeptide, triggering payload release. The tandem-cleavage component is a modular element, so is versatile and can be added to any cleavable linker system in order to impart stability and hydrophilicity.

Catalent SMARTag technology and stable linkers support growing ADC field

The SMARTag technology was conceived in Carolyn Bertozzi’s laboratory as part of the suite of bioorthogonal chemistry innovations that earned her the 2022 Nobel Prize in Chemistry, and combines site-specific conjugation and tandemcleavage linker technology.

In collaboration with licensed partners, several ADCs using Catalent’s SMARTag technology are currently in the development pipeline. The most advanced of these, Triphase Accelerator’s TRPH222, has completed Phase 1 studies for non-Hodgkin lymphoma. Additional SMARTag ADCs are in preclinical development, including Exelixis’ nextgeneration 5T4-targeting ADC (XB010) for various forms of solid tumours.

Catalent’s technology overcomes the limitations associated with conventional protein chemistries that produce heterogeneous products with variable conjugate potency, toxicity, and stability. It enables sitespecific, controlled drugprotein conjugation and uses only naturally occurring protein modifications requiring minimal cell-line engineering. SMARTag technology is agnostic to payload and is compatible with cytotoxic and non-cytotoxic payloads, including nucleic acids and peptides.

With recent approvals, ADCs are seeing a resurgence in popularity as potential treatments for cancer, as well as other diseases. The limitations associated with conventional protein chemistries that produce heterogeneous products with variable conjugate potency, toxicity, and stability are no longer inhibitory, and sitespecific, stable ADCs are filling development pipelines and show promise to deliver the therapies of the future.

Whilst the need for refrigeration or ultralow temperature storage for vaccines is not new, demand increased considerably during the pandemic, meaning the cold chain industry had to rapidly scale up production. The recent surge in the industry requiring reliable, ultra-low temperature medical cold chain solutions, is down to factors such as the rise in adoption of mRNA technology and the growing prominence of Cell & Gene Therapy (CGT). With this rise, we have seen increased support from the government for research activities and clinical trials focusing on immunisation, in turn increasing demand for vaccine transport carriers further.

Within the pharma industry, we are seeing the increased need for vaccine transportation boxes which are sustainable and can be used worldwide, particularly in ‘off-the-grid’ and remote locations. As with every industry, there is an increasing demand for brands to act sustainably and this is certainly true for the pharmaceutical industry.

At Secop, we draw on our experience and talented pool of people in order to create battery and solar-powered active cooling systems into our development to ensure we can offer the sustainable option so many are looking for. We have developed the innovative ultra-low temperature (ULT) cooling box, which is optimised for the last mile of distribution. The box has a compressor which can be used to precisely control temperature and, unlike passive systems, ensures that no valuable vaccine is rendered unusable and wasted. In addition, there is no dependence on dry ice which in turn produces tons of CO2. Not only offering environmental benefits, this creates advantages especially for distribution in remote areas where the availability of CO2 cannot be guaranteed.

Additionally, we recently partnered with B Medical to develop a new generation of medical transport boxes to safely store and transport vaccines, biospecimen and other temperature-sensitive specimens at ultra-low temperatures.

One trend we are also seeing in the pharma industry is an increase in partnerships to combine skill sets and technologies to support successful development of vaccine transportation products. For example, we partnered with Global Health Labs to develop a new Solar Direct Drive controller, which is tailored to the WHO Performance, Quality and Safety specifications. The controller meets demands for solar direct drive as well as range, unstable power grids and weak installations and enables vaccine refrigerator manufacturers to meet the latest voltage stabilisation requirements.

However, similar to other industries, both global and local disruptions have had a knock-on impact for the pharma industry, including disruption to the supply chain and shortages of drivers affecting the movement of goods around the world. Coupled with significant increases in fuel and freight costs, times are particularly challenging for the pharma sector.

The demand for effective and efficient last mile delivery of new generation vaccines is challenging the medical cold chain for reliable and robust solutions, to guarantee storage and distribution in safe conditions - with minimum waste of precious vials - and maximum control of transport conditions, including areas with poor grid connections or severe ambient conditions.