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Next-generation ADCs: better oncology treatments through innovation

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Penelope Drake, Ph.D., head of R&D, Bioconjugates, Catalent, highlights the potential of antibody-drug conjugates in paving a way to innovation in oncology treatments.

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Key insights:

Antibody-drug conjugates (ADCs) have the correct combination of tools to target and help eliminate tumour cells. They have three components: an antibody designed to identify tumour cells within a patient; a toxic payload to cause cell death; and a linker that connects the two. Initial success for ADCs came with the approval of Pfizer’s Mylotarg in 2000 for acute myeloid leukaemia. ADCs have since diversified from treating blood cancers into the realm of solid tumours, and have achieved commercial success over the last ten years, with 12 drugs currently being U.S. FDA-approved.

First-generation ADCs faced multiple challenges

The initial hope was that ADCs would be the “magic bullet” for cancer treatment. With their ability to selectively direct highly potent cytotoxic payloads to target cells, ADCs offered the prospect of more precise treatments, and a reduction in the off-target effects seen with chemotherapies. However, the first generation of ADC candidates were prone to early release of their payloads, indicating the need for improved linker chemistries.

Additionally, developers found that controlling the number and location of the payloads on the antibodies is critical to improving the pharmacological profile of ADCs and expanding the therapeutic window. New technologies that offer site-specific conjugation, as well as innovative linkers have since been developed and are enabling the next generation of ADCs to overcome some of these initial challenges.  

Site-specific conjugation of ADCs

There are numerous technologies available to engineer ADCs with site-specific 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 antibody. Through method development and optimisation, this process can be robust, reproducible, and high yielding. The limited number of steps and reagents keeps the cost-of-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: 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 valine-alanine 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 tandem-cleavage 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 TRPH-222, has completed Phase 1 studies for non-Hodgkin lymphoma. Additional SMARTag ADCs are in preclinical development, including Exelixis’ next-generation 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 site-specific, controlled drug-protein 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 site-specific, stable ADCs are filling development pipelines and show promise to deliver the therapies of the future.

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