De-risk your ADC development process with these stability considerations

Antibody-drug conjugates, or ADCs, are revolutionary therapeutics that represent a growing proportion of the biologics market. They combine the targeting power of a monoclonal antibody, which is highly specific to a given antigen, with the druggability of a small molecule.

This has the biggest implication on cancer treatments.

Many of the small molecule drugs used to treat cancer operate by shutting down some aspect of cellular transcription or metabolism, thereby killing the cancer cells. However, cytotoxic small molecules operate on a common principle – they must kill the cancer cells before they kill healthy cells. The unfortunate side effects of chemotherapy are commonly a result of the chemotherapeutic small molecules also having a negative impact on healthy cells.

However, ADCs offer an alternative to highly toxic chemotherapies. Monoclonal antibodies offer high specificity, targeting only their intended antigen. Since many cancer types express receptors that are unique to cancer cells, it’s possible to tether the chemotherapeutic small molecule to a cancer-targeting antibody, which essentially serves the cancer-destroying drug directly and only to the cancer cells. This offers truly amazing potential as the next generation of cancer therapeutics.

As with any biologic, there are many considerations around the stability, efficacy, and toxicity of ADCs.

Read on to learn how ADCs are built, what considerations are critical for their engineering, and how that impacts their stability characteristics.

ADCs are highly complex therapeutic molecules

There are three parts of an ADC, all of which present unique considerations when it comes to developability.

• Monoclonal antibody: This may be an already existing antibody with a known, well-characterized antigen interaction; or, you may be working to develop a new mAb for a new target or with different properties than an originator.
• Small molecule drug: As with the mAb, this small molecule may already be in use as a standalone treatment, or it may have been pulled from a synthetic library. It may also be built from a fragment library. There may be a need for additional modifications to the drug, to prevent interference with the antibody, or to provide a space for the linker.
• Linker: The linker must attach the small molecule to the antibody in a way that the active part of the small molecule is able to access the target protein. Length of linker, method of attachment to the antibody, and ability to release the small molecule are critical considerations when building the final ADC.

Many aspects of ADC engineering ultimately impact their stability characteristics

Developability profiling involves evaluating many CQAs, or critical quality attributes, to find candidates with the most optimal attributes. While here the focus will be on the developability characteristics of ADCs, particularly conformational and colloidal stability, it is worth noting there are many more characteristics of ADCs to consider.

Conjugation process

• There are many approaches within these conjugation types depending on where the linker is attached.
• Reactions often have to incubate for anywhere between 30 minutes and several hours, which means prolonged time in this environment. Conjugation buffers may be “harsh” to the protein-based mAb; but, longer incubations increase the attachment of the small molecule.

Drug-induced chemical environment change

• Consider the degree of conjugation. Here, a specific example makes it easier to understand why this is an important consideration:
  • • For drugs linked via amine reactions (the Lysine side chain), the reaction will occur between any solution-exposed lysine and the reactive end of the linker.
    • mAbs contain up to 80 Lysines per molecule, many of which are solution-exposed.
    • This means every individual molecule likely has multiple drugs conjugated to a single mAb.
    • It also means there is a risk of a drug binding a Lysine in the antigen-binding region, reducing or eliminating the targeting power of the mAb.
• Bringing a small molecule close to the antibody changes its chemical environment, which has an impact on its stability. If the drug interrupts the structure too much, the ADC could unfold or aggregate.
• Ensure full clean-up of free drug molecules after conjugation. Free-floating small molecules will have an impact on the conformational stability of the ADC.

Linkers

• One way to reduce some of the impact of the drug on the antibody’s structure is by altering the linker length.
• The linker must also not have any structural impact on the mAb base; further, the chemistry required for linking it to both the mAb and the small molecule must not destroy the structural or conformational integrity of either.
• There are many chemistry considerations with a linker, including ensuring it ultimately releases the drug into the target cell.

Formulation

• Once you’ve locked in the conjugation approach, exact linker, and drug to conjugate to your antibody, you must still optimize its buffer formulation.

A buffer that is stabilizing to a monoclonal antibody alone may not confer the same stability advantages to that mAb once it is part of an ADC.

Overall, the promise of ADCs is using two known, well-understood therapies – mAbs and small molecule drugs – and combining them to make an even better therapeutic. However, this means there is a lot more complexity when it comes to building the therapeutics and optimizing their stability characteristics, so that ultimately they make better therapeutics.

Summary: What to consider when building an ADC?

When getting started with building ADCs, consider:

• Conjugation approach: Determine if you have flexibility in the conjugation approach you’ll use, and experiment with alternatives
• Stability of the mAb alone: Formulate or test its stability in a buffer that is compatible with the desired conjugation reaction to ensure it will survive the conjugation process
• Exposure of the mAb to the conjugation conditions: If optimizing the conjugation buffer is not possible, and you know the buffer is destabilizing, aim to reduce the exposure of the mAb to the conjugation conditions
• Linker Length: Experiment with linker lengths; keeping in mind that the drug acts as a change to the chemical environment to the mAb, which will impact its stability characteristics

To learn more about ADCs and further considerations to de-risk your ADC development process, watch this webinar.