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Exploring the Potential of Antibody–Drug Conjugates

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Antibodydrug conjugates (ADCs) are a relatively new class of biological drugs – created by attaching a therapeutic agent to an antibody via a linker. ADCs are being designed as highly targeted therapies, delivering drugs with very high specificity to disease cells.

The UK registered and self-funding charity LifeArc has several ADC programs in the oncology and non-oncology space. Technology Networks recently had the pleasure of speaking with Dr Laura Murch, Scientist, Biology at LifeArc, to learn more about ADCs. She highlights some of the antibody characterization assays they have developed to identify candidate ADCs, and touches on the regulatory success of ADCs. 

Laura Lansdowne (LL):
What are the key benefits of ADCs?

Laura Murch (LM):
Antibodydrug conjugates (ADCs) are made up of 3 components: a monoclonal antibody, a payload (drug) and a linker. This combination of antibody and small molecule drug bestows several beneficial characteristics upon ADCs which the monotherapies alone do not possess. Firstly, the payloads used in many ADCs to date are highly toxic and therefore could not be used alone as single agents at an efficacious dose without severe off-target side effects. Secondly, the monoclonal antibody endows exquisite specificity to a given antigen, thereby providing targeted delivery of the payload to the tumor cell. Furthermore, ADCs are only “activated” once reaching the target, where the payload is released. Until this point, the cytotoxic payload is not free to damage healthy tissue and cells, which minimizes systemic exposure and therefore limits negative side effects. Due to this targeted delivery, a lower equivalent dose of small molecule for efficacy should be possible, improving the therapeutic window. Finally, ADCs can have a long half-life which in some cases can allow weekly dosing, which has a positive impact on patient quality of life. Essentially, an ADC combines the high specificity and long circulating half-life of an antibody with the potency of a highly cytotoxic small molecule, which gives ADCs an improved therapeutic index compared to either monotherapy. Of course, it should also be noted that there have been some difficulties with this modality to date. Off-target toxicity was an issue in some early formats of ADCs, where the payload was released before reaching its target, but recent developments in linker and conjugation technology are attempting to address this in novel ways. Additionally, developability of these complex molecules has proved challenging in the past as large-scale manufacturing of ADCs requires a combination of relatively high-cost facilities including high level containment (due to the toxic payload) and GMP level antibody manufacturing. This is improving due to a rise in CRO expertise dedicated to offering these services. As such, this is an exciting and rapidly evolving field with a growing knowledge base, built around designing highly specific and functional ADCs with the requisite facilities to produce these on a large scale.

LL: What is DAR and how does it impact the properties of the ADC and its efficacy?

The average Drug–Antibody Ratio (DAR) is an important value for an ADC. The DAR describes the number of drug molecules attached per antibody and varies depending on the conjugation method used. The DAR value has a significant impact on the properties of the ADC in vitro and in vivo. Often project teams will need to balance the potency of the payload with the DAR value of the ADC. High average DAR values usually (but not always) lead to efficacy of an ADC during in vitro studies, but this efficacy does not necessarily translate to the in vivo setting where the biophysical characteristics of the ADC dominate. For example, an ADC with high average DAR of a potent toxin will tend to induce clearance mechanisms from the host. So, whilst the high average DAR ADC may demonstrate better tumor killing potential as there is a large amount of potent payload present, the higher clearance rate can lead to liver toxicity. As such, DAR is a key attribute that requires careful assessment and consideration when developing an ADC.

It should be noted that the DAR is an average value and for many of the currently approved ADCs the conjugation methods used generate a highly heterogeneous ADC product. This has implications when considering the pharmacokinetics and pharmacodynamics (PK/PD) of an ADC therapy, as the different DAR species will have different attributes in vivo. Due to technology advances, ADCs generated using site-specific conjugations are becoming more common and enable production of a significantly more homogeneous ADC product. These more homogeneous ADCs have distinct advantages, with a more uniform PK/PD profile which can be more easily modelled and subsequently adjusted compared to the highly heterogeneous DAR ADCs. At LifeArc, we routinely determine the average DAR of ADCs using high-resolution mass spectrometry. Using our in-house Q-Exactive BioPharma platform, we can analyze ADCs at the intact level under denaturing or native conditions, and at the sub-unit level. We can also apply peptide mapping workflows to identify drug conjugation sites on the antibody, providing complete characterization of our ADCs.

LL: Could you touch on the regulatory success of ADCs?

The market for ADC therapies continues to expand, with three new approvals of ADCs in 2019. In total, there are currently seven ADCs approved by the FDA on the market (all for oncology indications) and over eighty ADCs currently under clinical development. The large number of next generation ADCs currently under clinical development highlights the growing appetite for ADCs in oncology and other therapeutic areas. Initial successes and challenges with the first-generation ADCs produced important learnings about the biopharmacology of ADCs. This has led to the development of new technologies to improve specificity and reduce toxicity. Next generation ADCs typically utilize humanized or fully human antibodies rather than murine or chimeric antibodies, significantly lowering the risk of immunogenicity. As mentioned earlier, site specific conjugation of payloads is now possible, allowing the production of single-DAR species ADCs rather than heterogeneous mixtures of DAR values. These single-DAR species have the benefit of delivering a consistent amount of payload to the target and a simpler PK/PD profile. The stability of linkers has also been improved, meaning less payload release prior to reaching the target antigen and therefore a lower off-target toxicity profile. The combination of these advances holds great promise for these next generation ADCs. Indeed, if the potential improvements in efficacy and safety are realized during current clinical development and evaluation, the field is likely to continue to expand over the next 5–10 years with many more regulatory approvals including for non-oncology indications.

LL: What makes a “good” ADC target?

Ideally good targets for ADCs are those which are overexpressed on the tumor/target cell surface, with low or absent expression in normal tissue, but it is important to understand each target in the context of the disease. For cytotoxic ADCs, targets should demonstrate good internalization properties, as intracellular tracking to the lysosome and endosomal compartments is key for many linker-release technologies to work well. It should be noted that if the complex is recycled to the cell surface and payload is released into the tumor microenvironment, there can be bystander effects upon surrounding tumor cells. Whilst this may be beneficial in the solid tumor setting, particularly if the tumor cells vary in target expression level, it needs careful monitoring on a target-by-target basis. It is also important to gather evidence that the toxin-linker combination selected can be effective at releasing payload and inducing cell toxicity in a range of relevant cell lines expressing the target, as this builds confidence in specificity and efficacy. Finally, ADCs bearing non-cytotoxic payloads are helping to redefine what makes a good ADC target, by considering biological problems which could be solved by selective re-targeting of a non-specific payload.

LL: Could you touch on some of the ADC programs LifeArc has within the oncology and non-oncology space?

LifeArc is involved in a multitude of projects across our three main therapeutic areas (oncology, neuroscience and anti-infectives) which include ADC projects as well as many other modalities. ADC programs are a natural fit for LifeArc as we have Chemistry, Biology and Biotherapeutics teams all based in the same building meaning these project teams can be interdisciplinary and immediately gain from this varied expertise. We have worked on two cytotoxic ADC programs and are looking to expand into programs involving non-cytotoxic payloads, where the resulting ADCs will have a different mode of action. For example, an early stage investigative project was carried out recently in-house based around a non-internalizing small molecule loaded ADC targeting MMP-9.1 This is certainly something that we are continuing to develop and expand upon.

LL: Can you highlight some of the key antibody characterization assays you have developed to identify candidate ADCs?

Due to the importance of selecting appropriate antibodies with good properties for the development of ADCs, an antibody characterization assay workflow was developed here at LifeArc.

Antibody clones are initially tested for binding to the target of interest, as well as for non-specific binding to a background cell line, in order to highlight positive clones. For this assay we use a flow cytometry-based assay on the Intellicyt® iQue Screener as this is a relatively quick way to analyze many clones simultaneously for specificity.

Antibodies which show specific binding to target are subsequently tested for their ability to internalize. We have two assay formats set up for this analysis, one which is a higher-throughput assay to test larger numbers of clones as a triage, and one which is low through-put, giving a more in-depth analysis and tracking antibody internalization to the lysosome. Both assays are image-based assays, set up using the IncuCyte® S3 and IN Cell Analyzer 6500HS respectively.

For the higher throughput assay using the IncuCyte® S3, antibody clones are labeled with a pH-sensitive dye which only fluoresces under acidic conditions (i.e. in the low-pH of the lysosome). Cells expressing the target of interest are treated with the pH-dye labeled antibody clones, and fluorescence is monitored over time on the IncyCyte® S3 over the course of 24 hours. Antibodies which internalize will show an increase in fluorescence over time.

The best performing antibody clones are then tested in the second assay, where they are directly conjugated to a fluorescent dye. Target cells are labeled with a nuclear marker as well as a fluorescent lysosome marker, and subsequently treated with the labeled test antibodies. Internalization is monitored at several time points using the IN Cell Analyzer 6500HS (Figure 1). The amount of internalization can be quantified by monitoring the fluorescently labeled antibody, and we can also look at the amount of colocalization of the antibody with the lysosome marker to gain an insight into whether the antibody tracks to the lysosome upon internalization. Those that do are promising candidates for use in ADC formats which often require internalization to the lysosome for payload release.

Figure 1: Antibody internalization. HEK293 cells overexpressing the target of interest were incubated with Alexa488 labeled test or isotype control antibody and internalization monitored at 0 and 48 hours on the IN Cell Analyzer 6500HS. Cells were stained for nuclei (Hoechst, shown in blue) and lysosomes (Lysotracker™ Deep Red, shown in red). Test antibody staining is mostly membrane restricted at 0hr, whereas by 48 hour punctate green and yellow staining can be observed showing antibody internalization. Yellow staining represents colocalization of the red lysosome dye with the Alexa488 labeled test antibody, indicating tracking to the lysosome.

Cell Toxicity:
Antibodies are also tested for their ability to induce cell kill by making use of a secondary antibody Fab fragment which is conjugated to a toxic payload, to mimic an ADC. Antibody clones are incubated with the secondary conjugated Fab-Tox and then tested in a dose-response experiment against cells which express our target of interest. Cell viability is monitored over the course of 48–72 hours on the IncuCyte® S3 and an endpoint viability reading is taken at 72 hours using Promega CellTitre-Glo®. In this way, we can gain an insight into how different antibody clones may compare in their ability to induce target cell death.

Due to our antibody expertise in-house, we routinely run promising antibody candidates through a suite of industry-standard biophysical assays. The data from all the characterization assays are collated and analyzed to determine which antibody clones should progress further for ADC development.

Laura Murch, Ph.D., was speaking with Laura Elizabeth Lansdowne, Senior Science Writer, Technology Networks. 

1. Love, et al. (2019) Developing an Antibody-Drug Conjugate approach to Selective Inhibition of an Extracellular Protein. Chembiochem. DOI: 10.1002/cbic.201800623