Dr. Liping Ma - Senior Study Director, DMPK Service Department, WuXi AppTec
Antibody-Drug Conjugates (ADCs) are an evolving therapeutic modality with an unprecedented ability to target and destroy cancer cells. They were developed to improve upon conventional chemotherapies’ toxicity and specificity challenges of targeted delivery. In many ways, ADCs combine the most significant advantages of chemotherapy and immunotherapy to fight cancer.
ADCs function by linking a highly potent cytotoxic drug (i.e., payload) to a tumor-specific antibody designed to ignore normal cells and latch onto cancerous ones. Their combination of high potency and laser-focused targeting has drawn comparisons to precision-guided missiles, eradicating tumors wherever they are found. As novel as ADCs may seem, the concept is not a new one.
After more than 40 years of research, scientists completed a successful clinical trial of the first ADC in 1983—using an anti-carcinoembryonic antigen antibody-vindesine conjugate—which they thought would trigger an industry boom.1 But the 1990s saw several ADCs developed and subsequently scrapped due to linker instability and patients receiving improper doses of the drug. Then in 2000, the U.S. FDA approved Gemtuzumab for CD33-posite acute myelogenous leukemia. Since then, the agency has approved 12 more ADCs designed to fight blood, breast, urethral and cervical cancers.
Scientists today use humanized or fully human monoclonal antibodies to deliver the cytotoxic payload. The payload disrupts DNA or exerts RNA polymerase inhibition, which causes cell death.2 The benefits of this approach include low off-site toxicity, minimal immunogenicity and a long-circulating half-life (i.e., the ADC drug stays in the bloodstream longer).
For these reasons, scientists, drug developers and sponsors are excited about ADCs as a therapeutic strategy. But great promise comes with some significant challenges, poor plasma stability and over-distribution of cytotoxin in non-target cells can derail the development of new ADCs and jeopardize clinical applications.3 Moreover, the mechanisms responsible for metabolizing and eliminating ADCs are complex and present their own unique challenges. Further testing is ongoing to optimize ADCs’ pharmacokinetic (PK) properties and support patient safety.
Challenges of Investigating ADCs
ADCs are a combination of large and small molecules that require ADMET (Absorption, Distribution, Metabolism, Elimination, Toxicity) studies to investigate the intact ADC as well as both constituent molecules. Understanding payload release and metabolism of ADCs is critical. The release of payload in target organs is directly related to ADCs’ efficacy, and the release of payload in non-target tissues determines ADCs’ toxicity. Drug-Drug Interaction (DDI) of ADCs is also associated with exposure, metabolism, and excretion of payload-related components. The complexity of designing and executing ADMET studies for ADC’s means developers will need to anticipate and plan for challenges and collaborative problem solving along the way.
Conducting the necessary preclinical ADMET studies for ADC drugs can take several months. Given the time it takes to screen and identify drug targets and then conduct IND-enabling studies, scientists must choose which tests are necessary for their drugs. The primary research focuses on payload release in ADCs. Knowing this will help determine which component will be tested for in vitro assays. In addition, it also helps to determine which component should be measured in PK/TK (pharmacokinetic (PK)/toxicokinetic (TK)) studies. Partnering with a laboratory to maximize scientific expertise when it comes to these unique studies can help anticipate and determine best paths forward in your test plan development.
Strategies and Unique Experimental Approaches to ADCs
ADC PK studies can be divided into four primary focuses: ADME, in vitro DDI, PK/TK and bioanalysis. These four study categories have different focus areas in the discovery, preclinical and clinical stages. The PK study strategy and protocols for each ADC project should be based on the structure of ADC.
A comprehensive review of data and summaries of studies conducted from newly marketed medicines has led to some anticipated requirements, and suggestions, for ADC PK studies for drug developers.
Here we break down each study category and identify the focus areas for each stage of development.
Monitoring Payload Release in ADCs
As mentioned above, ADCs’ basic structure includes a monoclonal antibody, a cytotoxic payload and a chemical linker. Linkers play a crucial role in releasing payloads at the correct times, so understanding the different ways they function is critical to success.
ADCs can contain two types of linkers: cleavable and non-cleavable. The former is the most common type of linker in ADC drugs and has a clear structure. When cleavable linkers encounter lysosomal enzymes or acidic conditions, they release their payloads. The cancerous cells are targeted, killed and excreted. Non-cleavable linkers, on the other hand, release their payloads by degrading antibodies. These released payloads bind to partial peptide chains or amino acids—they have an unknown structure and may be toxic if released in non-target tissue.
Scientists need to understand the structures of payload-related components, which refer to payload and payload-related metabolites, and the release process regardless of whether the linker is cleavable or non-cleavable. A metabolite identification study can help determine the ADC’s true active components. It can answer whether the ADC releases the payload itself or other payload-related metabolites. Combined with cytotoxicity test, this study determines which part is responsible for killing the tumor cells.
The release of payload can be studied by using different in vitro systems. First, a serum/plasma stability or metabolite identification study—using both human and animal plasma—demonstrates how different matrices affect ADC stability. These studies work to predict the ADC’s off-target toxicity by measuring how much payload or payload-related metabolites are released into the bloodstream. Another recommended study is to monitor the payload-related components in incubated tumor cells to see if the payload is released inside tumor cells and successfully eradicates them. Other in vitro systems such as lysosomal and acidified liver S9 fraction also can be used to investigate the release of payload in lysosome, which is the site of degradation of an ADC.
Drug Antibody Ratio (DAR) Analyses of ADC
In PK studies, bioanalysis is indispensable in helping scientists understand the results of each stage. DAR represents the amount of payload conjugated to each antibody. It is an important parameter to describe ADCs’ physiochemical properties, including their safety and effectiveness. For FDA approved ADC drugs, their DAR range is typically 3-4. An overly high DAR will increase drug clearance, decrease half-life, and lead to drug aggregation and increased toxicity.
DAR analysis can be divided into two aspects. DAR distribution characterizes the proportion of ADC molecules with different DARs in the total ADC molecules. The second aspect is the average DAR, which is the ratio of the molar concentration of the total payload molecules to that of the antibody molecule in the system. Using LC-MS to analyze DAR is highly recommended as it can reduce sample consumption and provide more accurate results.
Tissue Distribution for ADC
Qualitative Whole-Body Autoradiography (QWBA) helps confirm that the ADC drug is distributed to target tumor tissue and not others. This qualitative investigation uses color-stained pictures to track the ADC’s distribution throughout the body and ensure it interacts with the target cells. A dark stain means the drug shows strong distribution; a light stain suggests weak distribution. Ligand-binding and LC-MS approaches are more valuable if quantitative data is needed to confirm the ADC is distributed to the correct tissue.
Drug Interaction Consideration
The risk of payload-related components in the circulation system acting as a perpetrator of an enzyme or transporter is very low. However, scientists need to know which enzymes will be involved in metabolizing payload-related components. If the payload-related components are very potent and very toxic, their toxicity will increase significantly when co-administered with related metabolic enzyme inhibitors. Understanding how quickly the accumulation of payload-related components is eliminated in vivo is fundamental to understanding toxicity but also very challenging because of the drug’s long half-life. Using radiolabeled ADCs to conduct elimination studies is one of the most effective and time-sensitive ways to gather this information.
Additional ADC Hurdles Developers Must Overcome
ADCs have a bright future in a cancer treatment, but scientists face four primary hurdles in development.
First, achieving high clinical efficacy and low off-target toxicity are the biggest challenges for ADC drugs. Many ADC drug development efforts are terminated because they cannot be proven to be as effective or significantly better than existing treatments in clinical practice. In preclinical toxicological studies, off-target toxicity is common for ADC drugs. Developing an ADC drug that is stable in plasma but effectively releases sufficient toxic molecules in target tumor cells remains a problem.
Second, ADCs can degrade during storage and transportation. Increasing the concentration of the drug is an option, but that can lead to aggregation. To increase efficacy, a higher DAR value is adopted. Since most payloads are hydrophobic, high DAR values lead to increased risk of aggregation. Aggregates are more immunogenic and can also lead to the loss of ADC drugs.
The third hurdle is in achieving the correct DAR ratio. As mentioned, a typical DAR ratio is between three and four, meaning three or four payloads conjugated to a single antibody. A popular and recently developed ADC achieved a DAR ratio of eight, making it incredibly potent. However, a high DAR ratio can mean the ADC drug is eliminated quickly in vivo, reducing its efficacy. The final hurdle is achieving an acceptable half-life. An ADC drug that is active in vivo for a long time can be very effective, but given its potency, it can also raise concerns about dosage limit and toxicity.
A Final Word
The drug development industry is justified in its excitement around ADCs. They are highly effective cancer killers with clear advantages over conventional cancer-fighting strategies. But monitoring payload release and achieving the correct DAR ratio is the key to successfully developing safe and effective ADCs.
Many of the required ADC studies can be conducted simultaneously, so timelines for IND application are unlikely to be affected with well-managed programs. But it does take significant expertise and technological capabilities to test small and large molecules while also testing the intact ADC. Key considerations for success include:
- First, as both small and large molecule studies are needed, partnering with a lab experienced doing PK studies with both types of molecules is recommended.
- Because in vitro DDI can be a concern, in vitro and in vivo metabolite identification, and radiolabeled ADME is required. Working with a lab that has comprehensive ADME and bioanalysis capabilities for large and small molecules will support efficiency and comprehensive understanding of any unique challenges that are uncovered.
- Third, it is expected that developers specifically design their PK study strategies and protocols for each assay according to the unique structure of the ADC.
- Finally, ADC PK studies are extremely complex with many moving parts. The need for expert project management, cross-departmental organization and coordination cannot be underestimated.
Drug sponsors and developers that lack these in-house capabilities should consider working with a laboratory testing partner to ensure they have the most pertinent data and that their application is as complete as possible. This collaboration can also help prepare the drug candidate for regulatory submission when the time is right.
References
- Ford, C.H., Newman, C.E., Johnson, J.R., Woodhouse, C.S., Reeder, T.A., Rowland, G.F. and Simmonds, R.G., 1983. Localisation and toxicity study of a vindesine-anti-CEA conjugate in patients with advanced cancer. British journal of cancer, 47(1), pp.35-42.
- Tsuchikama, K. and An, Z., 2018. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein & cell, 9(1), pp.33-46.
- Kamath, A.V. and Iyer, S., 2015. Preclinical pharmacokinetic considerations for the development of antibody drug conjugates. Pharmaceutical research, 32(11), pp.3470- 3479
Author Biography
Dr. Liping Ma, PhD of Pharmaceutical Analysis, an expert in pharmacokinetic evaluation, is now a senior study director in DMPK Service Department of WuXi AppTec. She has over 10 years of research experience in the field of preclinical and clinical pharmacokinetics and extensive experience in the preclinical development of drugs. She successfully supported over 20 new drug IND applications globally.
Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered to your inbox!
Sign up now!