Optimization of Linker Chemistries for Antibody-Drug Conjugates

Published on: 
BioPharm International, BioPharm International, November 2023, Volume 36, Issue 11
Pages: 12-15

There is no such thing as a perfect linker.

Antibody-drug conjugate (ADC) safety and effectiveness relates directly to the functioning of the linker connecting the cytotoxic payload to the targeting antibody. The chemistries used to link the two crucial components of ADCs have evolved over time, leading to improved performance. Selection of the optimal linker chemistry is paramount to successful development of ADCs, however. The choice depends on the attributes of the antibody and the payload, as well as the nature of the target.

Efficiency and safety needs

Since the first ADC (Mylotarg, gemtuzumab ozogamicin, Pfizer) was introduced in 2000 (and withdrawn in 2010 for causing severe liver toxicity due to its unstable N-acylhydrazone linker, redesigned, and then reapproved in 2017), linker chemistries have evolved significantly to address issues with heterogeneity and instability. “First-generation ADCs struggled with heterogenous drug-to-antibody ratios (DAR) and unstable linkers, making them less effective and potentially more toxic,” says Campbell Bunce, chief scientific officer at Abzena. “Some of the early linkers used for ADCs were found to present a number of issues, including non-specific payload release in non-tumor tissues, leading to off-target toxicity and a limited therapeutic window,” agrees Sara Jenkins, ADC commercial manager with Sterling Pharmaceutical Services.

Specific examples include maleimide and lysine conjugation to antibodies. The former allows in vivo detachment of the linker with payload followed by reattachment to free cysteines, which can reduce ADC efficacy and impact the safety profile. The latter suffers from indiscriminate conjugation across the whole antibody, which can also interfere with binding to the cancer antigen or the Fc receptor functions. Both, according to Bunce, also suffer from variable DARs, making development of a robust manufacturing process challenging.

Much work has been undertaken to investigate the linker terminus to stabilize this reaction, find alternative methods of attaching the antibody via site-specific cysteines, or through use of enzymatic chemistry, Jenkins observes. Other efforts have been focused on ensuring that cleavable linkers cleave only in the target tumor tissue and at an appropriate rate, which can also be an issue for payload release, she adds. Increasing the number of accessible sites where linkers can be attached to payloads is another challenge currently being tackled by researchers who are, Jenkins says, exploring various functional groups on payloads and effector molecules.

Significant progress has been made on many fronts. In second-generation ADCs, advances in site-specific conjugation and cleavable linkers enable improved stability, homogeneity, and more controlled release, ensuring more targeted delivery to cancer cells and fewer off-target effects, Bunce notes. The third- and latest-generation of ADCs are further improved by using hydrophobicity-masking and solubility-enhancing elements for the payloads. “The new chemistries allow for improved PK properties; tandem or dual cleavage linkers, making payload release even more specific to the target cells; and multi-loaded linkers for increased therapeutic potency and a broader therapeutic index,” Bunce explains.

Cleavable and non-cleavable linkers

Linkers can be classified in two broad categories: cleavable and non-cleavable. Cleavable linkers, according to Nicolas Camper, senior director of chemistry with Abzena, release their payload when exposed to certain conditions in the target cell, such as low pH or high levels of a specific enzyme. Non-cleavable linkers, on the other hand, release the drug only upon catabolic degradation of the ADC.

There is still a great deal of interest in the classic approved peptide linkers such as Val-Cit-PAB (valine-citrulline-p-aminobenzylalcohol), which is used in Adcetris (brentuximab vedotin, Seagen) and more recently the GGFG (glycine-glycine-phenylanine-glycine) linker found in Enhertu (trastuzumab deruxtecan, Daiichi Sankyo), according to Jenkins.

Enzyme cleavable linkers have become increasingly popular, however, Bunce comments, as they tend to offer an optimal balance between systemic stability and efficient payload release inside cells. Jenkins points to glucuronidase triggered systems such as β-glucuronide as an example. “These linkers have a number of advantages, including the fact that lysosomal enzyme β-glucuronidase is abundant in lysosomes and over-expressed in some tumors, resulting in stable ADCs; and β-glucuronide is highly hydrophilic, leading to reduced ADC aggregation,” she says.

“The ultimate choice depends on the drug’s intended action and the biological context, although different payload release moieties deserve evaluation as part of the ADC lead-selection process,” Camper contends.

Conjugation chemistry

The conjugation chemistry used to connect the linker to the antibody is one of the important aspects of the linker that must be optimized. As the number of available technologies has increased, the trend has been to preferably leverage site-specific conjugation rather than stochastic (unpredictable or random) conjugation approaches, according to Camper.

Some site-specific conjugation technologies are compatible with native antibodies, Camper explains, while others require antibody engineering to introduce enzymatic conjugation tags or rare or non-natural amino acids into the antibody sequence. Not surprisingly, each approach presents its own challenges for manufacturing depending on the complexity of the process. All of these strategies, though, observes Camper, give better defined and more easily characterized ADCs while also allowing evaluation of different linker-payload conjugation locations on the antibody, which he notes can have a significant impact on biological activity.

Abzena offers its site-specific conjugation technology ThioBridge, which exploits the natural interchain disulfide bonds in antibodies, thus avoiding the need for extensive antibody reengineering. “Compared to older maleimide conjugation, ThioBridge produces uniform ADC constructs with homogenous drug loading. Its 3-carbon bridge attachment also enhances stability and improves pharmacokinetics,” Camper states. In addition to site-specific attachment, he says the technology is compatible with different linker designs and payload classes, has the potential to improve toxicity profiles, and is sufficiently flexible for DAR and hydrophilicity optimization.

New payloads require different chemistries

As research into ADCs has progressed, the number of types/classes of compounds that can serve as cytotoxic payloads has expanded. That has created the need for different attachment chemistries for these linkers other than those that must react with amines and alcohols, according to Jenkins.

Several chemistries have been reported in the literature and some have been put in practice. “Prominent among them,” says Jenkins, “is the addition of hydrophilicity to linkers using hydrophilic triggers or by adding pendant hydrophilic groups.” She also highlights different triggers under investigation by various groups, including stabilized maleimides that limit the retro-Michael elimination reaction, enzyme-independent triggers such as pH-dependent linkers that remain stable at physiological pH but unstable at the pH of the tumor microenvironment, and disulfide-based designs that take advantage of glutathione gradients to trigger release.

Optimal linker solutions

Determination of the optimal linker chemistry for a given antibody and payload combination depends on numerous factors. Specific considerations noted by Jenkins include stability at physiological pH and in serum; the ability to release the payload where desired, whether in the cell or the tumor microenvironment; sufficient hydrophilicity to counteract payload hydrophobicity while still reducing ADC aggregation; and a reasonable and practical manufacturing process.


Efficacy and safety are paramount, emphasizes Bunce. “The linker must ensure targeted payload delivery to maximize therapeutic impact and minimize off-target effects and toxicity. That is because from a design perspective, the choice of linker can affect ADC stability, DAR, and pharmacokinetics,” he elucidates. Manufacturability cannot be ignored, however, agrees Bunce. “The linker chemistry must align with scalable production methods to control costs and meet development timeframes,” he says.

Evaluation requires multifaceted approach

One of the main hurdles in optimizing linkers for ADCs is the number of parameters which can potentially be investigated. They include, says Camper, the antibody conjugation site and chemistry, the payload release mechanism, solubility enhancing elements, and architectures allowing accommodation of multiple copies of the payload, and positioning of these different elements in the linker. Adding to the challenge is the fact that there is a profuse offering of technologies to improve all the different elements of ADC linkers, all with their own advantages and drawbacks. “The number of possibilities to optimize an ADC can easily look overwhelming,” he concludes.

Scale up of ADC production processes can also present issues. “Advanced technical solutions working well at bench scale and providing ADCs with attractive pre-clinical therapeutic profiles may prove extremely complex to manufacture or turn into regulatory challenges due to the density of innovation introduced into the new ADC drug,” Camper observes. The result: complex decision-making for ADC linker optimization. Consequently, evaluating the performance of ADC linkers requires a multifaceted approach that combines physical experiments with analytical and predictive techniques, according to Camper.

Specific enzyme assays, such as cathepsin-B and β-glucuronidase, can be used to determine whether the payload is released at an appropriate rate and as expected, Jenkins observes. A linker’s stability in plasma can, meanwhile, be a key factor in establishing how resilient an ADC can be to unexpected cleavages or premature loss of payload when exposed to bio-fluids, she adds. In addition, cell and liver fractions such as lysosomes and S9 fractions can be utilized to see how linker-payloads, and even the toxins themselves, react to biological mixtures that are closer in nature to those found in tumors. Mass spectrometry, Jenkins continues, is a useful technique for these experiments because of its specificity and sensitivity in complex samples.

Specific methods highlighted by Camper include Incucyte FabFluor (Sartorius) labeling for confirmation of ADC internalization by the target cells and efficient intracellular tracking alongside the endo-lysosomal pathway, flow cytometry to assess on-target and off-target toxicity, EpiScreen and Cytokine Screen (Abzena) immunogenicity assays and in silico assays for safety, in vitro stability tests to ensure that the ADC remains stable in circulation and doesn’t release its payload prematurely, and pharmacokinetic and pharmacodynamic analyses to gain in vivo performance data that inform adjustments for optimal outcomes and allow selection of the best lead ADC design for further development and manufacture based on a favorable therapeutic index.

Bunce stresses, however, that as ADCs become more intricate—carrying multiple payloads or designed for controlled release mechanisms—these conventional analytical techniques may reach their limits. “Mass spectrometry, next-generation sequencing, and chemical modeling, or even novel technologies could fill this gap, providing a more comprehensive view of ADC characteristics,” he remarks. Bunce goes on to state, “these innovative techniques may well be the linchpin for overcoming the technical challenges ahead, offering a refined toolkit for the next generation of ADC linker optimizations.”

New analytical techniques

There is also potential for application of advanced digital tools in ADC linker selection and optimization. “As an industry, we’re delving deeper into the complexities of ADC linker chemistry, trying to get ahead of the next wave of optimizations that will hopefully deliver more effective therapies. One area often overlooked is the role of computational biology in predicting linker behavior,” Bunce contends.

Advanced simulations, Bunce adds, can provide insights into the dynamic interactions between the linker and the antibody, thereby reducing the need for exhaustive empirical tests. “If we can integrate machine learning algorithms, there is the possibility of bringing a greater degree of automation to the process of data analysis, making it easier for ADC experts to identify subtle but crucial trends in ADC performance,” he says.

Overcoming poor bioavailability issues

Cytotoxic payloads used in ADCs, like many other novel small-molecule APIs, often are hydrophobic compounds with poor water solubility and thus low bioavailability. As a result, chemists in the ADC field must often identify a solution to hydrophobicity problems associated with highly lipophilic payloads, according to Stepan Chuprakov, director of chemistry at Catalent. “Depending on the chosen bioconjugation method (site-specific, stochastic) and the desired DAR, the resulting conjugate may demonstrate poor biophysical properties, including high levels of aggregation and poor pharmacokinetics (rapid plasma clearance),” he explains.

Typically, some structural elements that mask the payload’s hydrophobicity are added, including discrete polyethylene glycol (PEG) chains, polyols, and charged groups, Chuprakov notes. As examples, Jenkins suggests that the cleavage trigger can be switched from Val-Cit to Val-Ala or a more hydrophilic β-glucuronide that can lower the logP of the molecule and increase the therapeutic index. “The approach works well if β-glucuronidase is abundant in the tumor cell to facilitate cleavage, but is not as beneficial if the trigger cleavage is dependent upon peptidase enzymes,” she notes.

With respect to the insertion of a long PEG unit into the linear chain to increase the hydrophilicity of the linker payload, Jenkins comments that it has become evident that, in some cases, adding long PEG units can actually have a detrimental effect on the molecule. Instead, she says that adding pendant hydrophilic units such as PEG or polysarcosine (pSar) in the correct positions has been shown to have a masking effect on the hydrophobic payload.

On the positive side, Chuprakov observes that the available potential chemical space for linker design is vast, and chemists have a variety of synthetic tools to incorporate those additional elements. “However, synthetic chemists may want to avoid the temptation of going too far beyond what nature dictates, as some exotic structural elements may come with their own concerns, for example, immunogenicity,” points out Chuprakov.

Developability assessments crucial

The best strategy for assuring selection of the optimal linker for any ADC is the use of performance of a rigorous pre-clinical developability assessment encompassing all the different components of an ADC, both taken individually and as a whole, Camper insists.

“Start from the selection of antibody clones optimized for target antigen binding specificity and affinity, internalization and trafficking properties, as a well as favorable immunogenicity and stability profiles. Then, coupling the lead antibody clones to a panel of payloads and linkers to determine the best combination of linker-payloads and antibodies to carry forward into in vivo evaluation on the basis of in vitro cell cytotoxicity and serum stability profiles, the therapeutic potential of lead candidate ADCs can be assessed from efficacy, PK, and toxicology studies,” Camper says.

Using this approach, liabilities can be identified and addressed, possibly by getting help from advanced linker technologies for optimization. “Early evaluation of conjugation efficiencies and biophysical stability also provides invaluable information on ADC manufacturability and is factored into the selection of the ADC candidate to take into clinical trials,” Camper observes.

“In addition to thoroughly reviewing how safe and effective an ADC is and how easily it can be manufactured, our team also uses ongoing data to make real-time adjustments to a drug’s design. By doing so, issues that could cause problems later on in the development process can be fixed or minimized in advance, making the drug more reliable and easier to produce. It’s all about starting smart so you can progress fast while balancing efficacy, safety, and ease of production,” says Bunce.

Cross-functional collaboration essential

Along with a multifaceted approach, successful linker optimization requires collaboration of many people with expertise in a wide range of fields. “The ADC field is complicated, and chemistry and biology teams need to work in parallel to create an efficacious drug. It is common for situations to occur when a chemistry problem is resolved, only to cause a new, unexpected biological problem,” comments Xiao Cai, senior scientist in chemistry at Catalent. He stresses that scientists need to bear in mind that an ADC has highly cross-functional composition, so timely and effective communication between chemists and biologists is imperative for their successful design.

Keep sustainability in mind

While much attention goes to the scientific and technical aspects of optimizing ADC linker chemistries, Bunce believes that ethical and environmental concerns also deserve a spotlight. “Not only the materials used in creating linkers could pose environmental risks if not managed properly. Some of the manufacturing processes are energy-intensive and may warrant exploration of more sustainable alternatives,” he suggests. On the ethical front, Bunce says it is critical to consider the accessibility and affordability of advanced ADC technologies, especially in low-resource settings.

“These aspects often go under the radar but could play a significant role in the broader acceptance and deployment of new ADC technologies,” Bunce contends. “A truly integrated approach that goes beyond just the science could be instrumental in shaping the future of ADC development,” he concludes.

No universal solution

One of the most important points about linker optimization that these experts all stress is the fact that there is no one solution that fits all ADCs. “Linkers will differ from case to case, and there is no one universal solution to their design, so an application-driven approach is needed,” states Chuprakov.

That is largely because, Chuprakov explains, a linker is not only a connector, but also has embedded engineered functionality, such as a desired level of stability during circulation, and an optimal rate of cleavage by intramolecular machinery after ADC internalization has released the drug. Once a linker’s functionality has been established, then additional design elements can be added, particularly those that address biophysical properties of the resulting conjugate, such as hydrophilicity, while simultaneously factoring in CMC considerations.

Jenkins agrees, stressing there is also no such thing as “the perfect linker.” “Both the antibody and payload will dictate what linker is ultimately chosen, while subsequent testing is likely to determine what further optimization needs to be undertaken,” she says.

About the author

Cynthia A. Challener, PhD, is a contributing editor to BioPharm International®.

Article details

BioPharm International
Vol. 36, Number 11
November 2023
Pages: 12-15


When referring Challener, C. A. Optimization of Linker Chemistries for Antibody-Drug Conjugates. BioPharm International 2023 36 (11).