Continuing Momentum in Bioconjugate Therapeutics with Advanced Linker Chemistry

Bioconjugate therapeutics, which unite biological principles with synthetic chemistry approaches to create powerful drugs, are among the most promising modalities in biopharma. The field encompasses an assortment of therapeutic classes sharing several core building blocks: carrier biomolecules such as lipids or proteins, small-molecule or macromolecule drugs, and chemical linkers. Antibody-drug conjugates (ADCs) are by far the most prominent category of bioconjugates, with fifteen products currently approved by the FDA and over 150 clinical trials underway.¹ Combining potent cytotoxic drugs with monoclonal antibodies that pinpoint cancer-associated biomarkers, ADCs show immense potential as a cancer treatment strategy that selectively targets tumor cells while sparing healthy tissues.

However, over a decade passed between the first FDA approval of ADCs in 2000 and the next in 2011, largely due to issues with off-target toxicity associated with early ADC candidates.² The basic model of simply attaching an antibody and a drug to build an efficacious and safe ADC has been repeatedly shown to be incredibly complex, with all three components contributing to the final characteristics and performance of the ADC. It’s often not until the drug moves beyond in vitro and in vivo studies to human testing that the liabilities are identified. A growing appreciation for strategic linker design and bioconjugation chemistry, coupled with advances in chemical biology, has ignited a new wave of efforts to optimize ADC performance and bring these groundbreaking drugs to market. By exploring linker design strategies beyond conjugation chemistry and site specificity to include the structure and composition of the linker itself, drug developers have more opportunities to manipulate features and improve the safety and efficacy of their ADC candidates.

Fine-Tuning ADC Safety and Efficacy with Strategic Linker Design

Maximizing the potential of an ADC candidate through strategic linker design requires a deep understanding of how linker parameters can be fine-tuned to impact the drug’s efficacy while minimizing off-target effects. The linker plays a vital role in ensuring an ADC remains stable enough in blood plasma to prevent premature payload release while enabling effective drug delivery once the target is reached. Additionally, the hydrophobicity of many drug payloads can lead to aggregation, resulting in pharmacokinetic issues and immunogenic effects. Drug developers and researchers can leverage linker design to modulate ADCs’ hydrophilicity, improve solubility, and overcome these limitations.³ Because a linker’s structure and the conjugation processes used to join ADC components can also impact the heterogeneity of the resulting product, these factors are also important for creating consistent ADC molecules with reliable pharmacological properties.

Several decades of accumulated research have better elucidated how specific linker characteristics and other ADC properties can be manipulated to optimize performance. These features must be selected for each ADC candidate to suit its unique combination of components, the mechanism of payload release, the disease state of interest, and other variables. Importantly, the tunable parameters of ADC components cannot be considered independent of one another and must be carefully balanced and assessed to maximize functionality without compromising key traits.

The following sections provide a brief overview of features to be considered in strategic linker design, as well as research findings illustrating their impact on ADC performance.

Mechanism of Payload Release

One of the first decisions in designing an ADC is selecting how a drug payload will be released from the antibody upon reaching its target. One broad distinction is the choice of a cleavable trigger versus a non-cleavable linker. While a non-cleavable linker relies on internalization via endocytosis and lysosomal processing to control payload release, cleavable linkers rely on a chemical or enzymatic trigger that typically responds to features of the tumor microenvironment, such as specific pH conditions or proteolytic enzymes overexpressed within a tumor cleavable linkers are employed in over 80% of currently approved ADCs, including inotuzumab ozogamicin (Besponsa) and brentuximab vedotin (Adcetris).^4,5

However, issues exist with nonspecific drug release following ADC uptake in non-cancerous tissues. Novel cleavable linker technologies aim to use more tumour-selective strategies to minimize ADC release in off-target tissues. For example, a linker prototype described by Spangler et al. leverages a ferrous iron-reactive trigger to enable selective payload release in response to accumulated ferrous iron in cancerous tissue.⁶ Other novel tissue-specific release strategies under exploration include oligonucleotide-based and UV-controlled cleavable linkers.^7,8

Linker Structure and Architecture

Structural factors of a linker can also be manipulated to alter the function of an ADC and overcome chemical and pharmacological limitations of drug payloads. As described previously, the highly hydrophobic nature of many drug payloads can present issues such as insolubility and aggregation, but increasing the hydrophilicity of the payload itself can compromise its function. Researchers have thus explored numerous linker-based strategies for modulating solubility and hydrophilic interactions, including the addition of negatively charged sulfonate groups or highly polar polyethylene glycol (PEG), polycarboxyl, or polyhydroxy groups. PEG-based linker backbones have become a valuable tool for enhancing the stability, drug loading, and performance of ADCs, but the configuration, length, and positioning of PEG segments significantly impact a therapeutic’s effectiveness.

For example, Lyon et al. demonstrated that an extended linear PEG spacer was insufficient as a means of reducing hydrophobicity to improve therapeutic index, as it increased the distance between antibody and payload and exposed the hydrophobic payload to the aqueous environment. Instead, using the same PEG oligomer in a branched rather than linear conformation effectively shielded the drug payload, resulting in enhanced antitumor activity in vivo.⁹ Additional studies have demonstrated that the “hydrophilicity reservoir” effect conferred by a branched, pendant-like PEG linker configuration can support a higher drug-to-antibody ratio (DAR) while maintaining desirable pharmacokinetic performance.¹⁰

A linker’s structure can also work in concert with its cleavable trigger function to control rates of payload release in an enzyme-specific manner. Just as linker architecture can be designed to shield a hydrophobic payload, a similar strategy can be used to affect enzymatic access to the cleavable trigger and minimize payload release in healthy tissues. Giese et al. demonstrated that manipulation of PEG linker architecture could effectively shield trigger payloads from enzymes with readily accessible binding sites.¹¹ While this approach alone cannot selectively limit the release to target tissues, it provides evidence that linker configuration could be used to tailor access to specific, tumor-associated proteins.

These and other findings illustrate the immense potential of manipulating linker composition and structure as a means of fine-tuning ADCs’ safety, efficacy, and pharmacokinetic attributes. Two currently approved ADCs, Zynlonta and Trodelvy, both leverage PEG8 linkers in their design. Ongoing ADC development efforts will likely continue to explore more sophisticated PEG-based architectures to optimize structure-function relationships.

Drug-to-Antibody Ratio (DAR)

The drug-to-antibody ratio (DAR) is another key determinant of ADC performance, directly influencing both efficacy and safety. A higher DAR typically enhances therapeutic potency by delivering more drug molecules per antibody, but increased drug loading also comes with risks. Overly high DAR can compromise the ADC’s stability, alter its pharmacokinetics, and increase off-target toxicity. Strategic choices in linker composition and architecture, such as those described above, can help to balance higher DARs while maintaining safety and efficacy. Using hydrophilic linkers and more complex architectures can enable higher drug loading of highly hydrophobic payloads without sacrificing stability. For instance, a 2019 study by Viricel and colleagues demonstrated that the synthesis and use of monodisperse polysarcosine compounds in a linker platform could effectively shield a hydrophobic payload, increasing maximum drug loading from DAR3-4 to DAR8.¹² Additionally, improvements in site-specific cleavable linkers can reduce the likelihood of off-target release and subsequent toxicity of high-DAR ADCs.

Conjugation Site and Method

Where and how an antibody is joined to a linker can also have significant implications for an ADC’s success. The placement of a linker and payload can influence binding efficacy, while the selection of antibody functional groups and linker reactive groups can determine maximum drug loading, manufacturing efficiency, and product homogeneity. Because antibodies contain multiple potential conjugation sites, depending on the conjugation method used, the resulting drug substance can contain a diverse mixture of ADC molecules with varying DARs as well as some portion of unconjugated antibody. Like drug loading, homogeneity has important implications for the in vivo efficacy of an ADC. For example, Anami et al. demonstrated a homogenous anti-EGFR ADC yielded a significant survival benefit and increased antitumor activity in a mouse brain tutumorodel relative to comparable but more heterogeneous ADCs.¹³

Advances in site-specific conjugation have enabled greater control over ADC homogeneity and performance. Leveraging engineered antibodies that incorporate tags for enzymatic ligation or unnatural amino acids (UAAs) with click chemistry functional groups can support

more specific and efficient bioconjugation reactions with compatible linkers. A promising example is Genetic Code Expansion (GCE), which manipulates translational machinery to insert tetrazine-containing UAAs in a site-directed manner. This enables rapid, site-specific “click-on” reactions with trans-cyclooctene (tCO)-containing functional groups on linkers. Studies employing this approach demonstrate high reaction efficiency and site specificity without compromising antibody functionality, although yield and homogeneity are still challenges to be overcome.¹⁴

Challenges and Considerations in Linker Design and Production

Advances in linker technologies and conjugation chemistry have been major drivers of the recently reignited interest in ADCs. In 2023, the majority of ADC-related investments were focused on technology platforms, with over a quarter of deals with named targets and technologies focused on linkers in particular.¹⁵ Robust linker design technologies and expertise are now in high demand among biopharma players looking to truly innovate in the ADC space, and specialized development and manufacturing partners are at the core of these efforts. Building a strong in-house foundation of expertise and infrastructure to design, manufacture, and purify linkers at scale may not be realistic for all organizations. While many manufacturers are now able to produce short, linear PEG-based linkers, synthesizing longer and more complex structures at necessary purity and scale remains a challenge for most.¹⁶

Deep, specialized expertise and infrastructure are necessary to realize the full potential of linker design and synthesis for optimizing ADC performance, leading many drug developers to turn to experienced, well-equipped providers of advanced linkers to supplement their in-house operations. SpeSpecialtyemical providers can be a more time- and resource-efficient approach for less established organizations to access a comprehensive range of design and synthesis capabilities. In addition to supporting sophisticated linker design and synthesis, an established linker manufacturing partner can ideally provide efficient and dependable synthetic processes, advanced manufacturing facilities, and comprehensive multi-method purity analyses, enabling drug developers to thoroughly characterize products and meet rigorous quality standards.

Fueling Innovation in the Path to the Clinic

While the important role of linkers in ADC function is now more widely appreciated, many researchers and early-stage drug developers are limited in their capacity to source different linker options to screen for their impact on ADC performance. The countless variables at play in selecting a linker design and conjugation strategy, as well as the complexity of an ADC’s target within a physiological context, make it difficult to accurately predict a candidate’s behavior in vivo. Exploring linker design options early on can help de-risk drug development by preventing a suboptimal linker choice from derailing an otherwise promising drug candidate in preclinical or even clinical stages. Working with a highly tunable scaffold to test a variety of linkers with distinct physicochemical properties early in discovery can help researchers thoroughly elucidate the impact of linker structure on ADC function and invest in only the most viable candidates.

In the two decades since the first FDA-approved ADC, the growing body of research demonstrating how linker design can influence virtually every aspect of ADC production and performance, from manufacturing efficiency to in vivo efficacy, has made it clear: for the ADC field to truly progress, linkers cannot be approached as a “one-size-fits-all” component. Whether investing in building internal capabilities or working with specialized chemical supply partners, exploring linker design will be vital for ADC developers to realize the full potential of this modality. Powered by deepening knowledge in chemical biology, specialized design capabilities, and more scalable, efficient methods for synthesizing increasingly complex linkers, continued innovation in ADC development will bring more life-changing therapeutics to the clinic.

References

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8. J. Li et al. (2021). Novel antibody-drug conjugate with UV-controlled cleavage mechanism for cytotoxin release. Bioorg. Chem. 111: 104475.
9. R.P. Lyon et al. (2015). Reducing the sphericity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nat. Biotech. 33(7): 733- 735.
10. T. Tedeschini et al. (2021). Polyethylene glycol-based linkers as hydrophilicity reservoir for antibody-drug conjugates. J. Controlled Release 337: 431-447.
11. M. Giese et al. (2021). Linker Architectures as Steric Auxiliaries for Altering Enzyme-Mediated Payload Release from Bioconjugates. Bioconj. Chem. 32(10): 2257-2267.
12. W. Viricel et al. (2019). Monodisperse polysarcosine-based highly-loaded antibody-drug conjugates. Chem. Sci., 10(14): 4048-4053.
13. Y. Anami et al. (2022). The homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors. Cell Reports, 39(8): 110839.
14. P. Huda et al. (2024). Click-on Antibody Fragments for Customisable Targeted Nanomedicines – Site-specific Tetrazine and Azide Functionalisation through Non[1]canonical Amino Acid incorporation. Chem. Methods, 4(2): e202300036.
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Author Details 

Pam James, Vice President, Product- Vector Laboratories

Publication Details 

This article appeared in American Pharmaceutical Review:

 Vol. 27, No. 6

Sept/Oct 2024

Pages: 28-30

 

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