Exolinkers: A New Strategy to Overcome Val-Cit Linker Drawbacks

Antibody-drug conjugates (ADCs) represent an innovative strategy in targeted therapy, enabling precise drug delivery to maximize therapeutic indices and reduce systemic toxicity by combining cytotoxic payloads with antibodies through linkers. The linker is a crucial component of ADCs, fundamentally determining their structural homogeneity, pharmacokinetics (PK), and safety margins, as well as their overall stability and efficacy. Linkers have to be stable in the bloodstream and healthy tissues while efficiently delivering payloads to tumors.

Despite the apparent proliferation of linker choices[1,2], there is a significant monotony in clinical adoption. Several FDA-approved ADCs are based on the ubiquitous Val-Cit linker or its derived Val-Ala linkers. The Val-Cit linker is a cleavable peptide linker that can be cleaved by cathepsin B, a protease highly expressed in cancer cells, which confers specificity of the ADCs to cancer cells. But it is still associated with several limitations, including:

Hydrophobicity-induced aggregation

Limited drug−antibody ratio (DAR)

Premature payload release

The hydrophobic nature of the Val-Cit p-aminobenzylcarbamate (PAB) linker restricts the quantity of payload that can be utilized[3]. Specifically, widespread payload linkers, like Mc-Val-Cit-PAB-MMAE, grapple with moderate drug-to-antibody ratios (DAR = 3-4), and efforts to achieve higher ratios are hindered by their hydrophobicity, leading to aggregation. Notably, a landmark publication by Pfizer highlighted the vulnerability of the Val-Cit linker to carboxylesterase Ces1C[4], which results in premature payload detachment Moreover, Zhao et al. revealed an additional issue associated with the aberrant cleavage of the Val-Cit bond involving human neutrophil elastase (NE)[5], and this implies potential ADC-associated off-target toxicity, possibly leading to neutropenia.

To address the hydrophobicity of the Val-Cit platform, various strategies have been developed, such as incorporating hydrophilic polymer scaffolds like PEG[6], polysarcosine[7], cyclodextrins[8], peptides[9], and polyacetals[10]. Significantly, a recent study[11] proposed a novel linker to address the intrinsic limitations of the Val-Cit linker (Figure 1). Breaking away from the conventional linear "Antibody-Linker-Payload" structure, the researchers developed an innovative design that repositions the peptide-cleavable linker Glu-Glu-Val-Cit (EEVC) or Glu-Val-Cit (EVC) at the exo position of the p-aminobenzylcarbamate moiety (Figure 1B).

Fig 1. Comparison of Val-Cit PAB (A) and exo-cleavable linkers (B)

In this design, the linker is positioned externally, bringing the payload closer to the antibody. This arrangement enhances shielding by the antibody, significantly improving hydrophilicity. Additionally, this hydrophilic, Glu-containing linker not only resists carboxylesterase Ces1C but also prevents premature payload detachment mediated by human neutrophil elastase (NE), contributing to an improved safety profile.

In vitro and in vivo evaluations showed that ADCs synthesized using the exolinker exhibited superior hydrophilic properties and significantly reduced aggregation. Further, the conjugation of the exolinker with established cytotoxic payloads such as MMAE and exatecan enhanced its potential. In summary, the introduction of exolinkers into ADCs represents a promising solution, addressing the inherent issues of traditional Val-Cit linkers while enhancing therapeutic efficacy and safety profiles.

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ChemExpress leads the field for ADC drug development providing one-stop CRO & CDMO services for both linkers, payloads and ADC conjugation. We have a substantial inventory of 80+ payloads and 400+ linkers, in addition to 1000+ successful linker compound syntheses. These include hydrophilic polymer scaffolds like PEG, polysarcosine, cyclodextrins, peptides, and polyacetals.

Notably, 12 ADC payloads and related intermediates are registered with the FDA DMF, including widely used payloads such as MMAE, Vc-MMAE, Exatecan, Eribulin, and more. Our diverse portfolio provides customers with flexible solutions. Over the past 12 months, ChemExpress has successfully completed more than 110 ADC payload-linker projects, supported clients with 5 projects in preparation for BLA submission. We also expanded our customized ADC conjugation, delivering over 80 ADC conjugate (DS) samples. With more than 10 years of experience and a team of over 230 experts, we have collaborated with more than 800 clients.

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Reference:

[1] FEBS J. 2020, 287, 1936−1969

[2] Acta Pharm. Sin. B 2023, 13, 4025−4059

[3] Clin. Cancer Res. 2004, 10, 7063−7070

[4] Mol. Cancer Ther. 2016, 15, 958−970

[5] Mol. Cancer Ther. 2017, 16, 1866−1876

[6] Bioconjugate Chem. 2021, 32, 2257−2267

[7] Chem. Sci. 2019, 10, 4048−4053

[8] Front. Pharmacol 2022, 13, 764540

[9] PLoS ONE 11(6): e0157193

[10] Mol. Cancer Ther. 2023, 22, 999−1012

[11] J. Med. Chem. 2024, 67, 18124−18138