Even though certain drugs can directly be attached to their carrier via their functional groups, linkers represent versatile tools for highly efficient conjugation. A well-chosen and designed linker as connective handle may allow to increase overall solubility, improve stability thus preventing premature drug release, and facilitate the release of the active drug at its target site. Thus, conjugating highly potent small molecules to vastly target specific biomolecules, such as antibodies, via a selected linker has become a frequently used strategy, particularly in the field of cancer therapy, enlarging the therapeutic window, thus increasing the amount of dosed drug reaching the target cell.

Typical mode of action of an ADC.

To fully suit the application’s needs, various linker systems have emerged, including permanent linkers as well as chemically or enzymatically cleavable ones. Amongst the latter ones, peptide linkers exert enhanced serum stability as their cleavage relies on lysosomal proteolytic enzymes that have very low activity in blood circulation while being overexpressed in certain tumor tissues.

First-generation peptide linkers include tetrapeptides such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu, which showed relatively slow drug release and a tendency for aggregation upon payload coupling. These limitations were circumvented by the development and intense investigation of dipeptide linkers.

Besides the peptidic core, additional parts can be added, e.g. to accelerate the drug release via self-immolation or to improve solubility by incorporation of a PEG moiety. The following scheme illustrates the cleavage of a Valine-Citrulline linked para-aminobenzyl alcohol self-immolative linker which triggers a 1,6-elimination of carbon dioxide and concomitant release of the free drug in its parent amine form.

Valyl-citrullyl dipeptide fragment serves as substrate for catephsin and undergoes cleavage by hydrolysis leading to a 1,6-elimination with fragmentation and traceless release of the drug molecule.

In a comparative study of dipeptide linkers (Debowchik et al.), the rate of doxorubicin release was measured by enzymatic hydrolysis. They report that Val-Cit shows the highest stability with a half-life of 240 min in contrast to Phe-Lys which was cleaved most rapidly with a half-life of 8 min.

Just like Val-Cit, Val-Ala is effectively cleaved by lysosomal proteolytic enzymes while being highly stable in human plasma. In an isolated cathepsin B-cleavage assay, the Val-Ala linker was cleaved at half the rate of the Val-Cit linker, but it also exhibited lower hydrophobicity, a trait that prevents aggregation and precipitation and represents one of its key advantages.

➔ Please see a selection of our related Val-Cit and Val-Ala linkers listed below! Interested in various linker lengths? Get in contact for your customized linker(s) of choice!

➔ Interested in more information on advanced linker technologies? See our Linkerology®* Booklet* which is available as free download on our homepage.

* Linkerology is a registered trademark of Iris Biotech GmbH.

References:

• Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry; J. R. McCombs, S. C. Owen; AAPS J. 2015; 17(2): 339-351. https://doi.org/10.1208/s12248-014-9710-8.

• Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. G. M. Dubowchik, R. A. Firestone, L. Padilla, D. Willner, S. J. Hofstead, K. Mosure, J. O. Knipe, S. J. Lasch, P. A. Trail; Bioconjugate Chem. 2002; 13(4): 855-869. https://doi.org/10.1021/bc025536j.

• Antibody-drug conjugates: Recent advances in linker chemistry; Z. Su, D. Xiao, F. Xie, L. Liu, Y. Wang, S. Fan, X. Zhou, S. Li; Acta Pharm. Sinica B 2021; https://doi.org/10.1016/j.apsb.2021.03.042.

• Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers; P. L. Salomon, E. E. Reid, K. E. Archer, L. Harris, E. K. Maloney, A. J. Wilhelm, M. L. Miller, R. V. J. Chari, T. A. Keating, R. Singh; Mol. Pharmaceutics 2019; 16(12): 4817-4825. https://doi.org/10.1021/acs.molpharmaceut.9b00696.

• Cleavable linkers in antibody-drug conjugates; J. D. Bargh, A. Isidro-Llobet, J. S. Parker, D. R. Spring; Chem. Soc. Rev. 2019; 48: 4361-4374. https://doi.org/10.1039/C8CS00676H.