The cytostatic antibody-drug-conjugate (ADC) Brentuximab-Vedotin (Adcetris®, Seattle Genetics) has been approved in 2011 for the treatment of CD30⁺ Hodgkin’s lymphoma and systemic anaplastic large cell lymphoma (ALCL). This drug is composed of a monoclonal antibody, which specifically binds to the tumor antigen CD30, conjugated to four molecules of monomethyl auristatin E (MMAE) via a self-immolative linker. The protease Cathepsin B cleaves this linker and liberates MMAE at the tumor site. Adcetris® is the first FDA-approved ADC and pioneered a bioconjugation concept which has been followed by many other examples. All of them consist of:

• a monoclonal antibody which is highly specific towards the target tissue or cells;
• a linker consisting of a reactive fragment, which is stable during circulation in the blood, but is cleaved after internalization by virtue of certain enzymes or a changed chemical environment, e.g., pH. Most popular are peptidic substrates, which undergo amide bond hydrolysis in the presence of proteases, and disulfides, which are cleaved under reductive conditions in the cytosol in the presence of glutathione (GSH) and other reducing agents;
  
  

Linker sequences which are known substrates for the cathepsin proteases. (*) “C” normally stands for cysteine within the peptide community. However, in the ADC literature it is common to abbreviate citrulline with “C”.

In 2020, Merck and Daiichi Sankyo have developed a novel, HER3 directed, experimental antibody-drug conjugate named Patritumab-Deruxtecan (U3-1402/MK-1022) for the treatment of patients with EGFR-mutated non-small-cell lung cancer and with HER3-positive metastatic breast cancer. Many other ADCs which are in clinical trials or on the market have copied the Val-Cit-PAB motif as in Adcetris®, sometimes exchanging citrulline by alanine. Patritumab-Deruxtecan is the first ADC which is using the tetrapeptide GGFG.

Adcetris® (Brentuximab-Vedotin) contains a valyl-citrullyl (Val-Cit) dipeptide which is followed by a p-aminobenzyl (PAB) moiety. Proteolytic hydrolysis leads to an aniline derivative of the effector molecule which then suffers 1,6-elimination and fragmentation to release 4-methylene-2,5-cyclohexadiene-1-imine, carbon dioxide, and the drug molecule MMAE.

Patritumab-Deruxtecan carries the tetrapeptide Gly-Gly-Phe-Gly, which is followed by the aminal of formaldehyde. Proteolytic hydrolysis leads to a hemiaminal which fragmentizes to formaldehyde and ammonia with the traceless release of the drug molecule Deruxtecan.

The payload release from a peptidyl-PAB-DMAE moiety happens initially by 1,6-elimination, yielding a free methylamine terminus, which then breaks the carbamate bond to the payload alcohol via the formation of a cyclic urea.

p-Aminobenzyl (PAB) is a very common fragmentation motif that is liberated after proteolytic hydrolysis of the corresponding peptide substrate. However, PAB can only be conjugated to a payload with a suitable amino group, as it is the case in MMAE. Several payloads exist which have alcohols (hydroxyl groups), such as Camptothecin, Doxorubicin, Dexamethasone, or Selumetinib. Direct conjugation of -OH terminated payloads to PAB will result in a carbonate moiety, which is not sufficiently stable while the drug is circulating in the plasma. This bottleneck has been overcome by inserting N,N’‑dimethylaminoethyl (DMAE) carbamate between PAB and the payload. However, potential drawbacks of the PAB-DMAE linker system are that the fragmentation happens in a two-step process delaying the release kinetics, that the synthesis of such constructs is more complex, and that the risk of immunogenicity is higher than in simpler constructs.

In 2022, phase I escalation studies of Patritumab-Deruxtecan have been published with promising results.

→  Read more about linker technologies in our Linkerology® brochure.

→  Interested in the custom synthesis of a linker or payload-linker conjugates? Please talk to us!

References:

Linker Technologies for Antibody–Drug Conjugates; B. Nolting; Antibody-Drug Conjugates; L. Ducry 2013; Springer Protocols 1045: 71-100. https://doi.org/10.1007/978-1-62703-541-5_5.

Efficacy and Safety of Patritumab Deruxtecan (HER3-DXd) in EGFR Inhibitor-Resistant, EGFR-Mutated Non-Small Cell Lung Cancer; P. A. Janne, C. Baik, W. C. Su, M. L. Johnson, H. Hayashi, M. Nishio, D. W. Kim, M. Koczywas, K. A. Gold, C. E. Steuer, H. Murakami, J. C. Yang, S. W. Kim, M. Vigliotti, R. Shi, Z. Qi, Y. Qiu, L. Zhao, D. Sternberg, C. YuH. A. Yu; Cancer Discov. 2022; 12(1): 74-89. https://doi.org/10.1158/2159-8290.CD-21-0715

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 Pharmaceutica Sinica B 2021; 11(12): 3889-3907. https://doi.org/10.1016/j.apsb.2021.03.042

Antibody-drug conjugates for non-oncological indications; R. Liu, R. E. Wang, F. Wang; Exp. Opin. Biol. Ther. 2016; 16(5): 591-593. https://doi.org/10.1517/14712598.2016.1161753