Linkerology® - An Example of Advanced Conjugation

Linkerology® - An Example of Advanced Conjugation

Published on 20.03.2024

The targeted delivery and traceless release of drugs is an important concept in pharmacology. Discover the possibilities of payload conjugation to carrier proteins shown for the example drug Elacestrant.

For anticancer drugs, many adverse effects are known, e.g., fatigue, musculoskeletal pain, decreased appetite, abdominal pain, upset stomach, nausea, vomiting, diarrhea, constipation, headache, hot flashes, increased cholesterol and triglycerides, elevated liver enzymes (e.g., ALT and AST), decreased hemoglobin, decreased sodium, and impaired kidney function (decreased creatinine clearance). They are not only a heavy burden for the patient, but also may limit the therapy itself. It is anticipated that the targeted delivery of the drug to the tumor ameliorates many of its side effects and may allow to increase the effective dose at the tumor site.

This can be achieved by conjugating the drug to a suitable antibody or protein. FDA-approved examples for antibody-drug conjugates are Sacituzumab govitecan (Trodelvy®) for the treatment of triple negative breast cancer or Trastuzumab emtansine (Kadcycla®) against HER2-positive metastatic breast cancer.

Elacestrant (Orserdu®) is a synthetic non-steroidal antiestrogen (antagonist and degrader of the estrogen receptor). It is taken orally and comes along with the many side effects as listed above. We picked Elacestrant as a model compound for targeted delivery, as it has several options for linker attachment allowing to design antibody- and peptide-drug conjugates (ADCs or PDCs) with appropriate targeting biologics, and to construct a next generation drug with improved specificity, efficacy, and less side effects. As vehicles, appropriate antibodies, single-chain fragments, nanobodies or peptide fragments can be used for delivering the payload to the tumor site. To recover its full activity, the drug is bound to its carrier protein via a self-immolative linker warranting the drug’s traceless release after reaching the target site.


Elacestrant (Orserdu®) possesses four nucleophilic positions, with the most nucleophilic group being its secondary aliphatic amine, which can react with p-nitrophenolcarbonate activated p-aminobenzyl (PAB) linkers. The phenolic function is less nucleophilic than the aliphatic amine. Conjugation can be achieved via Mitsunobu reaction. The nucleophilicity of the aromatic amine and the methoxy groups is much lower, hence, these positions cannot be used for conjugation.


Payload attachment and immolation of the PABC:

The linker consists most commonly of valine-alanine or valine-citrulline dipeptides, which are stable during circulation in plasma but degraded by the lysosomal serine protease Cathepsin B. For the conjugation to a drug with a p-aminobenzyl carbamate (PABC), a self-immolative linker is used in many ADCs. The C-terminal p-aminobenzylalcohol is activated with p-nitrophenylchloroformate, forming a stable and still reactive linker p-nitrophenol carbamate, which readily conjugates with primary and secondary amines of the cargo drug molecule.


Phenol attachment and immolation of PABE:

If the cargo molecule bears a phenol function, the linker-PAB compound requires activation, e.g., with a phosphine (Mitsunobu or related activation technologies) and will then form a p-aminobenzyl ether (PABE). Whenever both amines and phenols are present in a molecule, as in Elacestrant, both positions can be used for conjugation. Although initiated by the same trigger, the hydrolysis of the amide bond by Cathepsin B, the kinetics of the immolation are different for PABC and PABE. This flexibility allows to fine-tune both the reactivity and the pharmacokinetics of the drug conjugate and potentially influences even efficacy.


Mechanism of self-immolation and drug release by the degradation of PABC and PABE linker-drug conjugates. In both cases, the release of the drug molecule is initiated by the hydrolysis of the amide bond by the protease Cathepsin B, followed by a 1,6-elimination, which leads to liberation of the cargo molecule in a traceless manner. Kinetics of both releases are different, depending on the steric environment and on the electron density at the point of conjugation.


Nanobody design by cell-free synthesis and attachment of the cargo drug molecule:

In contrast to monoclonal antibodies, which have shown slow blood clearance and slow and low tumor uptake, antibody fragments, such as affibodies, single-chain fragments or nanobodies, demonstrated superior behavior. Novel cell-free synthesis technologies allow the fast and easy synthesis of any fragment, including the incorporation of non-canonical amino acids, like p-azidophenylalanine, enabling direct Click conjugation of any alkyne terminated linker-payload construct. CliCr® is an ideal moiety for this purpose, as it is a small, polar, and highly reactive alkyne fragment for strain-promoted copper-free Click chemistry.


→  For more information about linker technologies, download our brochure Linkerology®!

Interested in CliCr®? Check-out our Click Chemistry brochure or watch the recording of our online-workshop about “Innovations in Click Chemistry”! 



RAD1901: a novel, orally bioavailable selective estrogen receptor degrader that demonstrates antitumor activity in breast cancer xenograft models; F. Garner, M. Shomali, D. Paquin, C. Lyttle, G. Hattersley; Anticancer Drugs 2015; 26: 948-56.

"Orserdu Product information". Union Register of medicinal products. 18 September 2023. Retrieved 1 October 2023.

FDA approves elacestrant for ER-positive, HER2-negative, ESR1-mutated advanced or metastatic breast cancer.

Immolation of p-Aminobenzyl Ether Linker and Payload Potency and Stability Determine the Cell-Killing Activity of Antibody-Drug Conjugates with Phenol-Containing Payloads; D. Zhang, H. Le, J. Cruz-Chuh, S. Bobba, J. Guo, L. Staben, C. Zhang, Y. Ma, K. Kozak, G. Lewis Phillips, B. Vollmar, J. Sadowsky, R. Vandlen, B. Wei, D. Su, P. Fan, P. Dragovich, S. Khojasteh, C. Hop, T. Pillow; Bioconjug Chem. 2018; 29: 267-274.

Protease-mediated fragmentation of p-amidobenzyl ethers: a new strategy for the activation of anticancer prodrugs; B. Toki, C. Cerveny, A. Wahl, P. Senter; J Org Chem. 2002; 67: 1866-72.

HER2-directed antibodies, affibodies and nanobodies as drug-delivery vehicles in breast cancer with a specific focus on radioimmunotherapy and radioimmunoimaging; B. Altunay, A. Morgenroth, M. Beheshti, A. Vogg, N. Wong, H. Ting, H. Biersack, E. Stickeler, F. Mottaghy; Eur J Nucl Med Mol Imaging 2021; 48: 1371-1389.

A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells; X. Liu, L. Luan, X. Liu, D. Jiang, J. Deng, J. Xu, Y. Yuan, J. Xing, B. Chen, D. Xing, H. Huang; Frontiers in immunology 2023; 14: 1292839.

Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma; M. Keyaerts, C. Xavier, J. Heemskerk, N. Devoogdt, H. Everaert, C. Ackaert, M. Vanhoeij, F. Duhoux, T. Gevaert, P. Simon, D. Schallier, C. Fontaine, I. Vaneycken, C. Vanhove, J. De Greve, J. Lamote, V. Caveliers, T. Lahoutte; J Nucl Med. 2016; 57: 27-33.

Smart chemistry for traceless release of anticancer therapeutics; C. Prange, X. Hu, L. Tang; Biomaterials 2023; 303: 122353.


Verwandte Produkte
    1. Structure image for Boc-Val-Cit-PAB-PNP

      Art-Nr.: ADC1010

      Startet von 110,00 €

    2. Structure image for Fmoc-Val-Cit-PAB-PNP

      Art-Nr.: ADC1000

      Startet von 110,00 €

    3. Structure image for Boc-Val-Cit-PAB

      Art-Nr.: ADC1020

      Startet von 190,00 €

    4. Structure image for Fmoc-Val-Cit-PAB

      Art-Nr.: ADC1030

      Startet von 190,00 €

    5. Structure image for Boc-Val-Ala-PAB

      Art-Nr.: ADC1040

      Startet von 190,00 €

    6. Structure image for Boc-Val-Ala-PAB-PNP

      Art-Nr.: ADC1050

      Startet von 125,00 €

    7. Structure image for Fmoc-Val-Ala-PAB

      Art-Nr.: ADC1060

      Startet von 190,00 €

    8. Structure image for Fmoc-Val-Ala-PAB-PNP

      Art-Nr.: ADC1070

      Startet von 110,00 €

    9. Structure image for H-Val-Ala-pNA*AcOH

      Art-Nr.: LS-3580

      Startet von 135,00 €

    10. Structure image for Mal-Dap(Boc)-Val-Cit-PAB-PNP

      Art-Nr.: ADC1090

      Startet von 250,00 €

    11. Structure image for Mal-Dap(Boc)-Val-Ala-PAB-PNP

      Art-Nr.: ADC1080

      Startet von 500,00 €

    12. Structure image for 6-Azidohexanoyl-Val-Cit-PAB

      Art-Nr.: ADC1120

      Startet von 325,00 €

    13. Structure image for 6-Azidohexanoyl-Val-Cit-PAB-PNP

      Art-Nr.: ADC1130

      Startet von 375,00 €

    14. Structure image for Azido-PEG(4)-Val-Cit-PAB

      Art-Nr.: ADC1160

      Startet von 450,00 €

    15. Structure image for Azido-PEG(4)-Val-Cit-PAB-PNP

      Art-Nr.: ADC1170

      Startet von 500,00 €

    16. Structure image for 4-Pentynoyl-Val-Cit-PAB

      Art-Nr.: ADC1140

      Startet von 325,00 €

    17. Structure image for 4-Pentynoyl-Val-Cit-PAB-PNP

      Art-Nr.: ADC1150

      Startet von 375,00 €

    18. Structure image for Alkyne-PEG(5)-Val-Cit-PAB

      Art-Nr.: ADC1180

      Startet von 450,00 €

    19. Structure image for Alkyne-PEG(5)-Val-Cit-PAB-PNP

      Art-Nr.: ADC1190

      Startet von 500,00 €

    20. Structure image for Fmoc-Val-Cit-PAB-NMeCH2CH2NMe-Boc

      Art-Nr.: ADC1240

      Startet von 175,00 €

    21. Structure image for MC-Val-Cit-PAB

      Art-Nr.: ADC1100

      Startet von 300,00 €

    22. Structure image for MC-Val-Cit-PAB-PNP

      Art-Nr.: ADC1110

      Startet von 175,00 €

    23. Structure image for Mal-PEG(4)-Val-Cit-PAB

      Art-Nr.: ADC1200

      Startet von 500,00 €

    24. Structure image for Mal-beta-Ala-PEG(4)-Val-Cit-PAB

      Art-Nr.: ADC1220

      Startet von 450,00 €

    25. Structure image for Mal-beta-Ala-PEG(4)-Val-Cit-PAB-PNP

      Art-Nr.: ADC1230

      Startet von 500,00 €

    26. Structure image for 6-Azidohexanoyl-Val-Ala-PAB

      Art-Nr.: ADC1290

      Startet von 325,00 €

    27. Structure image for 6-Azidohexanoyl-Val-Ala-PAB-PNP

      Art-Nr.: ADC1300

      Startet von 375,00 €

    28. Structure image for Azido-PEG(4)-Val-Ala-PAB

      Art-Nr.: ADC1330

      Startet von 450,00 €

    29. Structure image for Azido-PEG(4)-Val-Ala-PAB-PNP

      Art-Nr.: ADC1340

      Startet von 500,00 €

    30. Structure image for 4-Pentynoyl-Val-Ala-PAB

      Art-Nr.: ADC1310

      Startet von 325,00 €

    31. Structure image for 4-Pentynoyl-Val-Ala-PAB-PNP

      Art-Nr.: ADC1320

      Startet von 375,00 €

    32. Structure image for Alkyne-PEG(4)-Val-Ala-PAB

      Art-Nr.: ADC1350

      Startet von 450,00 €

    33. Structure image for Alkyne-PEG(4)-Val-Ala-PAB-PNP

      Art-Nr.: ADC1360

      Startet von 500,00 €

    34. Structure image for Fmoc-Val-Ala-PAB-NMeCH2CH2NMe-Boc

      Art-Nr.: ADC1410

      Startet von 175,00 €

    35. Structure image for MC-Val-Ala-PAB-PNP

      Art-Nr.: ADC1280

      Startet von 175,00 €

    36. Structure image for Mal-beta-Ala-PEG(4)-Val-Ala-PAB

      Art-Nr.: ADC1390

      Startet von 450,00 €

    37. Structure image for Mal-beta-Ala-PEG(4)-Val-Ala-PAB-PNP

      Art-Nr.: ADC1400

      Startet von 500,00 €

    38. Structure image for Boc-Val-Ala-PAB-Cl

      Art-Nr.: ADC1660

      Startet von 750,00 €

    39. Structure image for Fmoc-Val-Ala-PAB-Cl

      Art-Nr.: ADC1670

      Startet von 750,00 €

    40. Structure image for 6-Azidohexanoyl-Val-Ala-PAB-Cl

      Art-Nr.: ADC1680

      Startet von 450,00 €

    41. Structure image for 4-Pentynoyl-Val-Ala-PAB-Cl

      Art-Nr.: ADC1690

      Startet von 450,00 €

    42. Structure image for MC-Val-Ala-PAB-Cl

      Art-Nr.: ADC1700

      Startet von 500,00 €

    43. Structure image for Azido-PEG(4)-Val-Ala-PAB-Cl

      Art-Nr.: ADC1710

      Startet von 500,00 €

    44. Structure image for Alkyne-PEG(4)-Val-Ala-PAB-Cl

      Art-Nr.: ADC1720

      Startet von 500,00 €

    45. Structure image for CliCr® base compound
      CliCr® base compound

      Art-Nr.: RL-4180

      Startet von 125,00 €

    46. Structure image for CliCr®-beta-Ala-NH2*TFA

      Art-Nr.: RL-4190

      Startet von 150,00 €

    47. Structure image for CliCr®-Suc

      Art-Nr.: RL-4200

      Startet von 150,00 €

    48. Structure image for CliCr®-OSu

      Art-Nr.: RL-4330

      Startet von 150,00 €

    49. Structure image for Mal-PhAc-Val-Ala-PAB

      Art-Nr.: ADC1730

      Startet von 300,00 €

    50. Structure image for Mal-PhAc-Val-Ala-PAB-PNP

      Art-Nr.: ADC1740

      Startet von 400,00 €

    51. Structure image for Mal-PhAc-Val-Cit-PAB

      Art-Nr.: ADC1750

      Startet von 350,00 €

    52. Structure image for Mal-PhAc-Val-Cit-PAB-PNP

      Art-Nr.: ADC1760

      Startet von 450,00 €

    53. Structure image for Mal-PhAc-PEG(4)-Val-Ala-PAB

      Art-Nr.: ADC1770

      Startet von 500,00 €

    54. Structure image for Mal-PhAc-PEG(4)-Val-Ala-PAB-PNP

      Art-Nr.: ADC1780

      Startet von 600,00 €