Products

Valine-Alanine-Based Enzymatically Cleavable Linkers

Peptidic bonds are expected to have a high serum stability, as lysosomal proteolytic enzymes show

reduced activities in blood due to endogenous inhibitors and the unfavorably high pH value of blood

compared to lysosomes. This was confirmed by preclinical in vivo studies which revealed half-lives of

seven to ten days for peptide linkers. Release of a drug conjugated via a peptidyl linker to monoclonal

antibodies (mAb) occurs specifically due to the action of lysosomal proteases (e.g., cathepsin and plasmin)

. These proteases may be present at even elevated levels in certain tumor tissues. Therefore, peptide linkers

combine greater systemic stability with rapid enzymatic release of the drug in the target cell. Besides Val-

Ala, Val-Cit and Phe-Lys, other sequences have been reported as lysosomally cleavable peptides, like Gly-

Phe-Leu-Gly and Ala-Leu-Ala-Leu.

Figure 1: Cyclobutane-1,1-dicarboxamide can replace valine in dipeptide linker systems, resulting in

improved ADC selectivity.

Peptide-based ADC linkers, like Val-Cit or Val-Ala, that are cleaved by lysosomal proteases have shown

sufficient stability in serum and effective payload-release in targeted cells. However, the use of peptide-

based linkers limits the ability to modulate protease specificity. Furthermore, if the linker can preferentially be

hydrolyzed by tumor-specific proteases only, safety margin may improve. In this context, a cyclobutane-

1,1-dicarboxamide-containing linker replacing valine in other sequences has been invented which is

hydrolyzed predominantly by cathepsin B, while the typical valine-citrulline dipeptide linker is rather less.

ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker.

Hence, the application of the peptidomimetic linker presents new opportunities for improving the selectivity of

ADCs.

6-Azidohexanoyl-Val-Ala-PAB

6-Azidohexanoyl-Val-Ala-PAB-PNP

Azido-PEG(4)-Val-Ala-PAB

Azido-PEG(4)-Val-Ala-PAB-PNP

4-Pentynoyl-Val-Ala-PAB