The goal of targeted tumor therapy is to eliminate the diseased cells efficiently with minimal side effects to healthy tissue.  Antibody-Drug-Conjugates are the method of choice to prevent such off-target effects and enrich cytotoxic medications at the desired site of action via selective release of the active drug. Once the antibody reaches its target, internalization of the receptor brings the conjugate inside the cell.

Various linker types for conjugation are available on the market, responding to and being – ideally tracelessly – cleaved by virtue of a certain trigger, e.g., peptidic linkers are cleaved in the presence of specific enzymes.

Thus, conditions solely present in the affected tissue need to be selected to avoid premature drug release. Compared to healthy cells, abnormal metabolism and proliferation in tumor cells lead to a lowered intracellular pH of around 5.0-6.0, while a lower pH (4.0-5.0) is reported for the lysosomes.

In today’s blog we introduce our novel pH-sensitive linker ADC1800 based on an alkyne-functionalized, para-nitrophenyl (PNP)-modified 5-(hydroxymethyl)-pyrogallol-orthoester (HMPO). 

Chemical structures of the pH-sensitive self-immolative alkyne-HMPO-PNP linker (ADC1800) and its precursor alkyne-HMPO-OH (ADC1790).

This construct is stable during circulation in plasma (tested for 24 hours at pH values of 7.4 and 6.6.) while being cleaved tracelessly via 1,6-elimination at pH 5.5. Payloads can be easily coupled: The HMPO-PNP linker ADC1800 reacts with base-activated amides while releasing the nitrophenyl residue.

Coupling of payload and linker. Reaction conditions: The drug (depicted in red) is dissolved in THF at
-78°C. The strong base potassium hexamethyldisilyzide (KHMDS) and then ADC1800 are added. For the workup, the temperature is allowed to rise to -5 °C over 4 hours. Ammonium chloride is added to quench excess linker, then the product is extracted and purified by chromatography.

While the payload is attached as carbamate, the connection to the carrier protein (usually a tumor-specific antibody) is realized via the propargyl residue through biorthogonal CuAAC click reaction, forming a 1,2,3-triazole. For this, the antibody must carry azido groups, which may be introduced via recombinant biosynthesis, utilizing pyrrolysyl-tRNA synthetase and a suitably modified lysine (e.g., HAA2080), or with an amino-reactive azido linker like, e.g., PEG1400 or RL-2980.

Combination of antibody and drug-linker adduct to generate the antibody-drug-conjugate (ADC).

At acidic pH, the linker gets protonated inducing a 1,6-elimination reaction. The oxocarbonyl moiety is released as carbon dioxide, leading to the irreversible and traceless linker cleavage and payload release.

Payload release at acidic pH in the tumor microenvironment.

While ADC1800 as the activated form of the linker reacts directly with the amide-containing drug molecule, we also offer the precursor ADC1790 (Alkyne-HMPO-OH) which can be further customized with your linker of choice.

→ Get in contact to inquire about or custom synthesis service! 

→ Download our brochure Linkerology® and discover our portfolio of self-immolative linker for ADCs and other linkers for conjugation.

→ For more information about Click Chemistry, see our booklet! 

References:

A pH-responsive crosslinker platform for antibody-drug conjugate (ADC) targeting delivery; F. Migliorini, E. Cini, E. Dreassi, F. Finetti, G. Ievoli, G. Macri, E. Petricci, E. Rango, L. Trabalzini, M. Taddei; Chem. Commun. 2022; 58(75): 10532-10535. https://doi.org/10.1039/D2CC03052G

A Self-Immolative Linker for the pH-Responsive Release of Amides; A. Petrini, G. Ievoli, F. Migliorini, M. Taddei, S. Siciliano; Molecules 2023; 28(6): 2445. https://doi.org/10.3390/molecules28062445

Conjugates of PSMA-binding moieties with cytotoxic agents; WO2024028258.

Compositions and methods for treatment of sexual dysfunction and related diseases, disorders, and conditions; WO2022251699.