Neutrophil elastase as a versatile cleavage enzyme for activation of αvβ3 integrin-targeted small molecule drug conjugates with different payload classes in the tumor microenvironment

Introduction: The development of bioconjugates for the targeted delivery of anticancer agents is gaining momentum after recent success of antibody drug conjugates (ADCs) in the clinic. Smaller format conjugates may have several advantages including better tumor penetration; however, cellular uptake and trafficking may be substantially different from ADCs. To fully leverage the potential of small molecule drug conjugates (SMDCs) with potent binding molecules mediating tumor homing, novel linker chemistries susceptible for efficient extracellular activation and payload release in the tumor microenvironment (TME) need to be explored. Methods: We designed a novel class of SMDCs, which target αvβ3 integrins for tumor homing and are cleaved by neutrophil elastase (NE), a serine protease active in the TME. A peptidomimetic αvβ3 ligand was attached via optimized linkers composed of substrate peptide sequences of NE connected to different functional groups of various payload classes, such as camptothecins, monomethyl auristatin E, kinesin spindle protein inhibitors (KSPi) and cyclin-dependent kinase 9 inhibitors (CDK-9i). Results: NE-mediated cleavage was found compatible with the diverse linker attachments via hindered ester bonds, amide bonds and sulfoximide bonds. Efficient and traceless release of the respective payloads was demonstrated in biochemical assays. The newly designed SMDCs were highly stable in buffer as well as in rat and human plasma. Cytotoxicity of the SMDCs in cancer cell lines was clearly dependent on NE. IC50 values were in the nanomolar or sub-nanomolar range across several cancer cell lines reaching similar potencies as compared to the respective payloads only in the presence of NE. In vivo pharmacokinetics evaluating SMDC and free payload exposures in rat and particularly the robust efficacy with good tolerability in triple negative breast and small cell lung cancer murine models demonstrate the utility of this approach for selective delivery of payloads to the tumor. Discussion: These results highlight the broad scope of potential payloads and suitable conjugation chemistries paving the way for future SMDCs harnessing the safety features of targeted delivery approaches in combination with NE cleavage in the TME.


General Procedures
All commercial reagents and catalysts were used as provided by the commercial supplier without purification.Solvents for synthesis, extraction and chromatography were of reagent grade and used as received.Moisture-sensitive reactions were carried out under an atmosphere of argon, and anhydrous solvents were used as provided by the commercial supplier. 1H NMR and 13 C NMR spectra were recorded at room temperature (RT) with Bruker Avance spectrometers.Chemical shifts (δ) are reported in ppm relative to TMS as an internal standard.
The descriptions of the coupling patterns of 1H NMR signals are based on the optical appearance of the signals and do not necessarily reflect the physically correct interpretation.In general, the chemical shift information refers to the center of the signal.In the case of multiplets, intervals are given.
Analytical mass spectrometry was performed on HPLC/MS (Waters, Agilent, Thermo Fisher) using Waters Time-of-Flight, Waters/Micromass Single Quadrupole, or Thermo Fisher Scientific Orbitrap mass spectrometers.Ionization methods were electrospray ionization (ESI) positive/negative or electron ionization (EI).
LC/MS analyses were performed using the respective method as noted.

Synthesis of Compound 2
The synthesis of Compound 2 has been described in WO2023/057813 as Compound B4b according to the scheme depicted below.

Synthesis of Compound 3
The synthesis of Compound 3 has been described in WO2023/057812 A1 as Example C48 according to the scheme depicted below.The synthesis of Compound 3a is described in the same patent application as Building Block 18.