Abstract 2722: Synthesis and characterization of novel small molecule drug conjugates with different payloads designed to be released in tumor microenvironment by neutrophil elastase
Authors: Lerchen Hans-Georg , Rebstock Anne-Sophie , Wiedmann Marieke , Stelte-Ludwig Beatrix , Wong Harvey , Zubov Dmitry , Izumi Raquel , Hamdy Ahmed 
Published: April 2023

Abstract
The development of small molecule drug conjugates (SMDCs) is gaining momentum after the 2018 approval of the small molecule radioconjugate lutetium-177 vipivotide tetraxetan. In contrast to the use of antibodies for tumor homing, small molecule binding ligands may result in a better tumor penetration. Also the potential targets for ADCs are limited to internalizing antigens so the ADC can be trafficked to the lysosomes for release and activation of the payload. We sought new mechanisms for extracellular activation of SMDCs using enzymes, which are present in the tumor microenvironment (TME). For example, neutrophil elastase (NE) belongs to a family of serine proteases that degrades elastin and other extracellular matrix proteins. NE also contributes to cancer progression by enhancing tumor evasion and metastasis. NE expression and neutrophil tumor infiltration have been correlated with metastatic potential and poor prognosis. Previous studies have shown that NE efficiently cleaves and activates camptothecin conjugates by cleaving an ester bond1.
Here we describe the successful use of NE for efficient activation and traceless payload release from several unique and proprietary SMDCs. Binding molecules such as a peptidomimetic αvβ3 ligand were linked via appropriate spacers to substrate tripeptides of NE. The C-terminal end was directly attached to different functional groups at various payloads such as camptothecins (CPT), monomethyl auristatin E, kinesin spindle protein inhibitors, and CDK9 inhibitors. Hindered ester bonds, amide bonds, and sulfoximide bonds were found to be efficiently cleaved by NE; conversely, those linkages were highly stable in culture medium and in circulation. Consequently, respective SMDCs are not or are moderately cytotoxic when incubated alone with cancer cell lines. However, in the presence of NE, cytotoxicity of the SMDCs increases by two orders of magnitude to the low nanomolar or subnanomolar range across several cell lines reaching similar potency as the respective payloads alone. In PK studies in mouse, rat, and dog, the SMDC with a CPT payload, shows a half-life of 3.6, 8.3, and 4.2 h, respectively, and excellent stability in circulation over 24 hr with low exposure levels (&lt;1% of SMDC) of cleaved payload detectable. In an MX1 triple-negative breast cancer xenograft model, tumor regressions were observed when the SMDC was administered in a 2 days on/5 days off schedule.
NE is capable of cleaving sterically hindered ester bonds, amides, and sulfoximides to release a variety of potent payloads from different SMDCs in a traceless manner. These molecules represents a versatile strategy for selective delivery of payloads to TME that does not require the tumor target to internalize.
1Lerchen HG, Stelte-Ludwig B, Kopitz C, et al. Cancers 2022
Citation Format: Hans-Georg Lerchen, Anne-Sophie Rebstock, Marieke Wiedmann, Beatrix Stelte-Ludwig, Harvey Wong, Dmitry Zubov, Raquel Izumi, Ahmed Hamdy. Synthesis and characterization of novel small molecule drug conjugates with different payloads designed to be released in tumor microenvironment by neutrophil elastase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2722.