Antibody-encapsulated actinomycin D for targeted cancer treatments.

Abstract

e13019 Background: HER2 is overexpressed in 15-20% of breast cancers and other cancers, and HER2-targeted therapies have revolutionized cancer treatment in the past 20 years. Trastuzumab (Tr) has demonstrated efficacy on HER2+ breast cancer and advanced gastric cancer. Recently, Tr-based antibody drug conjugates (ADCs), trastuzumab emtansine and trastuzumab deruxtecan, have emerged as a new class of anti-HER2 therapies by combining targeted antibodies with cytotoxic agents via linkers. However, ADC approach faces great challenges, in vivo instability, complicated manufacturing, limited cytotoxic payloads and suboptimal payload release, etc. Smarter and better designs are urgently needed. Methods: Our patented single protein encapsulation (SPE) platform, allowing encapsulation of small-molecule drugs by a single protein (albumins or globulins) without artificial nanoparticles and chemical modifications to drugs and proteins, has achieved great success in development of 2 drug products, SPEDOX-6 under clinical trial (NCT0764018) and SPESN38-8 (IND #: 164346) under IND-enabling study based on albumin, which have prompted us to utilize antibody, such as Tr, to encapsulate cytotoxic payload, like actinomycin D (ACT), forming antibody encapsulated drugs (AEDs). We have successfully developed Tr-ACT2 as a first-in-class AED drug, featuring each Tr molecule to encapsulate two ACT molecules without linkers. Tr-ACT2 was well characterized by UV, fluorescence, membrane dialysis, particle size distribution, and molecular docking. In vitro and in vivo anticancer efficacy of Tr-ACT2 against various HER2+ & HER2- cancers were evaluated. Results: Tr-ACT2 has been investigated in following. In vitro cytotoxicity of Tr-ACT2 against human breast cancers, HER2+ (SKBR3, JIMT1) and HER2- (BT549, MDA-MB-231) and A549 (NSCLC, HER2-), was evaluated, leading to a time-dependent and significant reduction in cell viability. Notably, Tr-ACT2's cytotoxic activity appeared to be independent of HER2 levels. Internalization study on Tr-ACT2 demonstrated that early-stage internalization of Tr-ACT2 is HER2 dependent, but rate of late-stage internalization of Tr-ACT2 is not limited by HER2 levels, confirming that Tr-ACT2 could be effectively internalized into both HER2+ and HER2- cancer cells. In vivo anticancer efficacy of Tr-ACT2 at 1 mg/kg vs irinotecan at 50 mg/kg using A549 mouse model has been evaluated, showing that Tr-ACT2 was significantly more effective in suppressing growth of A549 than irinotecan. Conclusions: We successfully developed first-in-class AED nanocomplex, Tr-ACT2, as a potent anticancer agent, demonstrating that the SPE technology can be applied to monoclonal antibody for encapsulating small-molecule drugs without covalent conjugation and marking a significant advancement in antibody-based therapeutics. Newly-created AEDs have great potentials for not only replacing, but also expanding ADC approaches due to SPE’s significant advantages.