Novel protein drug conjugate ANXV-chemo and cytotoxic and anti-proliferative effects against human triple-negative breast cancer in vitro.
Authors: Woodward Alexis Anne , Potrzebowska Katarzyna , Suchdev Susan , Thibault Alain , Frostegård Anna , Haegerstrand Anders 
Published: June 2024

e15024 Background: Targeted delivery of cytotoxic drugs to tumor cells and tumor microenvironment remains a fundamental challenge in the fight against cancer including triple-negative breast cancer (TNBC). The nonapoptotic externalization of the phospholipid phosphatidylserine (PS) on cancer cells is an attractive biomarker. Cancer cells have up to 90-fold more PS molecules externalized than healthy cells, and TNBC cells have high PS externalization. The Annexin A5 human protein has a high affinity to PS and has shown anti-cancer effects both in vitro and in vivo. Here we utilize the recombinant protein Annexin A5 (ANXV), currently in Phase 2a as a treatment of retinal vein occlusion, as a delivery vehicle to deliver a covalently linked chemotherapy (chemo) to TNBC cells via externalized PS. Methods: Chemo was conjugated to ANXV through a one-step SMCC crosslinking reaction. ANXV-chemo was analyzed via LC-MS and the weighted average of the drug-to-ANXV ratio (DAR) was determined. MDA-MB-231 TNBC cells were utilized for internalization, cytotoxic, and proliferation studies. Incucyte was used to monitor live cells for up to 96 hours (samples tested in quadruplicate). For internalization studies, ANXV-pHrodo red was incubated with the cells, and the fluorescent intensity was measured over 48 hours. For the cytotoxic and proliferation studies, ANXV-chemo (0-100 nM), sacituzumab govitecan-hziy (SG) (0-100 nM), or thefree chemo (0-200nM) were incubated with the cells for up to 96 hours. All concentrations refer to the amount of drug linked to the conjugates or free drug. All data analysis was completed in GraphPad Prism (10.1.1). Results: ANXV-chemo was successfully crosslinked and had a DAR of 2.4 ± 0.23 chemo molecules. ANXV was quickly internalized, and ANXV could be detected within the cells for 48 hours after initial incubation, indicating constant cell uptake of the protein. The ANXV-chemo was nearly 30 times more cytotoxic to the TNBC cells than the free chemo as indicated by the EC50 after 72 hours (ANXV-chemo = 1.2 nM; free chemo = 36.7 nM). When comparing equal molar drug concentrations (4 nM), ANXV-chemo had more significant anti-proliferative and cytotoxic effects than the standard of care SG. By 24 hours, ANXV-chemo significantly increased cell death (p < 0.0021). By 48 hours, ANXV-chemo significantly decreased proliferation (p < 0.0332). After 96 hours, ANXV-chemo inhibited proliferation by 5.5 times (p < 0.0001) and induced 6.5 times more cell death (p < 0.0002) when compared to SG. Conclusions: By covalently linking chemo to ANXV, an efficient and PS-targeted treatment for TNBC has been developed. ANXV-chemo demonstrated significantly better anticancer effects in vitro when compared to the free drug and current targeted standard of care treatments. An investigation of ANXV-chemo as a targeted anticancer agent is currently underway in a murine TNBC model.