Preclinical in vitro and in vivo evaluation of novel ANXV-chemo protein drug conjugate against human triple-negative breast cancer.

e15016 Background: The elevated nonapoptotic exposure of the phospholipid phosphatidylserine (PS) on cancer cells and tumor vasculature provides a unique site for the targeted delivery of chemotherapy agents. Annexin A5 is a human protein that binds to PS with a high affinity and has also shown anticancer immunomodulating effects in vitro and in vivo. Here we present a novel protein drug conjugate consisting of recombinant human Annexin A5 (ANXV) and a proprietary chemotherapy agent (chemo). The anticancer effects of the ANXV-chemo conjugate were tested in vitro and in vivo utilizing a syngeneic triple-negative breast cancer (TNBC) murine model. Methods: In vitro cytotoxic and anti-proliferative analysis of ANXV-chemo was tested against MDA-MB-231 TNBC cells utilizing Incucyte live cell monitoring for up to 96 hours. Cells were incubated with ANXV-chemo (0-100 nM) or thefree chemo (0-200nM) (n = 4). In vivo, the anticancer effect of the ANXV-chemo conjugate was evaluated in the immunodeficient NXG mouse model bearing orthotopic MDA-MB-231 cells. Mice were treated once every 3 days with 0, 2, or 5 mg/kg ANXV-chemo once the tumors reached 50-100 mm 3 (n = 7) or once the tumors reached 150 mm 3 (n = 3). After 4-6 treatments mice were sacrificed and tumors, kidneys, livers, and lungs were collected for further histopathologic examination. Results: The ANXV-chemo was significantly 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 tested in vivo, larger tumors benefited more from the ANXV-chemo treatment. When tumors were at least 150 mm 3 at the start of treatment, an immediate reduction in tumor volume was observed after 1 treatment in the 5 mg/kg ANXV-chemo group. Both 2 and 5 mg/kg resulted in vascular necrosis as indicated by tumor histology. Dose-limiting side effects were observed after 5 treatments of the 5 mg/kg ANXV-chemo as demonstrated by mouse posturing and effects on liver (histology). Mouse body weight was not affected by ANXV-chemo treatment and treatment did not affect the kidneys as indicated by histological analysis. Conclusions: The novel ANXV-chemo protein drug conjugate exploits TNBC and tumor vasculature upregulated PS externalization. ANXV-chemo demonstrated superior cytotoxic effects in vitro and reduced tumor volume after 1 treatment for larger tumors. Further treatment optimization for the ANXV-chemo conjugate is ongoing.