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Background Phosphatidylserine (PS) is abnormally externalized on tumor cells and vasculature. Persistent PS externalization has multiple immunomodulatory effects, such as exhaustion of T-cells, suppression in dendritic cell activation, promotion of M2 macrophage polarization, and activation of regulatory T-cells. 1 Here we present a novel protein drug conjugate (PDC) that exploits PS externalization in cancer. This PDC consists of the relatively small (36 kDa) recombinant human protein Annexin A5 (ANXV) and a chemotherapeutic agent (chemo). ANXV binds to PS with high affinity and is internalized efficiently. As a mono molecule, ANXV is a powerful immune checkpoint inhibitor after chemotherapy.2 The ANXV PDC is intended for targeted delivery of the chemotherapy to the tumor cells and vasculature. The immunomodulatory effect of ANXV itself is expected to minimize the immunosuppressive effects of chronic PS externalization.Methods Internalization, cytotoxic, and anti-proliferative effects of the ANXV-chemo conjugate were investigated in vitro using PSpositive human triple-negative breast cancer (TNBC) cell line (MDA-MB-231) utilizing Incucyte® live cell monitoring. In vivo anti-tumor effects and pharmacokinetics of the ANXV-chemo conjugate were evaluated in an immunodeficient NXG mouse model bearing orthotopic MDA-MB-231 tumors. Mice were treated IP and/or IV once every three days with 0-5 mg/kg ANXV-chemo. After 4-6 treatments, mice were sacrificed. Tumors, kidneys, livers, and lungs were collected for histopathologic examination.Results ANXV was rapidly internalized as observed in vitro over 48 hours. The ANXV-chemo conjugate was nearly 30 times more cytotoxic on MDA-MB-231 than the free chemo, as indicated by the EC50 after 72 hours. In vivo, ANXV-chemo was detectable in plasma of tumor-bearing mice in a dose-proportional manner. For mice with larger tumors (~120 mm3) at the start of treatment, 5 mg/kg ANXV-chemo reduced tumor volume by 12.4% after one treatment. After two treatments (1 IP and 1 IV), these larger tumors had significantly smaller tumor volumes than the untreated control. Additionally, 2 and 5 mg/kg ANXV-chemo significantly reduced lung weight compared to the untreated control, suggesting the PDC’s ability to prevent lung metastasis. Tumor histology revealed extensive vascular necrosis after the ANXV-chemo treatments. When PDC was administered IP, dose-limiting effects were observed in the 5 mg/kg treatment group as indicated by mouse posturing and liver histology.Conclusions These preclinical results demonstrate ANXV’s ability to deliver chemotherapeutic agents to tumor cells and tumor vasculature via PS targeting. Further work will focus on dosage optimization and immune profiling within the tumor microenvironment.References Pulica R, Aquib A, Varsanyi C, et al. Dys-regulated phosphatidylserine externalization as a cell intrinsic immune escape mechanism in cancer. Cell Commun Signal. 2025;23:131.Kang TH, Park JH, Yang A, et al. Annexin A5 as an immune checkpoint inhibitor and tumor-homing molecule for cancer treatment. Nat Commun. 2022;11:1137.Ethics Approval All procedures and experiments performed on animals were in accordance with regulations and guidelines approved by the local ethics committee.