Immunogenic Treatment for Metastatic Breast Cancer Using Targeted Carbon Nanotube Mediated Photothermal Therapy in Combination with Anti-PD-1.

The high prevalence of breast cancer is a global health concern, but there are no safe or effective treatments for it at its advanced stages. These facts urge the development of novel treatment strategies. Annexin A5 (ANXA5) is a natural human protein that binds with high specificity to phosphatidylserine, a phospholipid tightly maintained in the inner leaflet of the cell membrane on most healthy cells but externalized in tumor cells and the tumor vasculature. Here, we have developed a targeted photosensitizer for photothermal therapy (PTT) of solid tumors through the functionalization of single walled carbon nanotubes (SWCNTs) to ANXA5-the SWCNT-ANXA5 conjugate. The ablation of tumors through the SWCNT-ANXA5-mediated PTT synergizes with checkpoint inhibition, creating a systemic anti-cancer immune response. In vitro ablation of cells incubated with the conjugate promoted cell death in a dose-dependent and targeted manner. This treatment strategy was tested in vivo with the orthotopic EMT6 breast tumor model in female balb/cJ mice. Enhanced therapeutic effects were achieved by using intratumoral injection of the conjugate and treating tumors at a lower PTT temperature (45oC). Intratumoral injection prevented the accumulation of the SWCNTs in major clearance organs. When combined with checkpoint inhibition of anti-PD-1, SWCNT-ANXA5-mediated PTT increased survival and 80% of the mice survived for 100 days. Evidence of immune system activation by flow cytometry of splenic cells strengthens the hypothesis of an abscopal effect as a mechanism of prolonged survival. Significance Statement This study demonstrated a relatively high survival rate (80% at 100 days) of mice with aggressive breast cancer when treated with photothermal therapy using the SWCNT-ANXA5 conjugate injected intratumorally and combined with immune stimulation using the anti-PD-1 checkpoint inhibitor. Photothermal therapy was accomplished by maintaining the tumor temperature at a relatively low level of 45oC and avoiding accumulation of the nanotubes in the clearance organs by using intratumoral administration.

Annexin A5 as a targeting agent for cancer treatment.

Identifying a universal biomarker for cancer treatment remains a major challenge in cancer therapy. Extracellular exposure of phosphatidylserine (PS) is tightly regulated and is an "eat me" signal for phagocytosis in healthy cells. Although cancer cells and vasculature express high levels of externalized PS, they do not undergo apoptosis, making them a promising biomarker for cancer treatment. Annexin A5 (ANXA5) is the native binding partner of PS and can actively target and deliver chemotherapies to the tumor microenvironment (TME) via PS expression. ANXA5 acts as a bridge between the innate and adaptive immune systems and contributes to an immunostimulatory profile in the TME. ANXA5-enzyme prodrug therapies allow for systemic delivery of prodrugs and targeted killing at the tumor site. ANXA5-carbon nanotube conjugates have been used to physically ablate tumors via photothermal therapy. This review aims to explore the expression of PS in cancer cells and how ANXA5 has been used as a chemotherapeutic and targeting agent for cancer.

Anionic phospholipid expression as a molecular target in Listeria monocytogenes and Escherichia coli.

This study validates bacterial anionic phospholipids (APs) as a putative molecular target in a novel antibiotic treatment against the Gram-positive bacterium Listeria monocytogenes and the Gram-negative bacterium Escherichia coli. Bacterial AP expression was targeted with its associated protein-ligand partner, annexin A5 (ANXA5). This protein was functionalised with the covalent addition of the antibiotic ampicillin (AMP) and separately with the antibiotic moxifloxacin (MOX). Functionalised ANXA5 serves as a delivery vehicle, directing the antibiotic to bacterial AP expression. The results presented here suggest that this ANXA5-AMP bioconjugate participates in a positive feedback loop where APs, the target of the delivery vehicle ANXA5, are upregulated by the chemotherapeutic payload of the bioconjugate. Importantly, the ANXA5 delivery vehicle is non-toxic to bacterial cells by itself and neither is the ANXA5-antibiotic bioconjugate toxic to human vascular endothelial cells. As measured by the IC50, conjugation to ANXA5 resulted in increasing the antibiotic activity of AMP against L. monocytogenes and E. coli by more than 4 and 3 orders of magnitude, respectively, compared with free AMP, whilst the activity of MOX against L. monocytogenes is increased by 4 orders of magnitude. Given the conservation of AP expression in pathologies such as oncogenesis and other bacterial/viral/parasitic infections, we hypothesise that a therapeutic modality targeting AP expression may be a viable chemotherapeutic strategy in many infectious diseases.