Abstract
Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE−/− mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug.
Highlights
Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in eukaryotic membranes
Aortic root samples were cut into 5 μm tissue sections and, mounted on glass slides for conventional staining procedures and imaging mass spectrometry
For detection of XTEN sequence of 864 amino acids (XTEN864)-Annexin A5 (anxA5) and macrophages on the same section, tissue was incubated with MeCAT(Tm)labeled Mac-2 antibody (Cedarlane, Canada, labeling procedure described above) diluted in block solution at an optimized concentration (0.05432 μg/μl) for 1 h at RT
Summary
Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in eukaryotic membranes. Many enveloped viruses (e.g., vaccinia virus, dengue virus, Ebola virus, and pseudotyped lentivirus) rely on what is known as apoptotic mimicry to promote their infectious entry and replication in host cells as well as for immune evasion [20]. Such viruses use different strategies to acquire host cell phosphatidylserine and incorporate it into the viral membrane. These apoptotic body-like, PS-exposing viruses in turn can be recognized and internalized by host cells with PS receptors [20, 21]. For Ebola, anxA5 was hypothesized to cloak the PS-exposing envelop, preventing the pathologic inflammatory cytokine storm and hemorrhagic consumptive coagulopathy [23], which might be the case for other viruses including SARS-CoV-2 [24, 25]
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