title: Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease.
abstract: BACKGROUND
  Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component.
  
  
  OBJECTIVE
  To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation.
  
  
  RESULTS
  The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries.
  
  
  CONCLUSIONS
  This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.
authors.affiliation: Atherothrombosis and Vascular Biology, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.
authors.forename: H T
authors.initials: HT
authors.lastname: Ta
authors.forename: S
authors.initials: S
authors.lastname: Prabhu
authors.forename: E
authors.initials: E
authors.lastname: Leitner
authors.forename: F
authors.initials: F
authors.lastname: Jia
authors.forename: D
authors.initials: D
authors.lastname: von Elverfeldt
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Jackson
authors.forename: T
authors.initials: T
authors.lastname: Heidt
authors.forename: A K N
authors.initials: AK
authors.lastname: Nair
authors.forename: H
authors.initials: H
authors.lastname: Pearce
authors.forename: C
authors.initials: C
authors.lastname: von Zur Muhlen
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: K
authors.initials: K
authors.lastname: Peter
authors.forename: C E
authors.initials: CE
authors.lastname: Hagemeyer
mesh_terms: D019881:Aminoacyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism; D016466:CHO Cells; D002465:Cell Movement; D058948:Cell Tracking / Q000379:methods*; D002712:Chlorides; D003287:Contrast Media; D006224:Cricetinae; D003412:Cricetulus; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D005290:Ferric Compounds; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008279:Magnetic Resonance Imaging; D058185:Magnetite Nanoparticles; D051379:Mice; D008810:Mice, Inbred C57BL; D018715:Microscopy, Video; D015336:Molecular Probe Techniques; D015539:Platelet Activation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013927:Thrombosis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology*
chemical_list: D001426:Bacterial Proteins; D002712:Chlorides; D003287:Contrast Media; D005290:Ferric Compounds; D058185:Magnetite Nanoparticles; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; C024555:ferric chloride