title: A mutated anti-CA19-9 scFv-Fc for positron emission tomography of human pancreatic cancer xenografts.
abstract: OBJECTIVE
  Intact antibodies have a long serum persistence resulting in high background signal that inhibits their direct translation as imaging agents. Engineering of antibody fragments through the introduction of mutations in the fragment crystallizable (Fc) region can dramatically reduce serum persistence. We sought to develop a Fc-mutated, anti-CA19-9 antibody fragment (anti-CA 19-9 scFv-Fc H310A) to provide micro-positron emission tomography (microPET) imaging of pancreatic cancer xenografts.
  
  
  METHODS
  The anti-CA19-9 scFv-Fc H310A was successfully expressed and purified. Biochemical characterization included size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and flow cytometry. The antibody fragment was labeled with iodine-124 ((124)I) and injected into mice containing human pancreatic cancer xenografts. MicroPET/CT images were then obtained. Blood, organ, and tumor radioactivity was measured and expressed as the percent of injected dose per gram of tissue (%ID/g).
  
  
  RESULTS
  Biochemical characterization was consistent with the creation of a 105 kD dimer containing a human Fc region. Flow cytometry demonstrated antigen-specific binding, and cell-based ELISA further established a dissociation constant (K D) of 10.7 nM. (124)I-labeled scFv-Fc H310A localized to the antigen-positive tumor xenografts as detected by microPET. Objective confirmation of targeting was demonstrated by higher %ID/g in the antigen-positive tumor compared to the blood, antigen-negative tumor, and liver.
  
  
  CONCLUSIONS
  We successfully engineered and produced an anti-CA19-9 scFv-Fc H310A antibody fragment that retains similar affinity when compared to the parental intact murine antibody. Additionally, our engineered and mutated fragment exhibited antigen-specific microPET imaging of both subcutaneous and orthotopic pancreatic cancer xenografts at early time points secondary to decreased serum half-life.
authors.affiliation: Department of Surgery, UCLA, 570 Westwood Plaza, Bldg 114, CNSI, Rm4324E, Los Angeles, CA, 90095, USA, mrochefort@mednet.ucla.edu.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Rochefort
authors.forename: Mark D
authors.initials: MD
authors.lastname: Girgis
authors.forename: Scott M
authors.initials: SM
authors.lastname: Knowles
authors.forename: Jacob S
authors.initials: JS
authors.lastname: Ankeny
authors.forename: Felix
authors.initials: F
authors.lastname: Salazar
authors.forename: Anna M
authors.initials: AM
authors.lastname: Wu
authors.forename: James S
authors.initials: JS
authors.lastname: Tomlinson
mesh_terms: D000818:Animals; D002272:Carcinoembryonic Antigen / Q000235:genetics*; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008819:Mice, Nude; D009154:Mutation / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000000981:diagnostic imaging*; D049268:Positron-Emission Tomography; D011961:Receptors, Fc / Q000378:metabolism*; D057127:Single-Chain Antibodies; D014018:Tissue Distribution; D014057:Tomography, X-Ray Computed; D023041:Xenograft Model Antitumor Assays
chemical_list: D002272:Carcinoembryonic Antigen; D011961:Receptors, Fc; D057127:Single-Chain Antibodies