A Mutated Anti-CA19-9 scFv-Fc for Positron Emission Tomography of Human Pancreatic Cancer Xenografts



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.


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 (124I) 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).


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. 124I-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.


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.

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This study was supported in part by a VA Carreer Development Award (J Tomlinson) VA0002. Additionally, the authors would like to acknowledge the UCLA Jonsson Comprehensive Cancer Center grant P30 CA016042 and the CFAR grant 5P30 AI028697 for support of the flow cytometry assays performed in this study.

Conflict of Interest

The authors do not have any conflicts of interest to disclose for this manuscript.

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Correspondence to James S. Tomlinson.

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Rochefort, M.M., Girgis, M.D., Knowles, S.M. et al. A Mutated Anti-CA19-9 scFv-Fc for Positron Emission Tomography of Human Pancreatic Cancer Xenografts. Mol Imaging Biol 16, 721–729 (2014). https://doi.org/10.1007/s11307-014-0733-4

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Key Words

  • Pancreas
  • Cancer
  • CA 19-9
  • Imaging
  • Antibody
  • scFv-Fc