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Development, optimization, and validation of novel anti-TEM1/CD248 affinity agent for optical imaging in cancer.

Li C, Wang J, Hu J, Feng Y, Hasegawa K, Peng X, Duan X, Zhao A, Mikitsh JL, Muzykantov VR, Chacko AM, Pryma DA, Dunn SM, Coukos G - Oncotarget (2014)

Bottom Line: By characterizing avidity, stability, and pharmacokinectics, we identified one fusion protein, 78Fc, with desirable characteristics for immuno-imaging applications.The biodistribution of radiolabeled 78Fc showed that this antibody had minimal binding to normal organs, which have low expression of TEM1.From these results we conclude that further development and optimization of 78Fc as a TEM1-targeted imaging agent for use in clinical settings is warranted.

View Article: PubMed Central - PubMed

Affiliation: Ovarian Cancer Research Center, University of Pennsylvania; These authors contributed equally to this work.

ABSTRACT
Tumor Endothelial Marker-1 (TEM1/CD248) is a tumor vascular marker with high therapeutic and diagnostic potentials. Immuno-imaging with TEM1-specific antibodies can help to detect cancerous lesions, monitor tumor responses, and select patients that are most likely to benefit from TEM1-targeted therapies. In particular, near infrared(NIR) optical imaging with biomarker-specific antibodies can provide real-time, tomographic information without exposing the subjects to radioactivity. To maximize the theranostic potential of TEM1, we developed a panel of all human, multivalent Fc-fusion proteins based on a previously identified single chain antibody (scFv78) that recognizes both human and mouse TEM1. By characterizing avidity, stability, and pharmacokinectics, we identified one fusion protein, 78Fc, with desirable characteristics for immuno-imaging applications. The biodistribution of radiolabeled 78Fc showed that this antibody had minimal binding to normal organs, which have low expression of TEM1. Next, we developed a 78Fc-based tracer and tested its performance in different TEM1-expressing mouse models. The NIR imaging and tomography results suggest that the 78Fc-NIR tracer performs well in distinguishing mouse- or human-TEM1 expressing tumor grafts from normal organs and control grafts in vivo. From these results we conclude that further development and optimization of 78Fc as a TEM1-targeted imaging agent for use in clinical settings is warranted.

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Development, purification, and characterization of scFc78 fusion proteinsA, Schematic representation of the domain organization and the predicted molecular weights and valencies of scFv78 and the four scFv78 fusion derivatives. B, SEC analyses of affinity purified scFv78 and the four scFv78 fusion derivatives. C, multivalent status of scFv78 and the four scFv78 fusion derivatives by Western blot analysis with anti-HA antibody. R: reducing conditions, N: non-reducing conditions. * monomer, ** dimer, **** multimer.
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Figure 1: Development, purification, and characterization of scFc78 fusion proteinsA, Schematic representation of the domain organization and the predicted molecular weights and valencies of scFv78 and the four scFv78 fusion derivatives. B, SEC analyses of affinity purified scFv78 and the four scFv78 fusion derivatives. C, multivalent status of scFv78 and the four scFv78 fusion derivatives by Western blot analysis with anti-HA antibody. R: reducing conditions, N: non-reducing conditions. * monomer, ** dimer, **** multimer.

Mentions: Since the practical utility of many scFvs are often limited due to their small size, structural instability due to relatively weak variable domain interactions, and monovalency [1], we sought to construct novel oligomerised scFv78 variants more suitable for therapeutic and prognostic (theranostic) applications. To achieve this goal, we designed four multivalent scFv-Fc fusion proteins: 78F(ab`)2, 78CH2, scFv78-minibody (78mb), and scFv78-Fc (78Fc) (Fig 1A). While 78F(ab`)2 was generated by linking two scFv78 together via the IgG1 core hinge region (CPPCP), the other three variants were constructed by fusing different Fc regions to the C-terminal of scFv78. The calculated molecular weight of bivalent molecules of 78F(ab`)2, 78CH2, 78mb, and 78Fc are 65kDa, 90kDa, 90kDa, and 120kDa, respectively. A HA tag was added to the N terminus of the proteins for easy purification and detection, and upstream addition of the signal peptide from Ig KappaV enabled the fusion proteins to be secreted and easily purified from the media of the host 293T expression cells (sup Fig 1). Fusion proteins were purified by incubating the conditioned culture media with anti-HA affinity matrix beads. For all fusion proteins, we were able to purify 0.5-1mg/L protein at a purity >90%. Since the size exclusion HPLC (SE-HPLC) analysis of the purified proteins revealed additional peaks, suggesting the presence of aggregates/multimers of the proteins (Fig 1B), we further analyzed the quarternary status of the proteins by polyacrylamide gel electrophoresis. Under reducing conditions, the migration of all scFv derivatives appeared consistent with their calculated molecular weights (Fig 1C). Under non-reducing conditions, while scFv78 remained monovalent, we observed apparent oligomerisation of the fusion proteins: for 78Fc and 78F(ab`)2, the majority of protein appeared dimeric; for 78CH2, the majority (>90%) of protein migrated with an apparent mass consistent with a tetramer; and for 78mb, about 40% of the protein remained monomeric.


Development, optimization, and validation of novel anti-TEM1/CD248 affinity agent for optical imaging in cancer.

Li C, Wang J, Hu J, Feng Y, Hasegawa K, Peng X, Duan X, Zhao A, Mikitsh JL, Muzykantov VR, Chacko AM, Pryma DA, Dunn SM, Coukos G - Oncotarget (2014)

Development, purification, and characterization of scFc78 fusion proteinsA, Schematic representation of the domain organization and the predicted molecular weights and valencies of scFv78 and the four scFv78 fusion derivatives. B, SEC analyses of affinity purified scFv78 and the four scFv78 fusion derivatives. C, multivalent status of scFv78 and the four scFv78 fusion derivatives by Western blot analysis with anti-HA antibody. R: reducing conditions, N: non-reducing conditions. * monomer, ** dimer, **** multimer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196179&req=5

Figure 1: Development, purification, and characterization of scFc78 fusion proteinsA, Schematic representation of the domain organization and the predicted molecular weights and valencies of scFv78 and the four scFv78 fusion derivatives. B, SEC analyses of affinity purified scFv78 and the four scFv78 fusion derivatives. C, multivalent status of scFv78 and the four scFv78 fusion derivatives by Western blot analysis with anti-HA antibody. R: reducing conditions, N: non-reducing conditions. * monomer, ** dimer, **** multimer.
Mentions: Since the practical utility of many scFvs are often limited due to their small size, structural instability due to relatively weak variable domain interactions, and monovalency [1], we sought to construct novel oligomerised scFv78 variants more suitable for therapeutic and prognostic (theranostic) applications. To achieve this goal, we designed four multivalent scFv-Fc fusion proteins: 78F(ab`)2, 78CH2, scFv78-minibody (78mb), and scFv78-Fc (78Fc) (Fig 1A). While 78F(ab`)2 was generated by linking two scFv78 together via the IgG1 core hinge region (CPPCP), the other three variants were constructed by fusing different Fc regions to the C-terminal of scFv78. The calculated molecular weight of bivalent molecules of 78F(ab`)2, 78CH2, 78mb, and 78Fc are 65kDa, 90kDa, 90kDa, and 120kDa, respectively. A HA tag was added to the N terminus of the proteins for easy purification and detection, and upstream addition of the signal peptide from Ig KappaV enabled the fusion proteins to be secreted and easily purified from the media of the host 293T expression cells (sup Fig 1). Fusion proteins were purified by incubating the conditioned culture media with anti-HA affinity matrix beads. For all fusion proteins, we were able to purify 0.5-1mg/L protein at a purity >90%. Since the size exclusion HPLC (SE-HPLC) analysis of the purified proteins revealed additional peaks, suggesting the presence of aggregates/multimers of the proteins (Fig 1B), we further analyzed the quarternary status of the proteins by polyacrylamide gel electrophoresis. Under reducing conditions, the migration of all scFv derivatives appeared consistent with their calculated molecular weights (Fig 1C). Under non-reducing conditions, while scFv78 remained monovalent, we observed apparent oligomerisation of the fusion proteins: for 78Fc and 78F(ab`)2, the majority of protein appeared dimeric; for 78CH2, the majority (>90%) of protein migrated with an apparent mass consistent with a tetramer; and for 78mb, about 40% of the protein remained monomeric.

Bottom Line: By characterizing avidity, stability, and pharmacokinectics, we identified one fusion protein, 78Fc, with desirable characteristics for immuno-imaging applications.The biodistribution of radiolabeled 78Fc showed that this antibody had minimal binding to normal organs, which have low expression of TEM1.From these results we conclude that further development and optimization of 78Fc as a TEM1-targeted imaging agent for use in clinical settings is warranted.

View Article: PubMed Central - PubMed

Affiliation: Ovarian Cancer Research Center, University of Pennsylvania; These authors contributed equally to this work.

ABSTRACT
Tumor Endothelial Marker-1 (TEM1/CD248) is a tumor vascular marker with high therapeutic and diagnostic potentials. Immuno-imaging with TEM1-specific antibodies can help to detect cancerous lesions, monitor tumor responses, and select patients that are most likely to benefit from TEM1-targeted therapies. In particular, near infrared(NIR) optical imaging with biomarker-specific antibodies can provide real-time, tomographic information without exposing the subjects to radioactivity. To maximize the theranostic potential of TEM1, we developed a panel of all human, multivalent Fc-fusion proteins based on a previously identified single chain antibody (scFv78) that recognizes both human and mouse TEM1. By characterizing avidity, stability, and pharmacokinectics, we identified one fusion protein, 78Fc, with desirable characteristics for immuno-imaging applications. The biodistribution of radiolabeled 78Fc showed that this antibody had minimal binding to normal organs, which have low expression of TEM1. Next, we developed a 78Fc-based tracer and tested its performance in different TEM1-expressing mouse models. The NIR imaging and tomography results suggest that the 78Fc-NIR tracer performs well in distinguishing mouse- or human-TEM1 expressing tumor grafts from normal organs and control grafts in vivo. From these results we conclude that further development and optimization of 78Fc as a TEM1-targeted imaging agent for use in clinical settings is warranted.

Show MeSH
Related in: MedlinePlus