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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: 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.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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scFv78 fusion proteins demonstrate higher avidity to cell-surface TEM1A, The dose-dependent binding curves of scFv78 and its derivatives. Cell-surface binding activity in control (MS1) or TEM1-expressing cells (MS1-TEM1) was measured by a live cell-based ELISA assay. TEM1-specific binding was calculated as the difference between binding to the control and TEM1-expressing protein. The control anti-TEM1 IgG is a biotinylated humanized mouse IgG1. B, A summary of the calculated Kd and Bmax of the scFv78 and scFv78 derivatives. Anti-HA HRP was used as the secondary antibody in ELISA for all proteins except for the control anti-TEM1 IgG which required strepavidin-HRP. * indicates the different secondary antibody used.
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Figure 2: scFv78 fusion proteins demonstrate higher avidity to cell-surface TEM1A, The dose-dependent binding curves of scFv78 and its derivatives. Cell-surface binding activity in control (MS1) or TEM1-expressing cells (MS1-TEM1) was measured by a live cell-based ELISA assay. TEM1-specific binding was calculated as the difference between binding to the control and TEM1-expressing protein. The control anti-TEM1 IgG is a biotinylated humanized mouse IgG1. B, A summary of the calculated Kd and Bmax of the scFv78 and scFv78 derivatives. Anti-HA HRP was used as the secondary antibody in ELISA for all proteins except for the control anti-TEM1 IgG which required strepavidin-HRP. * indicates the different secondary antibody used.

Mentions: It is well established that increases in valency can improve the avidity of an antibody. To measure the avidities of scFv78 and its derivatives under conditions that are more relevant to in vivo settings, we established a live-cell ELISA assay to measure the binding of the fusion proteins to cell-surface TEM1. Briefly, we first modified Mile-Sven1 (MS1), a TEM1-negative endothelial cell line, to express human TEM1 at a moderate level, with the saturated maximal binding capacity (Bmax) of ~4 × 105 per cell. Different concentrations of scFv78 derivatives were then incubated with either control or TEM1 positive MS1 cells. Following washing, remaining molecules bound to the live cells at each concentration were detected by ELISA. Specific binding was observed when the concentration of fusion protein was as low as 0.1 nM, and non-specific binding was not observed below 10 nM (Fig 2A). While all samples tested have comparable Bmax, the fusion proteins all have lower apparent Kd values than scFv78, consistent with higher oligomeric avidities to TEM1 (Fig 2B). However, the apparent oligomerisation of 78CH2 does not translate into the expected avidity gain, suggesting that this species may have steric or structural issues. Among all antibodies tested, 78Fc demonstrated the lowest Kd value in sub-nanomolar range, which was ~15-fold lower than that of scFv78 (Fig 2B).


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)

scFv78 fusion proteins demonstrate higher avidity to cell-surface TEM1A, The dose-dependent binding curves of scFv78 and its derivatives. Cell-surface binding activity in control (MS1) or TEM1-expressing cells (MS1-TEM1) was measured by a live cell-based ELISA assay. TEM1-specific binding was calculated as the difference between binding to the control and TEM1-expressing protein. The control anti-TEM1 IgG is a biotinylated humanized mouse IgG1. B, A summary of the calculated Kd and Bmax of the scFv78 and scFv78 derivatives. Anti-HA HRP was used as the secondary antibody in ELISA for all proteins except for the control anti-TEM1 IgG which required strepavidin-HRP. * indicates the different secondary antibody used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: scFv78 fusion proteins demonstrate higher avidity to cell-surface TEM1A, The dose-dependent binding curves of scFv78 and its derivatives. Cell-surface binding activity in control (MS1) or TEM1-expressing cells (MS1-TEM1) was measured by a live cell-based ELISA assay. TEM1-specific binding was calculated as the difference between binding to the control and TEM1-expressing protein. The control anti-TEM1 IgG is a biotinylated humanized mouse IgG1. B, A summary of the calculated Kd and Bmax of the scFv78 and scFv78 derivatives. Anti-HA HRP was used as the secondary antibody in ELISA for all proteins except for the control anti-TEM1 IgG which required strepavidin-HRP. * indicates the different secondary antibody used.
Mentions: It is well established that increases in valency can improve the avidity of an antibody. To measure the avidities of scFv78 and its derivatives under conditions that are more relevant to in vivo settings, we established a live-cell ELISA assay to measure the binding of the fusion proteins to cell-surface TEM1. Briefly, we first modified Mile-Sven1 (MS1), a TEM1-negative endothelial cell line, to express human TEM1 at a moderate level, with the saturated maximal binding capacity (Bmax) of ~4 × 105 per cell. Different concentrations of scFv78 derivatives were then incubated with either control or TEM1 positive MS1 cells. Following washing, remaining molecules bound to the live cells at each concentration were detected by ELISA. Specific binding was observed when the concentration of fusion protein was as low as 0.1 nM, and non-specific binding was not observed below 10 nM (Fig 2A). While all samples tested have comparable Bmax, the fusion proteins all have lower apparent Kd values than scFv78, consistent with higher oligomeric avidities to TEM1 (Fig 2B). However, the apparent oligomerisation of 78CH2 does not translate into the expected avidity gain, suggesting that this species may have steric or structural issues. Among all antibodies tested, 78Fc demonstrated the lowest Kd value in sub-nanomolar range, which was ~15-fold lower than that of scFv78 (Fig 2B).

Bottom Line: 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.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