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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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Related in: MedlinePlus

The stability and pharmacokinetic profiles of scFv78 and its derivativesA, Thermal stability curves of the antibody variants. Differential scanning fluorimetry was used to measure protein aggregation and degradation. B, Serum stability assay of scFv78 and its derivatives. Purified antibody variants were incubated with 100% human serum for up to 7 days at 37°C. Samples taken at different time points were then analyzed by Western blot with anti-HA antibody. C, Pharmacokinetics of 125I-labeled anti-TEM1 variants in naïve nu/nu mice. The half-lives (fast and slow, in hours) deduced from the study is underneath.
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Figure 3: The stability and pharmacokinetic profiles of scFv78 and its derivativesA, Thermal stability curves of the antibody variants. Differential scanning fluorimetry was used to measure protein aggregation and degradation. B, Serum stability assay of scFv78 and its derivatives. Purified antibody variants were incubated with 100% human serum for up to 7 days at 37°C. Samples taken at different time points were then analyzed by Western blot with anti-HA antibody. C, Pharmacokinetics of 125I-labeled anti-TEM1 variants in naïve nu/nu mice. The half-lives (fast and slow, in hours) deduced from the study is underneath.

Mentions: Good biophysical stability and appropriate serum half-life are generally considered important prerequisites for antibodies or antibody products destined for clinical applications. To evaluate the stability of the scFv78 fusion proteins, we first measured their thermal stability by incubating the purified proteins with SYBR orange under a temperature gradient from 20°C to 99°C. Following incubation, the samples were analyzed by differential scanning fluorimetry (DSF), an assay employed to estimate protein degradation/unfolding and aggregation. The thermostability curves revealed that the melting temperatures (Tm) of scFv78 and its derivatives were comparable (Fig 3A).


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)

The stability and pharmacokinetic profiles of scFv78 and its derivativesA, Thermal stability curves of the antibody variants. Differential scanning fluorimetry was used to measure protein aggregation and degradation. B, Serum stability assay of scFv78 and its derivatives. Purified antibody variants were incubated with 100% human serum for up to 7 days at 37°C. Samples taken at different time points were then analyzed by Western blot with anti-HA antibody. C, Pharmacokinetics of 125I-labeled anti-TEM1 variants in naïve nu/nu mice. The half-lives (fast and slow, in hours) deduced from the study is underneath.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: The stability and pharmacokinetic profiles of scFv78 and its derivativesA, Thermal stability curves of the antibody variants. Differential scanning fluorimetry was used to measure protein aggregation and degradation. B, Serum stability assay of scFv78 and its derivatives. Purified antibody variants were incubated with 100% human serum for up to 7 days at 37°C. Samples taken at different time points were then analyzed by Western blot with anti-HA antibody. C, Pharmacokinetics of 125I-labeled anti-TEM1 variants in naïve nu/nu mice. The half-lives (fast and slow, in hours) deduced from the study is underneath.
Mentions: Good biophysical stability and appropriate serum half-life are generally considered important prerequisites for antibodies or antibody products destined for clinical applications. To evaluate the stability of the scFv78 fusion proteins, we first measured their thermal stability by incubating the purified proteins with SYBR orange under a temperature gradient from 20°C to 99°C. Following incubation, the samples were analyzed by differential scanning fluorimetry (DSF), an assay employed to estimate protein degradation/unfolding and aggregation. The thermostability curves revealed that the melting temperatures (Tm) of scFv78 and its derivatives were comparable (Fig 3A).

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