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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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Comparison of two purification methods for scale-up production of 78Fc fusion protein after codon-optimizationScale up production of fusion protein 78Fc was performed after codon optimized by adapting codon bias of Homo sapiens genes with GeneOptimizer (Life technology). A, Elution profiles on SEC columns after affinity purification with anti-HA (left panel) or Protein G (right panel) methods. 78Fc protein elution fractions which were pooled (red arrowhead) are indicated with dotted-lines. B and C, Comparison of the purification efficiencies of anti-HA and Protein G methods by Western blot analysis (B) and Coomassie blue staining (C). A yield of 15-20 mg/L were achieved post optimization from 293 Freestyle cells (Invitrogen) compare with 0.5-1mg/L before optimization with purity >95%.
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Figure 4: Comparison of two purification methods for scale-up production of 78Fc fusion protein after codon-optimizationScale up production of fusion protein 78Fc was performed after codon optimized by adapting codon bias of Homo sapiens genes with GeneOptimizer (Life technology). A, Elution profiles on SEC columns after affinity purification with anti-HA (left panel) or Protein G (right panel) methods. 78Fc protein elution fractions which were pooled (red arrowhead) are indicated with dotted-lines. B and C, Comparison of the purification efficiencies of anti-HA and Protein G methods by Western blot analysis (B) and Coomassie blue staining (C). A yield of 15-20 mg/L were achieved post optimization from 293 Freestyle cells (Invitrogen) compare with 0.5-1mg/L before optimization with purity >95%.

Mentions: The observation that 78Fc displays encouraging in vitro thermal and serum stability behavior, in addition to possessing the highest avidity to TEM1 and a favorable murine PK profile, prompted us to investigate further the suitability of 78Fc for imaging applications by extending the study into preclinical mouse models. As a first step, we sought to improve the HEK cell expression yield and purification efficiency of 78Fc. We first improved the Homo sapiens Codon Adaptation Index (CAI) score of the 78Fc coding sequence to 0.96 (GeneOptimizer; Life Technologies). Subsequently, cis-acting sites (such as splice sites, RNA instability motifs, TATA box, GC-hi and AT-hi regions, ribosomal entry sites) that could potentially inhibit protein expression were removed wherever possible. Finally, we adjusted the GC content to 63% to prolong mRNA half-life. These measures were sufficient to increase the yield of 78Fc from 0.5-1 mg/L for the un-optimized gene, to 15-20 mg/L for the optimized gene expressed in HEK 293 Freestyle cells. We next sought to compare the purification capture efficiencies of anti-HA and Protein G matrices. 78Fc protein was first enriched with anti-HA agarose or Protein G, and the purified proteins were then subjected to size exclusion chromatography (SEC) prior to concentration of peak fractions (Fig 4A). The yields and purities of the final concentrated proteins were compared by SDS-PAGE using Western blot analysis and Coomassie dye staining (Fig 4B, C). The results indicated that both affinity approaches were equally effective for purification with final purities exceeding 95%. Since purification using Protein-G is more cost-effective, we decided to use Protein-G capture combined with SEC to purify large quantities of 78Fc for in vivo testing.


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)

Comparison of two purification methods for scale-up production of 78Fc fusion protein after codon-optimizationScale up production of fusion protein 78Fc was performed after codon optimized by adapting codon bias of Homo sapiens genes with GeneOptimizer (Life technology). A, Elution profiles on SEC columns after affinity purification with anti-HA (left panel) or Protein G (right panel) methods. 78Fc protein elution fractions which were pooled (red arrowhead) are indicated with dotted-lines. B and C, Comparison of the purification efficiencies of anti-HA and Protein G methods by Western blot analysis (B) and Coomassie blue staining (C). A yield of 15-20 mg/L were achieved post optimization from 293 Freestyle cells (Invitrogen) compare with 0.5-1mg/L before optimization with purity >95%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Comparison of two purification methods for scale-up production of 78Fc fusion protein after codon-optimizationScale up production of fusion protein 78Fc was performed after codon optimized by adapting codon bias of Homo sapiens genes with GeneOptimizer (Life technology). A, Elution profiles on SEC columns after affinity purification with anti-HA (left panel) or Protein G (right panel) methods. 78Fc protein elution fractions which were pooled (red arrowhead) are indicated with dotted-lines. B and C, Comparison of the purification efficiencies of anti-HA and Protein G methods by Western blot analysis (B) and Coomassie blue staining (C). A yield of 15-20 mg/L were achieved post optimization from 293 Freestyle cells (Invitrogen) compare with 0.5-1mg/L before optimization with purity >95%.
Mentions: The observation that 78Fc displays encouraging in vitro thermal and serum stability behavior, in addition to possessing the highest avidity to TEM1 and a favorable murine PK profile, prompted us to investigate further the suitability of 78Fc for imaging applications by extending the study into preclinical mouse models. As a first step, we sought to improve the HEK cell expression yield and purification efficiency of 78Fc. We first improved the Homo sapiens Codon Adaptation Index (CAI) score of the 78Fc coding sequence to 0.96 (GeneOptimizer; Life Technologies). Subsequently, cis-acting sites (such as splice sites, RNA instability motifs, TATA box, GC-hi and AT-hi regions, ribosomal entry sites) that could potentially inhibit protein expression were removed wherever possible. Finally, we adjusted the GC content to 63% to prolong mRNA half-life. These measures were sufficient to increase the yield of 78Fc from 0.5-1 mg/L for the un-optimized gene, to 15-20 mg/L for the optimized gene expressed in HEK 293 Freestyle cells. We next sought to compare the purification capture efficiencies of anti-HA and Protein G matrices. 78Fc protein was first enriched with anti-HA agarose or Protein G, and the purified proteins were then subjected to size exclusion chromatography (SEC) prior to concentration of peak fractions (Fig 4A). The yields and purities of the final concentrated proteins were compared by SDS-PAGE using Western blot analysis and Coomassie dye staining (Fig 4B, C). The results indicated that both affinity approaches were equally effective for purification with final purities exceeding 95%. Since purification using Protein-G is more cost-effective, we decided to use Protein-G capture combined with SEC to purify large quantities of 78Fc for in vivo testing.

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