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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

78Fc as an NIR optical imaging tracer in a murine TEM1-expressing lung cancer xenograft modelA, Live cell ELISA analysis of 78Fc labeled with Vivotag750 via either maleimide (red) or NHS (green) linkage. Blue indicates the binding activity of unlabeled 78Fc. B, TC1-derived subcutaneous tumor grafts express high level of muTEM1. 104 TC1 cells were injected subcutaneously into B6 mice and tumors were harvested at the indicated time (up to 6 wks). muTEM1 mRNA levels in tumor and parental cells were measured by qRT-PCR, and 2H11 and MS1 cells were used as positive and negative controls, respectively. C, in vivo bioluminescence imaging (BLI) and 78Fc750-based live NIR optical imaging in a TC1 subcutaneous tumor model (n=5). 78Fc750 was given via retro orbital injection (5 mg/kg in 100 uL) and a longitudinal study of NIR imaging was performed up to 3 days post injection (n=5). Green circle, muTEM1+ positive tumor. 1. heart; 2.brain; 3. liver; 4. lung; 5. spleen; 6. kidney; 7. small intestine; 8. bladder; 9. ovary; 10. uterus; 11. thyroid; 12. TC1 sc. xenograft. Residual tracer was observed at the injection site (retro-orbital complexes). D, TC1 lung metastatic lesions express higher levels of muTEM1 than normal tissue in the lung. E, in vivo bioluminescence imaging (BLI) and 78Fc750-based live NIR optical imaging in a TC1 lung metastasis model. 78Fc750 was given via retro orbital injection (5 mg/kg in 100 uL) and a longitudinal study of NIR imaging was performed at 3 days post injection (n=5). From left: ventral view of mouse BLI showing TC1 lung metastasis at day0; live animal NIR at day2 p.i. of 78Fc750; ventral view at day3 p.i. (postmortem) of same animal with liver and kidneys removed to show lung signal; all harvested organs including liver and kidneys; close up view of the lung. Green box, muTEM1+ positive lung tumor. 1. heart; 2.brain; 3. liver; 4. lung; 5. spleen; 6. kidney; 7. small intestine; 8. bladder; 9. ovary; 10. uterus.
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Figure 6: 78Fc as an NIR optical imaging tracer in a murine TEM1-expressing lung cancer xenograft modelA, Live cell ELISA analysis of 78Fc labeled with Vivotag750 via either maleimide (red) or NHS (green) linkage. Blue indicates the binding activity of unlabeled 78Fc. B, TC1-derived subcutaneous tumor grafts express high level of muTEM1. 104 TC1 cells were injected subcutaneously into B6 mice and tumors were harvested at the indicated time (up to 6 wks). muTEM1 mRNA levels in tumor and parental cells were measured by qRT-PCR, and 2H11 and MS1 cells were used as positive and negative controls, respectively. C, in vivo bioluminescence imaging (BLI) and 78Fc750-based live NIR optical imaging in a TC1 subcutaneous tumor model (n=5). 78Fc750 was given via retro orbital injection (5 mg/kg in 100 uL) and a longitudinal study of NIR imaging was performed up to 3 days post injection (n=5). Green circle, muTEM1+ positive tumor. 1. heart; 2.brain; 3. liver; 4. lung; 5. spleen; 6. kidney; 7. small intestine; 8. bladder; 9. ovary; 10. uterus; 11. thyroid; 12. TC1 sc. xenograft. Residual tracer was observed at the injection site (retro-orbital complexes). D, TC1 lung metastatic lesions express higher levels of muTEM1 than normal tissue in the lung. E, in vivo bioluminescence imaging (BLI) and 78Fc750-based live NIR optical imaging in a TC1 lung metastasis model. 78Fc750 was given via retro orbital injection (5 mg/kg in 100 uL) and a longitudinal study of NIR imaging was performed at 3 days post injection (n=5). From left: ventral view of mouse BLI showing TC1 lung metastasis at day0; live animal NIR at day2 p.i. of 78Fc750; ventral view at day3 p.i. (postmortem) of same animal with liver and kidneys removed to show lung signal; all harvested organs including liver and kidneys; close up view of the lung. Green box, muTEM1+ positive lung tumor. 1. heart; 2.brain; 3. liver; 4. lung; 5. spleen; 6. kidney; 7. small intestine; 8. bladder; 9. ovary; 10. uterus.

Mentions: As a rapidly developing technology, optical imaging is becoming a promising diagnostic modality in cancer management. To evaluate 78Fc as an optical imaging tool, we first generated 78Fc-fluorochrome conjugates via two different coupling methods: N-hydroxysuccinimide (NHS)-esterification, and thiol (-SH, sulfhydryl) reactive maleimide chemistry. We chose VivoTag-S750 as the conjugating fluorochrome because the near infrared wavelength is ideal for both in vitro and in vivo biological imaging applications. In addition, with the fluorescence tomography (FMT) imaging systems, VivoTag-S750 can also provide quantitative, three-dimensional information of in vivo target tissues. While the fluorochrome-78Fc conjugate prepared with NHS chemistry effectively ablated the ability of 78Fc to bind to TEM1 (Fig 6A), the impact of maleimide coupling was far less dramatic, resulting in only a 3-fold reduction in apparent affinity for this conjugate (hereafter designated as 78Fc750) as compared to the underivatised 78Fc.


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)

78Fc as an NIR optical imaging tracer in a murine TEM1-expressing lung cancer xenograft modelA, Live cell ELISA analysis of 78Fc labeled with Vivotag750 via either maleimide (red) or NHS (green) linkage. Blue indicates the binding activity of unlabeled 78Fc. B, TC1-derived subcutaneous tumor grafts express high level of muTEM1. 104 TC1 cells were injected subcutaneously into B6 mice and tumors were harvested at the indicated time (up to 6 wks). muTEM1 mRNA levels in tumor and parental cells were measured by qRT-PCR, and 2H11 and MS1 cells were used as positive and negative controls, respectively. C, in vivo bioluminescence imaging (BLI) and 78Fc750-based live NIR optical imaging in a TC1 subcutaneous tumor model (n=5). 78Fc750 was given via retro orbital injection (5 mg/kg in 100 uL) and a longitudinal study of NIR imaging was performed up to 3 days post injection (n=5). Green circle, muTEM1+ positive tumor. 1. heart; 2.brain; 3. liver; 4. lung; 5. spleen; 6. kidney; 7. small intestine; 8. bladder; 9. ovary; 10. uterus; 11. thyroid; 12. TC1 sc. xenograft. Residual tracer was observed at the injection site (retro-orbital complexes). D, TC1 lung metastatic lesions express higher levels of muTEM1 than normal tissue in the lung. E, in vivo bioluminescence imaging (BLI) and 78Fc750-based live NIR optical imaging in a TC1 lung metastasis model. 78Fc750 was given via retro orbital injection (5 mg/kg in 100 uL) and a longitudinal study of NIR imaging was performed at 3 days post injection (n=5). From left: ventral view of mouse BLI showing TC1 lung metastasis at day0; live animal NIR at day2 p.i. of 78Fc750; ventral view at day3 p.i. (postmortem) of same animal with liver and kidneys removed to show lung signal; all harvested organs including liver and kidneys; close up view of the lung. Green box, muTEM1+ positive lung tumor. 1. heart; 2.brain; 3. liver; 4. lung; 5. spleen; 6. kidney; 7. small intestine; 8. bladder; 9. ovary; 10. uterus.
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Figure 6: 78Fc as an NIR optical imaging tracer in a murine TEM1-expressing lung cancer xenograft modelA, Live cell ELISA analysis of 78Fc labeled with Vivotag750 via either maleimide (red) or NHS (green) linkage. Blue indicates the binding activity of unlabeled 78Fc. B, TC1-derived subcutaneous tumor grafts express high level of muTEM1. 104 TC1 cells were injected subcutaneously into B6 mice and tumors were harvested at the indicated time (up to 6 wks). muTEM1 mRNA levels in tumor and parental cells were measured by qRT-PCR, and 2H11 and MS1 cells were used as positive and negative controls, respectively. C, in vivo bioluminescence imaging (BLI) and 78Fc750-based live NIR optical imaging in a TC1 subcutaneous tumor model (n=5). 78Fc750 was given via retro orbital injection (5 mg/kg in 100 uL) and a longitudinal study of NIR imaging was performed up to 3 days post injection (n=5). Green circle, muTEM1+ positive tumor. 1. heart; 2.brain; 3. liver; 4. lung; 5. spleen; 6. kidney; 7. small intestine; 8. bladder; 9. ovary; 10. uterus; 11. thyroid; 12. TC1 sc. xenograft. Residual tracer was observed at the injection site (retro-orbital complexes). D, TC1 lung metastatic lesions express higher levels of muTEM1 than normal tissue in the lung. E, in vivo bioluminescence imaging (BLI) and 78Fc750-based live NIR optical imaging in a TC1 lung metastasis model. 78Fc750 was given via retro orbital injection (5 mg/kg in 100 uL) and a longitudinal study of NIR imaging was performed at 3 days post injection (n=5). From left: ventral view of mouse BLI showing TC1 lung metastasis at day0; live animal NIR at day2 p.i. of 78Fc750; ventral view at day3 p.i. (postmortem) of same animal with liver and kidneys removed to show lung signal; all harvested organs including liver and kidneys; close up view of the lung. Green box, muTEM1+ positive lung tumor. 1. heart; 2.brain; 3. liver; 4. lung; 5. spleen; 6. kidney; 7. small intestine; 8. bladder; 9. ovary; 10. uterus.
Mentions: As a rapidly developing technology, optical imaging is becoming a promising diagnostic modality in cancer management. To evaluate 78Fc as an optical imaging tool, we first generated 78Fc-fluorochrome conjugates via two different coupling methods: N-hydroxysuccinimide (NHS)-esterification, and thiol (-SH, sulfhydryl) reactive maleimide chemistry. We chose VivoTag-S750 as the conjugating fluorochrome because the near infrared wavelength is ideal for both in vitro and in vivo biological imaging applications. In addition, with the fluorescence tomography (FMT) imaging systems, VivoTag-S750 can also provide quantitative, three-dimensional information of in vivo target tissues. While the fluorochrome-78Fc conjugate prepared with NHS chemistry effectively ablated the ability of 78Fc to bind to TEM1 (Fig 6A), the impact of maleimide coupling was far less dramatic, resulting in only a 3-fold reduction in apparent affinity for this conjugate (hereafter designated as 78Fc750) as compared to the underivatised 78Fc.

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