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Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies.

Iqbal U, Albaghdadi H, Luo Y, Arbabi M, Desvaux C, Veres T, Stanimirovic D, Abulrob A - Br. J. Cancer (2010)

Bottom Line: In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour.Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biological Sciences, National Research Council of Canada, 1200 Montreal Road, Ottawa, Ontario, Canada K1A 0R6.

ABSTRACT

Background: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications.

Methods: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe₃O₄ nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy.

Results: Surface plasmon resonance analyses revealed a medium affinity (K(D)=40-50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe₃O₄ NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.

Conclusions: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

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In vivo optical imaging of mice bearing orthotopic glioblastoma tumours after intravenous (i.v.) administration of anti-IGFBP7 sdAb labelled with the near-infrared fluorescent probe, Cy5.5. (A) Dorsal whole body in vivo optical images of mice bearing U87MG.EGFRvIII brain tumours at indicated time points after i.v. injection of 50 μg of either anti-IGFBP7 sdAb-Cy5.5 (upper panels), 100 × unlabelled anti-IGFBP7 sdAb followed by anti-IGFBP7 sdAb-Cy5.5 (competitive block, middle panels) or NC sdAb-Cy5.5 (lower panels) (arrows indicate the location of the brain tumour). (B) Graph illustrating changes in the average fluorescence concentration determined from an ROI in the brain tumour region in vivo at indicated times after the i.v. injection of sdAbs-Cy5.5. (C) Optical images of organs ex vivo and dissected brain tumours 24 h after injection of sdAbs-Cy5.5 (D) Graph illustrating the total fluorescence concentration per gram tissue in various organs and dissected tumours imaged ex vivo 24 h after the injection of sdAbs-Cy5.5. In B and D, the data are expressed as mean±s.e.m. for n=5 animals. *Indicates significant difference between anti-IGFBP7 sdAb and both competitively blocked anti-IGFBP7 sdAb and NC sdAb (P<0.01). #Indicates significant difference between competitively blocked anti-IGFBP7-sdAb and NC sdAb (P<0.05).
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fig3: In vivo optical imaging of mice bearing orthotopic glioblastoma tumours after intravenous (i.v.) administration of anti-IGFBP7 sdAb labelled with the near-infrared fluorescent probe, Cy5.5. (A) Dorsal whole body in vivo optical images of mice bearing U87MG.EGFRvIII brain tumours at indicated time points after i.v. injection of 50 μg of either anti-IGFBP7 sdAb-Cy5.5 (upper panels), 100 × unlabelled anti-IGFBP7 sdAb followed by anti-IGFBP7 sdAb-Cy5.5 (competitive block, middle panels) or NC sdAb-Cy5.5 (lower panels) (arrows indicate the location of the brain tumour). (B) Graph illustrating changes in the average fluorescence concentration determined from an ROI in the brain tumour region in vivo at indicated times after the i.v. injection of sdAbs-Cy5.5. (C) Optical images of organs ex vivo and dissected brain tumours 24 h after injection of sdAbs-Cy5.5 (D) Graph illustrating the total fluorescence concentration per gram tissue in various organs and dissected tumours imaged ex vivo 24 h after the injection of sdAbs-Cy5.5. In B and D, the data are expressed as mean±s.e.m. for n=5 animals. *Indicates significant difference between anti-IGFBP7 sdAb and both competitively blocked anti-IGFBP7 sdAb and NC sdAb (P<0.01). #Indicates significant difference between competitively blocked anti-IGFBP7-sdAb and NC sdAb (P<0.05).

Mentions: The in vivo and ex vivo biodistribution analyses of the systemically injected anti-IGFBP7 sdAb ‘tagged' with the near-infrared dye Cy5.5 alone or in combination with the 100-fold excess of unlabelled anti-IGFBP7-sdAb or NC sdAb-Cy5.5 were performed in mice bearing a 10-day-old U87MG.EGFRvIII orthotopic GBM tumour using prospective in vivo optical imaging (Figure 3A). The anti-IGFBP7 sdAb-Cy5.5 homed to the brain tumour as early as 10 min after injection (Figure 3A, upper panels; Figure 3C), with high signal in the tumour persisting up to 24 h after injection. In contrast, brain tumour signal in vivo was reduced at 4 and 24 h when animals were co-injected with the excess unlabelled anti-IGFBP7 sdAb (Figure 3A, middle panels; Figure 3B). NC sdAb-Cy5.5 (Figure 3A, lower panels; Figure 3B) was barely detectable in the brain tumour at any time. At 24 h after injection, brain tumour signal for the anti-IGFBP7 sdAb-Cy5.5 was three- and six-fold higher compared with competitively blocked anti-IGFBP7 sdAb-Cy5.5 and NC sdAb-Cy5.5, respectively (Figure 3B). As sdAbs have a blood half-lives in the range of 20–30 min and are completely cleared from the blood within the first few hours (Iqbal et al, 2010) after injection, the high tumour signal detected at 4 and 24 h with anti-IGFBP7 sdAb-Cy5.5, which was competitively attenuated in the presence of the excess of unlabelled anti-IGFBP7 sdAb, indicates retention of the antibody in the tumour due to its binding to the tumour-expressed antigen.


Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies.

Iqbal U, Albaghdadi H, Luo Y, Arbabi M, Desvaux C, Veres T, Stanimirovic D, Abulrob A - Br. J. Cancer (2010)

In vivo optical imaging of mice bearing orthotopic glioblastoma tumours after intravenous (i.v.) administration of anti-IGFBP7 sdAb labelled with the near-infrared fluorescent probe, Cy5.5. (A) Dorsal whole body in vivo optical images of mice bearing U87MG.EGFRvIII brain tumours at indicated time points after i.v. injection of 50 μg of either anti-IGFBP7 sdAb-Cy5.5 (upper panels), 100 × unlabelled anti-IGFBP7 sdAb followed by anti-IGFBP7 sdAb-Cy5.5 (competitive block, middle panels) or NC sdAb-Cy5.5 (lower panels) (arrows indicate the location of the brain tumour). (B) Graph illustrating changes in the average fluorescence concentration determined from an ROI in the brain tumour region in vivo at indicated times after the i.v. injection of sdAbs-Cy5.5. (C) Optical images of organs ex vivo and dissected brain tumours 24 h after injection of sdAbs-Cy5.5 (D) Graph illustrating the total fluorescence concentration per gram tissue in various organs and dissected tumours imaged ex vivo 24 h after the injection of sdAbs-Cy5.5. In B and D, the data are expressed as mean±s.e.m. for n=5 animals. *Indicates significant difference between anti-IGFBP7 sdAb and both competitively blocked anti-IGFBP7 sdAb and NC sdAb (P<0.01). #Indicates significant difference between competitively blocked anti-IGFBP7-sdAb and NC sdAb (P<0.05).
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fig3: In vivo optical imaging of mice bearing orthotopic glioblastoma tumours after intravenous (i.v.) administration of anti-IGFBP7 sdAb labelled with the near-infrared fluorescent probe, Cy5.5. (A) Dorsal whole body in vivo optical images of mice bearing U87MG.EGFRvIII brain tumours at indicated time points after i.v. injection of 50 μg of either anti-IGFBP7 sdAb-Cy5.5 (upper panels), 100 × unlabelled anti-IGFBP7 sdAb followed by anti-IGFBP7 sdAb-Cy5.5 (competitive block, middle panels) or NC sdAb-Cy5.5 (lower panels) (arrows indicate the location of the brain tumour). (B) Graph illustrating changes in the average fluorescence concentration determined from an ROI in the brain tumour region in vivo at indicated times after the i.v. injection of sdAbs-Cy5.5. (C) Optical images of organs ex vivo and dissected brain tumours 24 h after injection of sdAbs-Cy5.5 (D) Graph illustrating the total fluorescence concentration per gram tissue in various organs and dissected tumours imaged ex vivo 24 h after the injection of sdAbs-Cy5.5. In B and D, the data are expressed as mean±s.e.m. for n=5 animals. *Indicates significant difference between anti-IGFBP7 sdAb and both competitively blocked anti-IGFBP7 sdAb and NC sdAb (P<0.01). #Indicates significant difference between competitively blocked anti-IGFBP7-sdAb and NC sdAb (P<0.05).
Mentions: The in vivo and ex vivo biodistribution analyses of the systemically injected anti-IGFBP7 sdAb ‘tagged' with the near-infrared dye Cy5.5 alone or in combination with the 100-fold excess of unlabelled anti-IGFBP7-sdAb or NC sdAb-Cy5.5 were performed in mice bearing a 10-day-old U87MG.EGFRvIII orthotopic GBM tumour using prospective in vivo optical imaging (Figure 3A). The anti-IGFBP7 sdAb-Cy5.5 homed to the brain tumour as early as 10 min after injection (Figure 3A, upper panels; Figure 3C), with high signal in the tumour persisting up to 24 h after injection. In contrast, brain tumour signal in vivo was reduced at 4 and 24 h when animals were co-injected with the excess unlabelled anti-IGFBP7 sdAb (Figure 3A, middle panels; Figure 3B). NC sdAb-Cy5.5 (Figure 3A, lower panels; Figure 3B) was barely detectable in the brain tumour at any time. At 24 h after injection, brain tumour signal for the anti-IGFBP7 sdAb-Cy5.5 was three- and six-fold higher compared with competitively blocked anti-IGFBP7 sdAb-Cy5.5 and NC sdAb-Cy5.5, respectively (Figure 3B). As sdAbs have a blood half-lives in the range of 20–30 min and are completely cleared from the blood within the first few hours (Iqbal et al, 2010) after injection, the high tumour signal detected at 4 and 24 h with anti-IGFBP7 sdAb-Cy5.5, which was competitively attenuated in the presence of the excess of unlabelled anti-IGFBP7 sdAb, indicates retention of the antibody in the tumour due to its binding to the tumour-expressed antigen.

Bottom Line: In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour.Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biological Sciences, National Research Council of Canada, 1200 Montreal Road, Ottawa, Ontario, Canada K1A 0R6.

ABSTRACT

Background: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications.

Methods: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe₃O₄ nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy.

Results: Surface plasmon resonance analyses revealed a medium affinity (K(D)=40-50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe₃O₄ NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.

Conclusions: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

Show MeSH
Related in: MedlinePlus