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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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Related in: MedlinePlus

Protein sequence and surface plasmon resonance analysis of the anti-IGFBP7 sdAb 4.43. (A) Protein sequence of anti-IGFBP7 sdAb 4.43; CDR1, CDR2 and CDR3 are underlined. (B) Sensogram overlay showing 4.43 monomer binding to immobilised IGFBP7 at concentrations of 25, 25, 50, 50, 75, 75, 100 and 200 n. (C) Fitting of equilibrium binding data to a steady-state affinity model. (D) Scatchard plots of the equilibrium binding data.
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fig1: Protein sequence and surface plasmon resonance analysis of the anti-IGFBP7 sdAb 4.43. (A) Protein sequence of anti-IGFBP7 sdAb 4.43; CDR1, CDR2 and CDR3 are underlined. (B) Sensogram overlay showing 4.43 monomer binding to immobilised IGFBP7 at concentrations of 25, 25, 50, 50, 75, 75, 100 and 200 n. (C) Fitting of equilibrium binding data to a steady-state affinity model. (D) Scatchard plots of the equilibrium binding data.

Mentions: Based on phage ELISA results (data not shown), two clones showed binding to IGFBP7, clones 4.43 and 4.46. In SPR analyses, clone 4.43 showed strong binding to antigen but the affinity of clone 4.46 for IGFBP7 was in the low micromolar range (data not shown). As a result, only clone 4.43 was selected for further analysis both in vitro and in vivo studies. The binding data for 25 n 4.43 (Figure 1A) fit quite well to a 1 : 1 model, giving a ka of 6.6 × 104 m s−1, a kd of 1.3 × 10−3 s−1 and a KD of 20 n. However, at higher 4.43 concentrations the 1 : 1 fitting was poor with the dissociation phases being clearly biphasic (Figure 1A). Nevertheless, the equilibrium binding data showed good fitting to a steady state affinity model (Figure 1B) and gave a linear Scatchard plot (Figure 1C) from which KD's of 40 and 50 n, respectively, were derived. Inexplicably, the observed Rmax for the equilibrium data was several fold higher that the theoretical Rmax for a 1 : 1 interaction in which monovalent sdAb binds to an immobilised antigen without repeating sequences, an observation that may compromise the kinetic and affinity constant calculations.


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)

Protein sequence and surface plasmon resonance analysis of the anti-IGFBP7 sdAb 4.43. (A) Protein sequence of anti-IGFBP7 sdAb 4.43; CDR1, CDR2 and CDR3 are underlined. (B) Sensogram overlay showing 4.43 monomer binding to immobilised IGFBP7 at concentrations of 25, 25, 50, 50, 75, 75, 100 and 200 n. (C) Fitting of equilibrium binding data to a steady-state affinity model. (D) Scatchard plots of the equilibrium binding data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990581&req=5

fig1: Protein sequence and surface plasmon resonance analysis of the anti-IGFBP7 sdAb 4.43. (A) Protein sequence of anti-IGFBP7 sdAb 4.43; CDR1, CDR2 and CDR3 are underlined. (B) Sensogram overlay showing 4.43 monomer binding to immobilised IGFBP7 at concentrations of 25, 25, 50, 50, 75, 75, 100 and 200 n. (C) Fitting of equilibrium binding data to a steady-state affinity model. (D) Scatchard plots of the equilibrium binding data.
Mentions: Based on phage ELISA results (data not shown), two clones showed binding to IGFBP7, clones 4.43 and 4.46. In SPR analyses, clone 4.43 showed strong binding to antigen but the affinity of clone 4.46 for IGFBP7 was in the low micromolar range (data not shown). As a result, only clone 4.43 was selected for further analysis both in vitro and in vivo studies. The binding data for 25 n 4.43 (Figure 1A) fit quite well to a 1 : 1 model, giving a ka of 6.6 × 104 m s−1, a kd of 1.3 × 10−3 s−1 and a KD of 20 n. However, at higher 4.43 concentrations the 1 : 1 fitting was poor with the dissociation phases being clearly biphasic (Figure 1A). Nevertheless, the equilibrium binding data showed good fitting to a steady state affinity model (Figure 1B) and gave a linear Scatchard plot (Figure 1C) from which KD's of 40 and 50 n, respectively, were derived. Inexplicably, the observed Rmax for the equilibrium data was several fold higher that the theoretical Rmax for a 1 : 1 interaction in which monovalent sdAb binds to an immobilised antigen without repeating sequences, an observation that may compromise the kinetic and affinity constant calculations.

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