Table 1.
DNA sequences of oligonucleotides used in this study.
Fig 1.
Identification of a GD2-specific binding peptide and binding characteristics of WHWRLPS-phage in vitro.
A) Individual phage clones (n = 12) derived from a phage display screen were tested for binding to immobilized human GD1b, immobilized human GD2, or to BSA-coated surfaces. Phage clone 7 displayed the peptide sequence WHWRLPS and bound with high affinity to human GD2, and to a lesser extent, to human GD1b. B) Binding affinity of WHWRLPS-phage (black bars) to GD3, GD2, GD1b, GT1, BSA or polystyrol was tested and compared to control phage (white bars). “*” = p < 0.05 vs. GD2 binding of the WHWRLPS-phage. C) Mutations in the displayed heptapeptide and resulting affinities to GD2. * p < 0.01 vs. unmutated WHWRLPS-phage.
Fig 2.
The WHWRLPS-peptide inhibits IMR32 cell viability, binds specifically to human GD2 and displays tumour homing in vivo.
A) Cell viability of human neuroblastoma cells (IMR32) in the presence of the WHWRLPS peptide (1 μg [black] or 10 μg [dark grey]), a control peptide (1μg [white] or 10 μg [light grey]) normalised to buffer control (PBS). “*” = p<0,05 vs. PBS control, “†” = p<0,05 vs. control peptide. B) Competition experiments were performed in vitro to evaluated phage binding in the presence of soluble WHWRLPS-peptide. Shown are phage output titers using different peptide concentrations for competition. “*” = p < 0.05 vs. 0 μg peptide. C) WHWRLPS-phage or control phage (GHGRLPS) were injected i.v. mice bearing xenografts induced by s.c. injection of IMR32 cells. After 10 minutes of circulation the mice (n = 6 in each group) were sacrificed, the tumours and the organs were explanted and the number of phage present was determined. Results are shown as fold change compared to control phage (mean values ± SEM, “*” = p < 0.05 vs. control phage). D) WHWRLPS-phage and 200 μg WHWRLPS-peptide were coinjected i.v. in tumour-bearing mice (n = 6). Mice were sacrificed 10 minutes later, the tumours and the organs were explanted and the number of phage present was determined. Representative results of 3 independent experiments are shown (mean values ± SEM,”*” = p < 0.01 vs. control phage).
Fig 3.
Tumour-homing of the 68Ga and 111In -labelled DOTA-peptide WHWRLPS compared to 124I-MIBG.
A) The WHWRLPS-DOTA peptide was labelled with 111In and i.v. injected into mice (n = 3) bearing xenografts induced by s.c. injection of IMR32 cells. Mice were sacrificed at indicated time-points, tumours and organs were explanted and their activity concentration measured. Results are presented as mean values ± SEM. B) Tumour/blood ratio (black bars) and tumour/muscle ratio (white bars) of 111In- labelled WHWRLPS peptide using the data obtained in 3A, p.i. = post injection. C) The 68Ga- or 111In-labelled WHWRLPS-DOTA peptides (black bars and grey bars, respectively) were injected i.v. in IMR32 tumour-bearing mice (n = 3). As a control, 124I -MIBG (white bars) was administered to two mice. Mice were sacrificed after one hour, the tumour and the organs were explanted and their activity concentration was quantified. Results are presented as mean values ± SEM.
Fig 4.
PET imaging of tumour using the 68Ga -labelled DOTA-peptide WHWRLPS.
A) For kidney protection, a mixture of Gelofundine, lysine and arginine was i.v. injected in tumour-bearing mice prior to i.v. injection of the 68Ga-labelled WHWRLPS peptide. After one hour, mice were sacrificed, tumours and organs were explanted and their radioactive content was quantified. All values were corrected for tissue weight and radioactive decay. Results are presented as mean values ± SEM (black bars: no kidney protection, white bars: with kidney protection as described above). B)-D) The 68Ga-labelled WHWRLPS peptide was i.v. injected in IMR32 tumour-bearing mice. After one hour, peptides were detected using a PET/MRT scanner. B) PET image, C) MRI image and D) merge of B) and C).