STRATEGIES OF REGIOSELECTIVE RADIOLABELING
OF NANOFITIN BINDER FOR IMAGINGGoux M.[1,2,*], Dammicco S.[1], Becker G.[1], Cinier M.[3], Plenevaux A.[1], Tellier C.[2] and Luxen A.[1]
[1] Cyclotron Research Center – University of Liege B30 – 4000 Liège – Belgium ; [2] UFIP UMR CNRS 6286 – University of Nantes B09 – 44322
Nantes – France ; [3] Affilogic SAS – 2 rue de la Houssinière – 44322 Nantes – France ; *contact: [email protected]
Introduction
Conclusions and perspectives
We succeeded to generate a phosphorylatable tag able to chelate terbium(III). Through competition studies, we have shown evidence for a capacity of chelation of zirconium(IV) and
gallium(III). Radiolabeling studies with gallium-68 are on going to evaluate the powerfulness of such a strategy for the chelation of radionuclides. We have also obtained an hypothetic
ADME profile of the Nanofitin NF2 and we are currently making use of its specific binding to a cell-surface receptor to target a very precise cell population by using a new animal
model. Once the phosphorylatable tag optimized for regioselective radiolabelling and the Nanofitin targeting validated in an animal model, the next steps will be to combine these two
approaches: we will fuse genetically the tag to the specific Nanofitin, radiolabel it with gallium-68 and perform the biokinetic study of this new radiopharmaceutical product.
Labelling with fluorine-18
Recently, new strategies emerged in the field of monoclonal antibodies radiolabeling for PET imaging with the use of positron
emitters such as zirconium-89 or gallium-68. Despite their important role in the therapeutic world, antibodies have many
disadvantages related to their structure. Moreover, conjugation of chelating agent often occurs on lysines, which is non-
regioselective and leads to a heterogeneous mixture of products. In addition, the slow clearance of antibodies can be a problem to
obtain a good contrast when they are used in imaging.
To address these different limitations, we developed a chemistry-free chelating system consisting of a phosphorylatable
peptide tag. A specific phosphorylation step can generate a nanocluster of phosphate moieties that can interact strongly with
metal ions like zirconium[1]. We used a peptide sequence which has been selected for its capacity to chelate lantanide ions such as
terbium(III) to optimize this peptide tag and fuse it genetically to a Nanofitin, a protein scaffold developed as an alternative to
antibodies, to ensure an efficient targeting of the radionuclide.
1) Adapt the labeling tag to the stereoselective chelation of rgallium-68 for PET imaging.
2) Validate the use of Nanofitin as a potent alternative tool for in vivo imaging.
Objectives:
Small Protein: 10kDa
pH stability: 0-12
Temperature Tm≈80°C
Stability:
Production: Generated in bacteria
Affinity: nM
What are Nanofitins ?
BioForum 2015May 13, 2015
Liège, Belgium
Thesis project funded by “Région
Pays de la Loire”, into the Erasmus
Mundus programme NanoFar
Method:
Fluorescence ?+
UV
+ +
[Zr(NTA)2]2-
Fluorescence ?+
UV
+Ga3+
In vitro
phosphorylation
+LBT
KD(Tb3+) ≈ nMNanofitin
UV
Fluorescence
Gene fusion
Mutation
UV
Fluorescence ?
LBT 1S LBT 1SP
Tb3+
NB : Terbium emits fluorescence intrinsically. In aqueous solution, water molecules quench fluorescence and chelation prevents this quenching by expelling water molecules.
0
5
10
15
20
25
30
35
%ID
/g
(g
-1)
Uptake of the [18F]-FBEM-NF2 2h40 p.i. (n=4)
Method:
PET
2h
MRISacrifice and organ
harvesting
10 MBq
18F-FBEM-S
PBS pH7.4 10 min
1) Automatic synthesis of [18F]-FBEM and radiolabeling of Nanofitin NF2 (Dammicco S. et al.)
2) Injection of the Nanofitin radiolabeled in balb/c mice and PET/MRI imaging
0
10
20
30
40
50
60
70
80
90
100
0 0,5 1 1,5 2 2,5 3 3,5 4
Flu
orescen
ce 5
44
nm
(%
)
[Tb3+] (µM)
Terbium(III) titration of protein and competitionwith Zr(NTA)2 in HEPES buffer pH7
LBT (1µM) (n=4) LBT + Zr4+ (1:1) (1µM) (n=4)
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50
Flu
orescen
ce 5
44
nm
(%
)
[Tb3+] (µM)
Terbium(III) titration of protein and competition with GaCl3 in MES buffer pH5.5
LBT (1µM) (n=2) LBT (1µM) + Ga3+ (1:1) (n=2)
LBT (1µM) + Ga3+ (1:1000) (n=2)
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8
Flu
orescen
ce 5
44
nm
(%
)
[Tb3+] (µM)
Terbium(III) titration of protein and competition with Zr(NTA)2 in HEPES buffer pH7
LBT 1SP (1µM) (n=4) LBT 1SP + Zr4+ (1:1) (1µM) (n=2) LBT 1SP + Zr4+ (1:4) (1µM) (n=3)
Chelation with gallium
0
5
10
15
20
25
30
35
0 20 40 60 80 100 120
%ID
/g
(g
-1)
Time (minutes)
Uptake kinetic of the [18F]-FBEM-NF2 (n=4)
Kidney Liver
18F-FBEM-S
30 ± 2 % (n=4) 37 ± 10 % (n=4)
(0,046 ± 0,014 % of radiolabeled Nanofitin)
Cys
10 nmol
After radiolabeling of the Cys-tagged Nanofitin NF2 with [18F]-FBEM, the protein
is injected in mice to evaluate its biokinetic. It seems that the Nanofitin is metabolized
by the liver and reabsorpted in the cortical area of the kidneys. The metabolites
obtained are excreted by kidneys through urine and by the liver via biliary excretion to
the gut with feces.
To increase the affinity for radionuclide, we worked on a sequence derived from
calcium-binding proteins to chelate specifically lanthanides[2]. We optimized this
sequence by incorporating a phosphate nanocluster to improve the chelation with
radionuclides [3].
- Affinity for terbium(III) is in the sub-micromolar range for the lanthanide-binding tag
fused to the Nanofitin and in the micromolar range for the mono-phosphorylated.
- Chelation of zirconium and gallium by the peptide tag was observed by a
competition study.
References : [1] Cinier M. et al. (2012), Journal of Biological Inorganic Chemistry, 17, pp.399–407 ; [2] Martin L. J. et al. (2007), Journal of American Chemistry Society, 129(22),7106–7113 ; [3] Pardoux R. et al. (2012), PLoS ONE, 7(8).
kidney
Liver
Intestine
Bladder
Coregistred coronal sections of MRI and two-hours duration PET after injection of the NF2 radiolabeled
Analytical HPLC (λ = 254 nm) of the siRNA [18F]10