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