Transcript Figure 1

Peptidic and amino-acid tracers labelled with fluor-18
for PET Imaging
Laïque Salma [1], Egrise Dominique [1], Lemaire Christian [2], Lazarova Elena [3], Monclus Michel [1], Schmitz Frédéric [1], Flamand Véronique [3], Jacquemotte
Françoise [4], Luxen André [2], Communi David [5], Goldman Serge [1]
[1]
PET/Biomedical Cyclotron Unit, ULB, Hopital Erasme, Route de Lennik 808, 1070 Anderlecht, BELGIUM
[2] Cyclotron Research Center, ULG, Bldg 30 Building, 4000 Liège, BELGIUM
[3] Institute for Medical Immunology, ULB, Rue A. Bolland, B 6041 Gosselies, BELGIUM
[4] Département des Substances Naturelles, Institut Meurice, 50 Avenue Emile Gryzon, 1070 Anderlecht, BELGIUM
[5] I.R.I.B.H.M. , Bldg. C, ULB, Route de Lennik 808, 1070 Anderlecht, BELGIUM
Study of amino acid tracers labelled with fluor-18, in order to better differentiate tumor from inflammation in PET Scan imaging
Aim : Among labelled amino-acids, FET and FT are transported across the cellular membrane by the exchanger l–system. In vitro utilisation of FET and FT, after preload or prior to after load of non-radioactive L-aminoacids, was evaluated to measure the effects of intra- and extra cellular amino-acid content on the differential tracers uptake in tumor (ROS 17/2.8) and inflammatory cells (human leukocytes).
FET Chemistry :
FT : 2-[18F] fluoro-L-Tyrosine :
incorporated into proteins
HO
NH2
FET : O-(2-[18F] fluoroethyl)-L-Tyrosine :
not incorporated into proteins
HO
O
OH
NH2
O
O
F
F
d) L-phenylalanine load after FET administration on ROS 17/2.8 and leukocytes : see table 1
Biology
a) L-tyrosine preload prior to FET administration on ROS 17/2.8 and leukocytes : see figure 1
Figure 1 : preload effect = ratio between the tracer
uptake into preloaded cells and control cells
b) L-methionine preload prior to FET administration on ROS 17/2.8 and leukocytes : see figure 1
Table 1 : differenciation between ROS 17/2.8 and leukocytes = ratio
between FET content in ROS 17/2.8 and in the inflammatory cells
Conclusions : L–tyrosine preload, prior to FET administration, can help in the differentiation
between tumours and inflammatory lesions : see figure 3
c) L-tyrosine preload prior to FT administration on ROS 17/2.8 and leukocytes : see figure 2
Figure 2 : preload effect = ratio between the tracer
uptake into preloaded cells and control cells
Figure 3 : tracer uptake = % of loaded activity
Study of the synthesis of new peptidic tracers : the 323-339 and 257-264 fragments of Ovalbumine, radiolabeled with fluor-18
Aim : Ovalbumin 323-339 is recognised by DC MHC II complex and induces T-lymphocyte proliferation. We propose to radiolabel this fragment with [18F]Fluoroethyl to allow pulsed-DC PET-tracking in mice. We here present
biological results obtained with the non-radioactive derivative, N-[19F]fluoro-ethyl-OVA323-339 (FEthOVA323-339).
OVA
: NH – Ile – Ser – Gln – Ala – Val – His – Ala – Ala – His – Ala – Glu – Ile – Asn – Glu – Ala – Gly – Arg – OH
323-339
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Chemistry : fluoroethyltosylate was prepared from fluoroethanol and tosyl chloride. The peptide
was build on solid phase made up with Wang resin. OVA323-339, protected on its side chains and
carboxylic function, was N-alkylated by fluoroethyltosylate. The protecting groups were then
removed and the final product was purified by preparative RP-HPLC. The peptide and the neo
peptide, were anaylsed on MS and RP-HPLC.
Results : FEthOVA323-339-pulsed-DC induced T-cell proliferation (see figure 1), at a lower rate, however, than with
OVA323-339 pulsed DC. It did not induce detectable IFN-g production (see figures 2 and 3)
Figure 1 :
Figure 3 :
Figure 2 :
In vitro comparaison
IN VIVO production of INFg by T-lymphocytes
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after immunisation with DC pulsed with commercial OVA and OVA-F
and restimulation in vitro with commercial OVA
IN VITRO producton of INFg after 48h of proliferation of
T-Ly from DO11.10/BALB/c mice
in the presence of DC from BALB/c mice
loaded with commercial OVA-50µg and
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OVA-F in two concentrations-50 and 100 µg
between commercialy available OVA (323-339)*NEOSYSTEM and
the neo-synthetisized OVA (323-339)-CH2-CH2-F19
measured by 3H-thymidin incorporation after 48h MLR
1.4e+5
1800
400
1600
1.2e+5
1400
1.0e+5
IFNg (pg/ml)
8.0e+4
6.0e+4
IFNg production(pg/ml)
1200
1000
800
600
4.0e+4
400
2.0e+4
200
200
100
0
0.0
Biology : FEthOVA323-339 was pulsed on BALB/c-DC and then mixed with DO11.10 T-lymphocytes.
T-cell proliferation was measured by 3H thymidine incorporation. In vitro and in vivo production of
IFN-g was tested by ELISA tests.
300
0.0
0.2
0.4
0.6
0.8
0.00
0.05
0.10
0.15
0.20
0.30
0.35
0
DC/T ratio
DC-Wild Type
DC-LPS activated
WT-OVA-F19 loaded
WT-OVA loaded
LPS act.-OVA-F19 loaded
LPS act.-OVA loaded
0.25
DC-WT
DC WT,OVAc loaded
DC WT,OVA-F19-50µg loaded
DC WT,OVA-F19,100µg loaded
DC-LPS activated
DC+LPS, OVAc loaded
DC+LPS,OVA-F19-50µg loaded
DC+LPS,OVA19-100µg loaded
0
1
2
3
4
5
6
7
different groups of mice
1-non injected mice
2-mice injected with non pulsed DC non restimulated with OVA
3-mice injected with non pulsed DC restimulated with OVA
4-OVA pulsed DC non restimulated with OVA
5-OVA pulsed DC restimulated with OVA
6-OVA-F19 pulsed DC non restimulated with OVA
7-OVA-F19 pulsed DC restimulated with OVA
Conclusions: FEthOVA323-339 holds antigenic properties of OVA323-339. The F-18-labeled form of this
peptide should therefore be evaluated for PET imaging of pulsed-DC migration.
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