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of the radiolabelled reactive small molecule [68Ga]-5.1 Cell uptake
studies were performed using high (A431) and low (HCT116)
EGFR-expressing cells, showing a significant and concentration-
dependent activity retention in A431 cells pre-incubated with the
TCO-modified Cetuximab 7, followed by incubation with [68Ga]-5
(Fig. S8, ESI†). Pre-incubation with non-modified Cetuximab 6
neutralised cell activity retention, and overall no detectable
activity above background was found in HCT116 cells, highlight-
ing the specificity and sensitivity of the pretargeting strategy.
These data were comparable to the direct labelling setting with
[68Ga]-8 (Fig. S8, ESI†). We then performed in vivo PET imaging
in A431-xenograft bearing mice, where the animals were pre-
treated with TCO-modified Cetuximab 7 for 3 or 23 h, followed
by injection of B1.85 MBq of [68Ga]-5. The PET image analysis
indicated high retention of the activity in the liver, which is
consistent with the characteristic pharmacokinetic profile of a
mAb (Fig. S6, ESI†). Monoclonal antibodies, while having high
affinity for targets of interest, also have inherently slow phar-
macokinetics with high initial delivery to liver. The pretargeting
strategy aims at conducting imaging studies when background
distribution of the macromolecule in non-tumour tissue has
decreased sufficiently to permit tumour-specific contrast. This
aim is achieved in the 23 h protocol compared to the 3 h protocol.
The PET images and derived data indicated no tumour detection
with the 3 h treatment protocol, most probably attributed to the
high concentration of antibody present in circulation at this
time point, leading to the subsequent quenching of the injected
[68Ga]-5 in circulation and possibly lower concentration of the
antibody at the tumour site, consequently tumour radioactivity
was lower in the 3 h compared to 23 h treatment protocol
(0.86 %ID mLÀ1 and 3.48 %ID mLÀ1 at 60 min, respectively,
Fig. 3; Fig. S6 and S7, ESI†). Moreover, we showed the significant
superiority of the pretargeting approach over the traditional direct
labelling method, through the analysis of the tumour to liver ratio,
indicating a key high tumour to background signal when using
the 1-day pretargeting protocol (T/L ratio 2.64, Fig. 3). It was noted
that tumour uptake of 68GaCl3 (from g-counting) was 5 %ID gÀ1
compared to that of [68Ga]-5 and pretargeting at 1.8 and
3.34 %ID mLÀ1, respectively. While the time points of measurement
are different, this observation initially questions the specificity of the
pretargeting strategy. 68GaCl3 is a small molecule and its associated
high non-specific uptake in some tumours has been reported,
although the mechanism is not entirely clear.20 In our study, this
high uptake is only relevant if the metal is rapidly eliminated from
the [68Ga]-5-complex. However, tumour uptake of [68Ga]-5 was found
to be low precluding this possibility. The higher tumour uptake of
radioactivity with the pretargeting strategy, compared to [68Ga]-5,
therefore infers specific localisation.
Fig. 2 (a) Synthesis of TCO-modified Cetuximab 7 and (b) radiolabelling
via the IeDDA reaction with [68Ga]-5 to form dihydropyrazine [68Ga]-8.
imaging studies with either 68GaCl3 or [68Ga]-5 to investigate
their corresponding pharmacokinetic profiles. 68GaCl3 accumu-
lated in plasma and peripheral tissues, notably in relation to the
small size of the tracer, resulting in overall high background activity
(Fig. S4a and S5, ESI†). [68Ga]-5 was characterised by a rapid tissue
distribution and subsequent clearance through the urinary route,
yielding a low overall background activity (Fig. S4b and S6, ESI†).
Neither 68GaCl3 nor [68Ga]-5 achieved significant accumulation in
the tumour (Fig. 3; Fig. S4, S6 and S7, ESI†). We then performed
PET studies where [68Ga]-8 was injected into animals and allowed
to distribute for 3 h, followed by 60 min PET imaging; this showed
sustained retention of the tracer and significant tumour detection
(3.34 %ID mLÀ1 at 60 min, compared to 0.60 %ID mLÀ1 for
[68Ga]-5, Fig. 3 and Fig. S7, ESI†). As anticipated, the PET image
analysis also indicated a high retention of the activity in the
liver, which is characteristic of the pharmacokinetic profile of a
mAb (Fig. S6, ESI†).
We further assessed an in vivo pretargeting strategy, where
the delivery of a radionuclide is separated from that of the mAb, and
has the anticipated advantage of allowing for the TCO-modified
Cetuximab to achieve optimal tumour accumulation and sufficient
blood and non-tumour tissue clearance, before administration
In summary, we have developed a rapid and efficient method
for the 68Ga-labelling of a clinically relevant mAb, using the IeDDA
reaction, and in high radiochemical yield. This represents a mild
and efficient strategy for radiometal incorporation when compared
to existing methods, which rely on direct labelling of mAbs
containing functional chelates. We have demonstrated the utility
of the labelling strategy through in vivo application for non-
invasive PET imaging of EGFR, highlighting the potential utility
Fig. 3 ‘Direct’ and ‘pretargeted’ PET imaging of EGFR-expressing A431
tumours. (a) Representative axial PET images of animal injected with
[
68Ga]-5 (n = 3), [68Ga]-8 (n = 5), TCO-modified Cetuximab 7 for 3 or
23 h followed by [68Ga]-5 (‘‘pretargeting’’, n = 3 and 6, respectively). All
scans were 60 min dynamic acquisition following a bolus injection of
B1.85 MBq of activity. The white arrowheads indicate the tumours. (b) PET
extracted variables are shown: normalised tumour uptake at 60 min, area
under the tumour TAC from 30 to 60 min.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 9557--9560 | 9559
Evans, Helen L.
