An average of 5.2±0.2, 4.1±0.2 and 1.9±0.1 molecules of 1,4,7,10-tetraazacyclododecane-N, N',N'',N'''-tetraacetic acid (DOTA) were conjugated to the IgG, F(ab')2
and Fab forms of cG250. This represents a w/w DOTA loading of 1.3, 1.3 and 1.2%. Specific activities of 370 MBq/mg were easily achieved with labeling efficiencies of >90% and chemical purity of >99.9%. The immunoreactivity of these radiolabeled antibodies was preserved at >90% (Figure S1
). The binding affinity for CAIX expressed by SKRC-38 cells was 2.5 and 1.8 nM for the IgG and F(ab')2
forms and 14 nM for the Fab fragment. The saturation binding data for all three forms of cG250 gave around 1.2×106
sites/cells (Figures S1A, S1B and S1C
The biodistribution of 111
In-DOTA-cG250 in nude mice with HT29 tumors () showed an initial high uptake of radioactivity (20.1±4.8%ID/g) at 2 days p.i.. However, the biodistribution of two similarly-labeled irrelevant antibodies (J591 and 3S193) also showed relatively high uptakes of 7.4±2.8 and 8.1±2.7%ID/g (data not shown). The biodistribution in all other organs was similar for all three radiolabeled IgGs. The uptake of the cG250 IgG increased marginally to 26.9±6.8 and 26.4±5.7%ID/g at days 4 and 7 p.i. (). By comparison the uptake by the irrelevant antibodies decreased to 1.7±1.1 and 3.8±0.8 for 111
In-J591 and 111
In-3S193. The activity slowly cleared from all organs except the liver and spleen and this clearance mimicked the overall blood clearance. Similarly the tumor/non tumor ratios increased over the 7 day period for all organs except liver and spleen. By 7 days post injection, the tumor/muscle ratio was 69 (Figure S2A
). The tumor/blood ratio was moderate at 6.6.
Biodistribution of 111In labeled intact and fragmented cG250 in athymic mice with hypoxic HT29 colorectal tumors.
The radiolabeled F(ab')2
fragment of cG250 showed lower absolute tumor uptakes of 7.6±1.4 and 9.3±2.1%ID/g at 6 and 24 hours post injection (). The tumor/muscle ratio was only 8.9 at 24 hours p.i. compared to 69 for the IgG at 7 days p.i.. The tumor/blood ratios were lower than the IgG and were 0.9 and 4.6 at 6 and 24 hours p.i. (Figure S2B
The Fab fragment had the lowest tumor uptake of 3.6±1.3 and 3.5±1.7%ID/g at 6 and 24 hours post injection (). This low tumor uptake is due to its much faster clearance from circulation (0.20±0.02%ID/g blood at 24 h p.i.). At 6 hours p.i. tumor/muscle ratios were 4.8, but the tumor/blood ratios were still poor at 2.8 (Figure S2C
) and similar to the IgG at 4 days p.i. At 24 hours p.i. tumor/muscle ratios had increased to 6.7., but the tumor/blood ratios were 16.6 (Figure S2C
) and higher than either the IgG at 7 days or the F(ab)2
at 24 hours p.i..
Macroscopic examination of the tumor sections at 2 days post injection of 111In-cG250 () shows areas of high perfusion around the rim of the tumors as well as discrete areas within the core (blue). The pimonidazole and endogenous CAIX staining (green and red) are generally in good agreement with some minor variations which presumably arise from short-lived fluctuations in tumor perfusion and slow rates of CAIX degradation. The graphical correlation of endogenous CAIX and pimonidazole staining in was linear with a slope of 1 (c.f. ) illustrating that the endogenous CAIX expression is restricted to hypoxic regions of the tumor. Similarly there was a negative correlation between the tumor perfusion and CAIX expression ().
Images of the same HT29 tumor section showing blood perfusion, in vivo targeting of CAIX by 111In-DOTA-cG250, endogenous CAIX expression and hypoxia at 2 days p.i..
Binned pixel by pixel analysis of 111In-DOTA-cG250 distribution, tumor hypoxia, endogenous CAIX expression and blood perfusion in single HT29 tumor sections.
These images also show an initial heterogenous and focal uptake of radioactivity at 2 days p.i. () which progressively becomes more diffuse over the 7 day period (see Figures S3
). The enlarged microscopic views () show the anti-correlation between the Hoechst perfusion marker (blue) and the endogenous CAIX (red). The pixel by pixel analysis of this tumor section () graphically illustrates the correlation of 111
In activity and perfusion (Hoechst) in the tumor with a slope approaching 1 at 2 days post injection. The correlation between 111
In and CAIX (and pimonidazole) is quite flat at high 111
In levels indicating that there is some targeting of CAIX fairly close to the blood vessels, but this uptake is not directly proportional to the endogenous CAIX expression. At 4 and 7 days post injection the sections show a more diffuse pattern of 111
In activity (Figure S3
). The digital analysis () clearly show the progressive clearance of 111
In from well perfused areas of the tumor and the slope of the curve approached -1 at 7 days postinjection. The correlation of 111
In with endogenous CAIX improves over time and the wider spacing between the binned 111
In data points is indicative of a more diffuse distribution of radioactivity within the tumor section. These findings confirm the generally accepted view that IgGs and large molecules can have early high uptakes at the tumor due to the tumor vasculature being permeable to such large molecules. However these data show that by 7 days post injection the antibody has diffused away from the tissue adjacent to the vasculature and moved to the poorly perfused hypoxic regions rich in CAIX.
The two control antibodies at 7 days p.i. (Figures S5
) showed some activity in well perfused tumor regions, little activity in hypoxic CAIX regions, but also significant amounts of radioactivity in tumor regions which were furthest away from the vasculature. The pixel by pixel analysis of the data (Figure S7
) showed an inverse relationship between 111
In uptake and CAIX immunofluorescence which was the inverse of that seen with the CAIX-specific cG250 (). The correlation between 111
In activity and tumor perfusion was comparable to the early cG250 data at 2 and 4 days p.i. (). because most of the activity was still in the well perfused tumor regions.
Binned pixel by pixel analysis of 111In-DOTA-F(ab')2-cG250 distribution, endogenous CAIX expression and blood perfusion in single HT29 tumor sections.
The analysis of the fluorescent images for the tumor sections removed from animal injected with 111
-cG250 at 6 and 24 hours post injection (Figures S8
) is shown in . For the 6 hour p.i. image, the distribution of 111
In and the correlation with perfusion and endogenous CAIX is similar to that seen with the intact IgG at 2 day p.i.. At 24 hours p.i., the 111
In is almost ideally coregistered with the endogenous CAIX and there is almost an inverse correlation with Hoechst staining, except for the highest 111
In data points. These data show that the distributions of 111
-cG250 at 24 hours p.i. is comparable to the intact IgG at 7 days post injection with excellent targeting of CAIX, but some residual activity in well perfused and non-hypoxic areas. These data reflect the fact that the F(ab')2
fragmented antibody clears more rapidly from the blood and can more rapidly diffuse into hypoxic area rich in CAIX than the intact IgG, but at the optimum time the absolute tumor uptake of the F(ab')2
fragment is around 3 times lower and non specific uptake by the liver, spleen and kidneys is considerably higher.
This fast macroscopic blood clearance observed in the biodistribution studies is clearly shown on the microscopic tumor localization of the Fab at 6 (Figure S10
) and 24 h p.i. () where there is a diffuse uptake in the central poorly perfused region of the tumor, which has high CAIX expression. The corresponding digital analysis () show much more rapid blood clearance and specific uptake in CAIX rich regions. In particular the 24 hour data () gives a slope of around -1 for the perfusion correlation and +1 for the CAIX correlation. These images, and the digital analysis, clearly show that the Fab fragment is the most rapid and reliable indicator of CAIX expression in hypoxic tumor areas. However this comes at the cost of around a tenfold reduction in the absolute tumor uptake and a tenfold increase in renal retention of radioactivity.
HT29 tumor sections showing blood perfusion, in vivo targeting of CAIX by 111In-DOTA-Fab-cG250 and endogenous CAIX expression at 24 hours pi.
Binned pixel by pixel analysis of single HT29 tumor sections showing the correlation of in vivo targeting of CAIX by 111In-DOTA-Fab-cG250 with endogenous CAIX expression and blood perfusion.