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Human antibodies targeting all subtypes of mesothelioma could be useful to image and treat this deadly disease. Here we report tumor targeting of a novel internalizing human single chain antibody fragment (scFv) labeled with 99mTc (99mTc-M40) in murine models of mesothelioma of both epithelioid (M28) and sarcomatoid (VAMT-1) origins. 99mTc-M40 was taken up rapidly and specifically by both subtype tumor cells in vitro, with 68–92% internalized within 1h. The specificity of binding was evidenced by blocking (up to 95%) with 10-fold excess of unlabeled M40. In animal studies, tumors of both subtypes were clearly visualized by SPECT/CT as early as 1h post-injection of 99mTc-M40. Tumor uptake measured as percent of injected dose per gram tissue (%ID/g) at 3h was 4.38 and 5.84 for M28 and VAMT-1 tumors respectively, significantly greater than all organs or tissues studied (liver, 2.62%ID/g; other organs or tissues <1.7%ID/g), except the kidneys (130.7%ID/g), giving tumor-to-blood ratios of 5:1 and 7:1 and tumor-to-muscle ratios of 45:1 and 60:1, for M28 and VAMT-1 respectively. The target-mediated uptake was confirmed by a nearly 70% reduction in tumor activity following administration of 10-fold excess of unlabeled scFv. Taken together, these results indicate that M40 can rapidly and specifically target epithelioid and sarcomatoid tumor cells, demonstrating the potential of this agent as a versatile targeting ligand for imaging and therapy of all subtypes of mesothelioma.
Malignant mesothelioma (MM), caused primarily by exposure to asbestos, is a highly aggressive tumor arising from serosal surfaces of the pleura, peritoneum and pericardium (1, 2). MM has three major subtypes; epithelioid (EM) that is more likely to respond to treatment and accounts for 50–70% of all cases, sarcomatoid (SM) that rarely responds to any treatment and represents 7–20%, and mixed/biphasic for the remaining 20–30 %. MM has a median survival time of 8–14 months (3). There is an urgent unmet need to develop new diagnostics and therapeutics for MM (4) as the disease has a long latency period with past and ongoing exposure to asbestos contributing to the development of new cases worldwide.
One approach to detect and treat cancer is to conjugate imaging and/or therapeutics to molecules which can recognize internalizing antigens, receptors or cell surface markers that are over-expressed on tumor cells, leading to efficient localization and tumor cell killing (5, 6). However, presently there are very few MM-associated cell surface antigens that are over-expressed by all subtypes of MM, especially the SM (7). One well established marker—mesothelin, a 40kDa cell surface glycoprotein, has been reported to be expressed by EM cells (8), but not SM (9). Recently we have identified a panel of internalizing human scFv antibodies by phage display selection that target cell surface antigens associated with both EM and SM (6). The selected scFvs bind to human mesothelioma cells in situ, thereby recognizing clinically represented tumor antigens (6) and thus offer the potential to deliver high levels of imaging probes to tumor cells but not to non-target normal tissues based on intracellular delivery strategies. By screening the yeast surface human cDNA display library with mesothelioma targeting phage antibody, we have further identified MCAM/CD146/MUC18 as one of the target antigens for MM cells that was over-expressed in >80% of EM and SM tissues, but not other tissues (10). In the present study, we investigated both the in vitro and in vivo tumor targeting and imaging potential of an additional scFv (M40) for both EM (M28) and SM (VAMT-1) subtypes.
The scFv was radiolabeled as previously reported (14, 16). The carbonyl-kit (IsoLink® Tyco/Mallinckrodt) was used to prepare the [99mTc(CO)3] moiety. An aliquot (40–60 μg) of M40 was mixed with 100–500 μL of [99mTc(CO)3(OH2)3]+ solution and the mixture was heated at 37°C for 60 min. The product was purified using a PD-10 column. Labeling efficiency and purity were determined by size-exclusion- HPLC and thin-layer-chromatography (TLC).
The M40 was labeled with Cy5.5 by incubation with 3:1 molar excess of Cy5.5-NHS ester in a carbonate/bicarbonate buffer (pH 7.2–8.5) for 1 h followed by purification using PD-10 column.
Internalization experiments were performed as described previously (12, 15, 16). Briefly, 1 million VAMT-1, M-28 or BPH-1 cells (control) were incubated with 0.05–2 μCi 99mTc-M40 in various concentrations for 1 h or 3 h. All cell lines had been tested for mycoplasma contamination and characterized by cell proliferation and morphology evaluation (6). The cells were washed to determine cell surface–bound (acid releasable) and internalized (acid resistant) radioligand/radioactivity expressed as the percentage of applied activity normalized to 1 million cells. For nonspecific uptake, the above procedure was repeated after addition of 10 fold excess unlabeled M40. For microscopy study, 1 μM (10 μl) of Cy5.5-M40 was incubated with tumor cells for 1 and 3 h. The cells were washed with PBS and then imaged using a TE2000-E Fluorescence Microscope (Nikon Inc., Japan) at 20× magnification.
Animal procedures were performed according to a protocol approved by the UCSF Animal Care and Use Committee. Six-week-old male nu/nu mice were purchased from Charles River Laboratories. For tumor inoculation, 106 M-28 and VAMT-1 cells in 200 μL of PBS were administered subcutaneously into the right and left shoulders of the animal respectively. The mice were studied when tumor size reached ~3–5 mm in diameter (~20–60 mm3 in volume). Tumor mice in groups of 4 animals were injected each with 18.5–37 MBq (0.5–1.0 mCi) of 99mTc-M40 containing ~2–4 μg of scFv. A control group (blocking study) were injected with 10 fold excess unlabeled M40 1h before 99mTc-M40. The biodistribution at 1, 3 or 6 h of the study group was determined and compared with that of the control group.
Mice were imaged with a small animal SPECT/CT system (GE healthcare). For anatomical correlation, CT was first performed after injection. SPECT imaging was initiated 1h and 3h after injection.
Mice were fasted overnight prior to 18F-FDG injection of 3.7–7.4 MBq [100–200 μCi] and imaged with a microPET/CT system (Siemens Medical Solutions USA, Inc., PA) 1 h after injection for a 20 min acquisition time.
The 99mTc labeling yield of M40 (from [99mTc(CO)3(OH2)3]+) was 70–85%. The radiochemical purity of the 99mTc-M40 was greater than 95%. The final 99mTc-M40 had a high specific activity of ~9.3 MBq/μg (6.3 ×105 Ci/mol). The 99mTc radiolabeling was stable in phosphate buffer for at least the duration investigated (24 h), as reported previously (14, 16).
The binding affinity of 99mTc-M40 to the mesothelioma cells exhibited an apparent Kd of 20–21 nM and the antigen density for M40 derived from the saturation cell binding assay was comparable on both tumor cells (Figure 1A, 1B). M40 was rapidly internalized over the concentrations tested with about 68–92% of total cell-associated following 1h incubation at 37°C (Figure 1C) whereas the binding and uptake in the control BHP cells was much less (Figure 1C). Specificity was further demonstrated by blocking of uptake/internalization into both M28 and VAMT-1 (>95%) cells with a 10-fold excess of unlabeled M40 (Figure 1C).
As shown in Figure 1D, Cy5.5-M40 was rapidly internalized into mesothelioma cells within 1h after incubation at 37°C, whereas there was negligible uptake in the control (BPH-1) cells under identical conditions.
Mice were imaged with small animal SPECT/CT at 1h and 3h after injection with concomitant assessment of ex vivo biodistribution at 1h, 3h and 6h (Figure 2A). Imaging with SPECT/CT showed high tumor uptake (Figure 2B) as early as 3h post injection. At 3h, tumor uptake was 4.38 %ID/g and 5.84 %ID/g for M28 and VAMT-1 tumors respectively (Table 1), greater than for all organs/tissues studied (liver 2.62%ID/g, other organs/tissues <2 %ID/g) except the kidney (130.7%ID/g), giving M-28 and VAMT-1 tumor/muscle ratios of 45:1, 60:1 and tumors/blood ratios of 5:1 and 7:1 respectively (Figure 2A). In contrast, the control blocking study with 10-fold excess of unlabeled M40 at 3h reduced by more than 70% of tumor uptake of the 99mTc-M40 to 0.9 %ID/g and 1.6%ID/g for M28 and VAMT-1 respectively (Table 1 and Figure 2C).
18F-FDG PET imaging was performed to confirm the distinct identity of the two subtypes in single animal. As shown in Figure 2D, the 18F-FDG PET/CT image at 1h detected SM (VAMT-1) preferentially.
We report here the in vitro and in vivo tumor targeting and imaging potential of a rapidly internalizing human scFv (M40) selected from a panel of scFvs targeting internalizing epitopes present on both EM and SM cells (6).
Several important findings have been uncovered in this study. Firstly, the 99mTc-M40 showed strikingly rapid and selective binding and internalizing ability in vitro into the mesothelioma tumor cells but not into the control non-tumorigenic cells, consistent with our previous findings (6). Although we did not perform the Lindmo assay (17) to determine what percentage of our labeled antibodies retained reactivity (the immunoreactive fraction), our results nonetheless demonstrate that the M40 binds with high affinity to both subtypes of mesothelioma cells and that either the site-specific labeling of M40 with 99mTc through the hexahistidine tag or conjugation to Cy5.5 does not significantly affect its targeting ability. Secondly, while the M40 accumulated in the tumors, its clearance from blood and other normal organs was rapid (except from the kidney which is the site for all scFv clearance) making it feasible to label the M40 with positron-emitting residualizing radiometal isotopes with short half-lives such as gallium-68 (68Ga) for quantitative PET imaging. Finally, unlike other available antibodies against MM, this antibody has the ability to target both EM and SM. If such dual targeting can be maintained in the clinical setting, this antibody would have a major imaging and therapeutic potential.
The 99mTc-M40 had a high uptake in the kidneys as observed with other 99mTc-scFvs (10, 16), consistent with the route of scFv clearance in vivo. Nonetheless, additional engineering of M40 may further increase the kidney clearance rate and improve the contrast. In addition, developing other forms of the antibody such as diabody, minibody, or Affibody (Affibody AB) could further improve the tumor binding and homing efficiency as well as overall pharmacokinetic profile (10, 18).
The 99mTc-M40 rapidly accumulated into both M28 and VAMT-1 cells as early as 3h with comparable tumor uptake at 4.38 ±0.39%ID/g) and 5.84±0.72ID%/g respectively. Because we observed the difference of 18F-FDG uptake in our system between the two cell types, M28 and VAMT-1, with approximately 8%ID/g and 16%ID/g respectively similar to other reports (19, 20), 18F-FDG was used to confirm the identity of the two subtypes implanted in a single mouse and thus to highlight the dual targeting by M40. The characteristics of 99mTc-M40 are representative of a promising new class of radiotracers that recognize cell surface markers highly expressed at comparable level on both types of mesothelioma (Figure 1A & 1B). As such, they may find utility for tumor characterization and staging as well as treatment planning and monitoring complementary to 18F-FDG.
In conclusion, we demonstrated that 99mTc-M40 selectively binds and internalizes into both M28 and VAMT-1 cells in vitro, and furthermore rapidly and specifically targets both subtypes of mesothelioma in vivo, demonstrating its potential as a novel agent for imaging and therapy of all subtypes of mesothelioma.
The authors are grateful to Dr. Mary E. Dyszlewski at Tyco/Mallinckrodt for providing the carbonyl-kit (IsoLink®). This work is supported partially by NIH R01 CA135358 and the American Cancer Society (IRG-97-150-10) to Jiang He, and R01 CA118919 and R01 CA129491 to Bin Liu.