Miniaturized antibodies for imaging membrane type-1 matrix metalloproteinase in cancers

Abstract

Since membrane type-1 matrix metalloproteinase (MT1-MMP) plays pivotal roles in tumor progression and metastasis and holds great promise as an early biomarker for malignant tumors, a method of evaluating MT1-MMP expression levels would be valuable for molecular biological and clinical studies. Although we have previously developed a (99m) Tc-labeled anti-MT1-MMP monoclonal IgG ((99m) Tc-MT1-mAb) as an MT1-MMP imaging probe by nuclear medical techniques for this purpose, slow pharmacokinetics were a problem due to its large molecular size. Thus, in this study, our aim was to develop miniaturized antibodies, a single chain antibody fragment (MT1-scFv) and a dimer of two molecules of scFv (MT1-diabody), as the basic structures of MT1-MMP imaging probes followed by in vitro and in vivo evaluation with an (111) In radiolabel. Phage display screening successfully provided MT1-scFv and MT1-diabody, which had sufficiently high affinity for MT1-MMP (KD = 29.8 and 17.1 nM). Both (111) In labeled miniaturized antibodies showed higher uptake in MT1-MMP expressing HT1080 cells than in non-expressing MCF7 cells. An in vivo biodistribution study showed rapid pharmacokinetics for both probes, which exhibited >20-fold higher tumor to blood radioactivity ratios (T/B ratio), an index for in vivo imaging, than (99m) Tc-MT1-mAb 6 h post-administration, and significantly higher tumor accumulation in HT1080 than MCF7 cells. SPECT images showed heterogeneous distribution and ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles in tumors corresponded to MT1-MMP-positive areas. These findings suggest that the newly developed miniaturized antibodies are promising probes for detection of MT1-MMP in cancer cells.

Figure 1

(a) Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of MT1‐scFv (1) and MT1‐diabody (2). (b) Chromatograms of MW‐marker; 1 (7.93 mL), glutamate dehydrogenase 290 kDa: 2 (8.43 mL), lactate dehydrogenase 142 kDa: 3 (9.15 mL), enolase 67 kDa: 4 (10.98 mL), myokinase 32 kDa: 5 (13.18 mL), cytochrome C 12.4 kDa. (c) Chromatogram of ¹¹¹In‐MT1‐scFv (11.0 mL). (d) Chromatogram of ¹¹¹In‐MT1‐diabody (9.25 mL). (e) Chromatogram of ¹¹¹In‐NC‐scFv (11.0 mL).

Figure 2

The radioactivity of HT1080 cells and MCF‐7 cells after incubation for 1 and 3 h with ¹¹¹In‐MT1‐scFv (scFv), ¹¹¹In‐MT1‐diabody (diabody) and ¹¹¹In‐NC‐scFv (NC). Data are expressed as radioactivity per cell protein (mg) (mean ± standard deviation [SD]). Comparison between HT1080 and MCF‐7 cell groups was performed with the Mann–Whitney U‐test (*P < 0.005, **P < 0.0001 versus MCF‐7).

Figure 3

Tumor to blood radioactivity ratio (T/B) of ¹¹¹In‐MT1‐scFv and ¹¹¹In‐MT1‐diabody. Comparisons between ¹¹¹In‐MT1‐scFv and ¹¹¹In‐MT1‐diabody were performed with the Mann–Whitney U‐test (*P < 0.05 versus ¹¹¹In‐MT1‐diabody).

Figure 4

Tumor accumulations of ¹¹¹In‐MT1‐scFv (1, 24 h) and ¹¹¹In‐MT1‐diabody (3, 24 h) in HT1080 and MCF7 tumors. Comparisons of accumulations between HT1080 and MCF7 were performed with the Mann–Whitney U‐test (*P < 0.05 versus MCF7).

Figure 5

Transverse, sagittal and coronal images of tumor‐bearing mice 3 h after administration with ¹¹¹In‐MT1‐scFv (a–c) and ¹¹¹In‐MT1‐diabody (d–f). Yellow arrows indicate tumors.

Figure 6

Representative images of MT1‐MMP immunostainings and autoradiograms of ¹¹¹In‐MT1‐scFv (a,b), ¹¹¹In‐MT1‐diabody (c,d), and ¹¹¹In‐NC‐scFv (e,f). Black arrowheads indicate areas of MT1‐MMP expression.