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 (K D = 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. (Cancer Sci 2013; 104: 495-501) T umor metastasis is the most frequent cause of death for cancer patients. In order to metastasize, tumor cells must acquire the ability to break through the basement membrane and invade dense networks of interstitial ECM proteins.(1) Matrix metalloproteinases (MMPs) are a family of enzymes responsible for degrading the various ECM components. While most MMPs are secreted as soluble zymogens, members of the subfamily of membrane-type MMPs (MT-MMPs) anchored to the cell membrane are suited for pericellular proteolysis. MT1-MMP is the major pericellular protease involved in processing triple helical collagen type I.(4) In addition, MT1-MMP activates MMP zymogens such as proMMP-2 and proMMP-13 that have significant involvement in tumor cell invasion and metastasis.(5,6) As MT1-MMP has a close relationship with tumor malignancy and holds great promise as an early biomarker of malignant tumors, (7,8) in vivo monitoring and ⁄ or quantitation of MT1-MMP expression could be valuable tools for molecular biological and clinical studies.Recently, in the course of focusing on nuclear medical techniques for noninvasive quantitative evaluation of biological molecules deep within the body, we developed a 99m Tc-labeled anti-MT1-MMP monoclonal IgG ( 99m Tc-MT1-mAb) as a radiolabeled...

Radioiodinated Peptidic Imaging Probes for <i>in Vivo</i> Detection of Membrane Type-1 Matrix Metalloproteinase in Cancers

“…In this regard, nuclear medical imaging would provide more accurate quantitation; therefore, several protein-based radiolabeled probes for MT1-MMP have been developed. 10,11,26) However, such high molecular weight probes have other problems such as probe heterogeneity 13) and low apparent specific radioactivity. 27) In particular, one issue concerning MT1-MMP imaging probes is that the MT1-MMP expression level in cancer was reported to be low (about 1.6×10…”

“…Furthermore, radioiodinated peptide probes could be completely separated from their precursors by RP-HPLC, so that the probes could be administered with a much higher specific radioactivity (>260 MBq/µg) than was achieved for our previously reported radiolabeled antibody probes (37 kBq/ µg). 11) Although [ 123/125 I] I-DC showed relatively low affinity for MT1-MMP (K D =810 nM) compared to our previously developed antibody probes 11) (K D <40 nM for MT1-MMP) and well-established peptide probes such as RGD peptide probes 30) and 90 Y-DOTATOC 31) (K D <10 nM), the radioactivity distribution profiles nonetheless clearly corresponded to MT1-MMPpositive areas (Fig. 5), likely because of the high specific radioactivity of this probe.…”

“…32,33) In fact, we reported that an 111 In-labeled diabody, a dimer of scFv, could bind to MT1-MMP in a bivalent manner with higher affinity and increased tumor accumulation compared with scFv. 11) Optimization of labeling could also be an effective strategy because our newly developed [ ). Thus it appears that the additional moiety in the DC used for radiolabeling somewhat hindered the interaction between the MT-loop and the peptide sequence.…”

“…Since nuclear medical techniques are optimal for noninvasive quantitative evaluation of biological molecules deep within the body, we recently developed 99 m Tc-labeled anti-MT1-MMP monoclonal immunoglobulin G (IgG) 10) and 111 In-labeled miniaturized antibodies (scFv and diabody) 11) as radiolabeled probes for nuclear medical imaging of MT1-MMP. Although these probes could be used for imaging MT1-MMP in cancer, antibody derivatives have several undesirable characteristics when used as molecular imaging probes.…”

<i>In Vivo</i> Molecular Imaging

“…After allowing sufficient time for POS to degrade in normal tissues, We also developed probes using anti-MT1-MMP antibody or its reduced-molecular-weight form for the target recognition unit and succeeded in imaging of MT1-MMP. [46][47][48][49][50][51] In addition, we performed research on structure-activity-distribution rela- tions of an asymmetric urea compound that binds to prostatespecific membrane antigen (PSMA) expressed in prostate cancer cell membrane, and developed a nuclear medical molecular imaging probe for PSMA-positive tumor. [52][53][54] We are currently preparing a clinical study using this probe.…”