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TIMP-2 zymography for the development of bone metastases
MT1-MMP, TIMP-2 RT-PCR, ELISA, WB, - MNNG Inhibition of Rho prevented LPA-mediated reduction in MT1-MMP and TIMP-2 [27]
zymography, IF
MT1-MMP, MMP-2 - SJSA-1 A dose-dependent decrease in MT1-MMP and MMP-2 expression was observed [57]
following exposure to Dz13
MT1-MMP, MMP-2 - MG-63 Increasing the concentration of clodronate reduced MT1-MMP and MMP-2 mRNA [62]
and protein expression in MG-63 cells

ChIP: Chromatin immunoprecipitation; ELISA: enzyme-linked immunoassay; IF: immunofluorescence; IHC: immunohistochemistry; MMP: matrix metalloproteinase; MT-MMP: membrane-type MMP; NB: Northern
blotting; RT-PCR: reverse transcription polymerase chain reaction; WB: Western blotting.

which were attributed to a lack of understanding of the biological complexities of MMPs, there had been some recent clinical successes. MMPIs have wide-
ranging applications, from the treatment of HER2-negative adenocarcinomas to diabetic foot ulcers. Doxycycline and minocycline, which are broad-spectrum
MMPIs, have also shown promise in the treatment of patients with relapse-remitting multiple sclerosis. Both were shown to reduce brain lesions and serum
levels of MMP-9 and were considered to be effective, safe and well-tolerated. A potent, selective MMPI could make a significant clinical impact on OS therapy.

An alternative approach is to exploit the proteolytic activity of MT1-MMP to activate a prodrug (a drug administered in an inactive form, which is
subsequently activated at the target site) in the tumour microenvironment. By adopting this approach, there is potential to overcome the off-target toxicity
seen in many chemotherapeutic agents like HD-MTX, since the drug would only be released in the tumour microenvironment, thereby sparing normal tissues
of exposure to the drug, while simultaneously enhancing its therapeutic index.

Two such examples of MT1-MMP targeted agents are BT1718 and ICT2588 (see Figure 3), which are currently being evaluated in other cancers, but have
significant potential for OS therapy. BT1718, which was developed by researchers at Bicycle Therapeutics Ltd, is currently in Phase I/II clinical trials for the
treatment of advanced solid tumours (Clinical Trials. gov Identifier: NCT03486730). The drug conjugate is comprised of a bicyclic peptide covalently linked to
DM1, a potent anti-tubulin agent. The peptide sequence has been designed to specifically bind to MT1-MMP through a disulphide linker - taking advantage of
its expression profile in many solid tumour models (including various sarcomas, non-small cell lung cancer and triple-negative breast cancer) - thereby
[75]
targeting the delivery of DM1 . Pre-clinical models have shown that in both cell-derived and patient-derived xenograft tumours, DM1 is released in an MT1-
[76]
MMP dependent manner, which results in partial or complete tumour regression . Although the clinical outcome is not yet known, this validates MT1-MMP
as a target. ICT2588, which is currently being prepared for Phase I clinical trials, consists of 3 components; azademethylcolchicine, a vascular disrupting agent;
an MT1-MMP activatable peptide sequence, Arg-Ser-Citrulline (Cit)-Gly-Homophenylalanine (Hof)-Tyr-Leu-Tyr; and a fluorescein isothiocyanate (FITC)
based end cap. The release of the azademethylcolchicine warhead is achieved through (1) the selective cleavage of the peptide conjugate at the scissile bond
between Gly and Hof; and (2) tumour exopeptidase activity removing the remaining amino acids. In tumour xenograft models, ICT2588 was shown to reduce
tumour vasculature by up to 90% and, when co-administration with doxorubicin, led to a significant reduction in relative tumour volume . Furthermore,
[77]