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Airway epithelial cells (AECs) participate in allergic airway inflammation by producing mediators in response to allergen stimulation. Whether ovalbumin (OVA) challenge promotes exosome release from AECs (OVA-challenged AEC-derived exosomes (OAEs)), thereby affecting airway inflammation, as well as the underlying mechanisms, is unknown. Our study showed that AECs released an increased number of exosomes after OVA challenge, and the expression of Plexin B2 (PLXNB2; a natural CD100 ligand) was increased by a massive 85.7-fold in OAEs than in PBS-treated AEC-derived exosomes (PAEs). CD100 + F4/80 + macrophages engulfed OAEs to trigger the transcription of pro-inflammatory chemokines and cytokines. Plxnb2 transcripts increased in asthmatic lungs, and similarly, PLXNB2 protein was highly enriched in exosomes purified from asthmatic bronchoalveolar lavage (BAL) fluid. Furthermore, aspiration of PLXNB2 or OAEs increased the recruitment of lung neutrophils, monocytes, eosinophils and dendritic cells in OVA-challenged mice. Mechanistically, OAE aspiration enhanced the cleavage of CD100 by MMP14, which manifested as an increase in the soluble CD100 (sCD100) level in BAL fluid and lung homogenates. Knockdown of Mmp14 in macrophages prevented the cleavage of CD100 and reduced Ccl2, Ccl5 and Cxcl2 transcription. These data indicate that PLXNB2-containing OAEs aggravate airway asthmatic inflammation via cleavage of CD100 by MMP14, suggesting potential therapeutic targets of OAE-mediated asthma exacerbations., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.

Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli -an antibody clone, a protease of interest, and a β-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified β-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), β-secretase 1 (BACE-1, an aspartic protease in Alzheimer's disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aβ 40 ) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice., Competing Interests: The authors declare no conflict of interest.

Targeting effectual epitopes is essential for therapeutic antibodies to accomplish their desired biological functions. This study developed a competitive dual color fluorescence-activated cell sorting (FACS) to maturate a matrix metalloprotease 14 (MMP-14) inhibitory antibody. Epitope-specific screening was achieved by selection on MMP-14 during competition with N-terminal domain of tissue inhibitor of metalloproteinase-2 (TIMP-2) (nTIMP-2), a native inhibitor of MMP-14 binding strongly to its catalytic cleft. 3A2 variants with high potency, selectivity, and improved affinity and proteolytic stability were isolated from a random mutagenesis library. Binding kinetics indicated that the affinity improvements were mainly from slower dissociation rates. In vitro degradation tests suggested the isolated variants had half lives 6-11-fold longer than the wt. Inhibition kinetics suggested they were competitive inhibitors which showed excellent selectivity toward MMP-14 over highly homologous MMP-9. Alanine scanning revealed that they bound to the vicinity of MMP-14 catalytic cleft especially residues F204 and F260, suggesting that the desired epitope was maintained during maturation. When converted to immunoglobulin G, B3 showed 5.0 nM binding affinity and 6.5 nM inhibition potency with in vivo half-life of 4.6 days in mice. In addition to protease inhibitory antibodies, the competitive FACS described here can be applied for discovery and engineering biosimilars, and in general for other circumstances where epitope-specific modulation is needed., (© 2018 Wiley Periodicals, Inc.)

Inhibiting individual MMPs of biomedical importance with high selectivity is critical for both fundamental research and therapy development. Here we describe the methods for discovery of inhibitory monoclonal antibodies from synthetic human antibody phage display libraries carrying convex paratopes encoded by long complementarity-determining region (CDR)-H3 segments. We demonstrate the application of this technique for isolation of highly specific and potent antibody inhibitors of human MMP-14.

Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC 50 values of 10-4,000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions. Biotechnol. Bioeng. 2017;114: 1140-1150. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Gelsolin amyloidosis is a dominantly inherited, incurable type of amyloidosis. A single point mutation in the gelsolin gene (G654A is most common) results in the loss of a Ca2+  binding site in the second gelsolin domain. Consequently, this domain partly unfolds and exposes an otherwise buried furin cleavage site at the surface. During secretion of mutant plasma gelsolin consecutive cleavage by furin and MT1-MMP results in the production of 8 and 5 kDa amyloidogenic peptides. Nanobodies that are able to (partly) inhibit furin or MT1-MMP proteolysis have previously been reported. In this study, the nanobodies have been combined into a single bispecific format able to simultaneously shield mutant plasma gelsolin from intracellular furin and extracellular MT1-MMP activity. We report the successful in vivo expression of this bispecific nanobody following adeno-associated virus serotype 9 gene therapy in gelsolin amyloidosis mice. Using SPECT/CT and immunohistochemistry, a reduction in gelsolin amyloid burden was detected which translated into improved muscle contractile properties. We conclude that a nanobody-based gene therapy using adeno-associated viruses shows great potential as a novel strategy in gelsolin amyloidosis and potentially other amyloid diseases., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Matrix metalloproteinase-14 (MMP-14) plays important roles in cancer metastasis, and the failures of broad-spectrum MMP compound inhibitors in clinical trials suggested selectivity is critical. By grafting an MMP-14 specific inhibition motif into complementarity determining region (CDR)-H3 of antibody scaffolds and optimizing other CDRs and the sequences that flank CDR-H3, we isolated a Fab 1F8 showing a binding affinity of 8.3 nM with >1000-fold enhancement on inhibition potency compared to the peptide inhibitor. Yeast surface display and fluorescence-activated cell sorting results indicated that 1F8 was highly selective to MMP-14 and competed with TIMP-2 on binding to the catalytic domain of MMP-14. Converting a low-affinity peptide inhibitor into a high potency antibody, the described methods can be used to develop other inhibitory antibodies of therapeutic significance., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 10 9 individual variants) that carried the extended, 23- to 27-residue, complementarity-determining region (CDR)-H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes., Competing Interests: The authors declare no conflict of interest.

Background: A critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models., Methods: An anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for 89Zr labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments., Results: 89Zr labeling of DFO-LEM2/15 was achieved with high yield (>90%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that 89Zr-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent 89Zr-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP- MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. 89Zr-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models., Conclusion: A new anti MT1-MMP-mAb tracer, 89Zr-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM.

Objective: In rheumatoid arthritis (RA), destruction of articular cartilage by the inflamed synovium is considered to be driven by increased activities of proteolytic enzymes, including matrix metalloproteinases (MMPs). The purpose of this study was to investigate the therapeutic potential of selective inhibition of membrane type 1 MMP (MT1-MMP) and its combination with tumor necrosis factor (TNF) blockage in mice with collagen-induced arthritis (CIA)., Methods: CIA was induced in DBA/1 mice by immunization with bovine type II collagen. From the onset of clinical arthritis, mice were treated with MT1-MMP selective inhibitory antibody DX-2400 and/or TNFR-Fc fusion protein. Disease progression was monitored daily, and serum, lymph nodes, and affected paws were collected at the end of the study for cytokine and histologic analyses. For in vitro analysis, bone marrow-derived macrophages were stimulated with lipopolysaccharide for 24 hours in the presence of DX-2400 and/or TNFR-Fc to analyze cytokine production and phenotype., Results: DX-2400 treatment significantly reduced cartilage degradation and disease progression in mice with CIA. Importantly, when combined with TNF blockade, DX-2400 acted synergistically, inducing long-term benefit. DX-2400 also inhibited the up-regulation of interleukin-12 (IL-12)/IL-23 p40 via polarization toward an M2 phenotype in bone marrow-derived macrophages. Increased production of IL-17 induced by anti-TNF, which correlated with an incomplete response to anti-TNF, was abrogated by combined treatment with DX-2400 in CIA., Conclusion: Targeting MT1-MMP provides a potential strategy for joint protection, and its combination with TNF blockade may be particularly beneficial in RA patients with an inadequate response to anti-TNF therapy., (© 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Tuberculosis (TB) remains a global pandemic and drug resistance is rising. Multicellular granuloma formation is the pathological hallmark of Mycobacterium tuberculosis infection. The membrane type 1 matrix metalloproteinase (MT1-MMP or MMP-14) is a collagenase that is key in leukocyte migration and collagen destruction. In patients with TB, induced sputum MT1-MMP mRNA levels were increased 5.1-fold compared with matched controls and correlated positively with extent of lung infiltration on chest radiographs (r = 0.483; p < 0.05). M. tuberculosis infection of primary human monocytes increased MT1-MMP surface expression 31.7-fold and gene expression 24.5-fold. M. tuberculosis-infected monocytes degraded collagen matrix in an MT1-MMP-dependent manner, and MT1-MMP neutralization decreased collagen degradation by 73%. In human TB granulomas, MT1-MMP immunoreactivity was observed in macrophages throughout the granuloma. Monocyte-monocyte networks caused a 17.5-fold increase in MT1-MMP surface expression dependent on p38 MAPK and G protein-coupled receptor-dependent signaling. Monocytes migrating toward agarose beads impregnated with conditioned media from M. tuberculosis-infected monocytes expressed MT1-MMP. Neutralization of MT1-MMP activity decreased this M. tuberculosis network-dependent monocyte migration by 44%. Taken together, we demonstrate that MT1-MMP is central to two key elements of TB pathogenesis, causing collagen degradation and regulating monocyte migration., (Copyright © 2015 The Authors.)

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., (© 2013 Japanese Cancer Association.)

Forensic identification of body fluids is a crucial aspect of the discipline. To date, there are no robust tests to make a distinction between menstrual blood and peripheral blood. Past techniques have fallen short of producing clear and consistent results while present mRNA techniques are still in their infancy. The aim of this study was to develop of an accurate and rapid immunoassay based identification method. Three different targets were evaluated: matrix metalloproteinase 14 (MMP14), oestrogen receptor α (ERα), and fibrinogen. Cellular or membrane bound antigens were analyzed using immunocytochemical staining while soluble antigens were analyzed by indirect ELISA. Results showed that ERα and MMP14 are present in the endometrial cells of menstrual blood but absent in peripheral blood. At a total protein concentration of 10 μg/mL or lower, fibrinogen was detected in menstrual blood but absent in peripheral. If evaluated on a larger scale, immunoassays may prove to be beneficial in discriminating menstruum and peripheral blood., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The membrane associated MMP, MT1-MMP, is a critical pericellular protease involved in tumour cell invasion and angiogenesis and is highly up-regulated in numerous human cancers. It therefore represents an exciting new therapeutic cancer-specific target. We have generated recombinant human scFv antibodies against the non-catalytic, hemopexin domain of MT1-MMP that modulate its interactions with collagen. One of these is an effective inhibitor of the invasive capacity of cancer cells and of angiogenesis in model systems. This demonstrates that targeting sites outside the catalytic domain presents a potential novel approach to proteinase inhibition that could have applications in cancer therapeutics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To reach the lymphatics, migrating dendritic cells (DCs) need to interact with the extracellular matrix (ECM). Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and ECM. The role of heparanase in the physiology of bone marrow-derived DCs was studied in mutant heparanase knock-out (Hpse-KO) mice. Immature DCs from Hpse-KO mice exhibited a more mature phenotype; however their transmigration was significantly delayed, but not completely abolished, most probably due to the observed upregulation of MMP-14 and CCR7. Despite their mature phenotype, uptake of beads was comparable and uptake of apoptotic cells was more efficient in DCs from Hpse-KO mice. Heparanase is an important enzyme for DC transmigration. Together with CCR7 and its ligands, and probably MMP-14, heparanase controls DC trafficking.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a protease that activates pro-MMP-2 and pro-MMP13, which are related to tumor malignancy. Therefore, probes that specifically image MT1-MMP would be useful for malignant tumor diagnosis. In the present study, we prepared rhodamine X-conjugated anti-MT1- MMP antibody (anti-MT1-MMP mAb-ROX) as an activatable fluorescent probe and evaluated its usefulness for MT1-MMP-specific imaging. Anti-MT1-MMP mAb-ROX was obtained in a quenched form with approximately three ROX molecules per mAb. Its fluorescence intensity increased approximately 14-fold in the presence of detergent, which is suitable for activatable systems. C6 glioma cells and MCF-7 human breast adenocarcinoma cells were used as MT1-MMP-positive and MT1-MMP-negative models, respectively. The fluorescence intensity of C6 cells treated with anti-MT1-MMP mAb-ROX, but not ROX-conjugated isotype control antibody (NC Ab-ROX), increased with time and was significantly higher than that of MCF-7 cells at 6 h (P < 0.001). The fluorescence intensity of cells treated with anti-MT1-MMP mAb-ROX was also suppressed by pre-treatment with a MT1-MMP endocytosis inhibitor (P < 0.05). Furthermore, the probes were intravenously administered to C6 and MCF-7 xenografted mice. The tumor-to-muscle (T/M) ratio of the anti-MT1-MMP mAb-ROX group was 15.1 ± 3.2 at 48 h and was significantly higher than that of the NC Ab-ROX group (T/M ratio = 4.6 ± 3.0, P < 0.05) in C6 xenografted mice, while the T/M ratio of the anti-MT1-MMP mAb-ROX and NC Ab-ROX groups was not different in MCF-7 xenografted mice. These findings suggest that anti-MT1-MMP mAb-ROX is a promising probe for specifically detecting MT1-MMP-expressing tumors.

Purpose: Membrane type 1 matrix metalloproteinase (MT1-MMP) activates pro-MMP-2 and pro-MMP-13 to their active forms and plays important roles in the destabilization of atherosclerotic plaques. This study sought to determine the usefulness of (99m)Tc-labelled monoclonal antibody (mAb), recognizing MT1-MMP, for imaging atherosclerosis in a rabbit model (WHHLMI rabbits)., Methods: Anti-MT1-MMP monoclonal IgG(3) and negative control IgG(3) were radiolabelled with (99m)Tc after derivatization with 6-hydrazinonicotinic acid (HYNIC) to yield (99m)Tc-MT1-MMP mAb and (99m)Tc-IgG(3), respectively. WHHLMI and control rabbits were injected with these radio-probes. The aorta was removed and radioactivity was measured at 24 h after the injection. Autoradiography and histological studies were performed., Results: (99m)Tc-MT1-MMP mAb accumulation in WHHLMI rabbit aortas was 5.4-fold higher than that of control rabbits. Regional (99m)Tc-MT1-MMP mAb accumulation was positively correlated with MT1-MMP expression (r = 0.59, p < 0.0001), while (99m)Tc-IgG(3) accumulation was independent of MT1-MMP expression (r = 0.03, p = NS). The highest (99m)Tc-MT1-MMP mAb accumulation was found in atheromatous lesions (4.8 ± 1.9, %ID×BW/mm(2) × 10(2)), followed in decreasing order by fibroatheromatous (1.8 ± 1.3), collagen-rich (1.6 ± 1.0) and neointimal lesions (1.5 ± 1.5). In contrast, (99m)Tc-IgG(3) accumulation was almost independent of the histological grade of lesions., Conclusion: Higher (99m)Tc-MT1-MMP mAb accumulation in grade IV atheroma was shown in comparison with neointimal lesions or other more stable lesions. Nuclear imaging with (99m)Tc-MT1-MMP mAb, in combination with CT and MRI, could provide new diagnostic imaging capabilities for detecting vulnerable plaques, although further investigations to improve target to blood ratios are strongly required.

Engagement of tumor cell surface MHC class I chain-related molecule A (MICA) to NKG2D stimulates NK and T cell antitumor immunity. Shedding of MICA by tumor cells facilitates tumor immune evasion, which may in part contribute to tumor progression. Thus, elucidating the mechanisms by which tumors shed MIC is of great importance for therapy to reinforce NK and T cell antitumor immunity. In this study, we report that the membrane type matrix metalloproteinase (MMP)14 mediates MICA shedding. Suppression of MMP14 expression blocks MICA shedding. Concomitantly, overexpression of MMP14 enhances MICA shedding. The regulation of MICA shedding by MMP14 is independent of the activity of a disintegrin and metalloproteinases, which have been reported to mediate MICA shedding. Finally, MMP14 expression in MICA-positive tumor cells regulates the sensitivity of tumor cells to NK cell killing. These findings suggest that MMP14 may be a new target for tumor immune therapy.

Background: Liposomes, phospholipids, nanosized bubbles with a bilayered membrane structure, have drawn a lot of interest as pharmaceutical carriers for drugs and genes. In particular, liposomes are widely used for drug delivery into tumors., Objective: In many cases, to enhance the efficacy of the liposomal drugs, drug-loaded liposomes are targeted to the tumors by means of different specific ligands, such as monoclonal antibodies. Thus, this review analyzes the application of antibody-targeted liposomes loaded with various chemotherapeutic agents and various liposomal products under development at experimental and preclinical level., Methods: The papers published on the subject of cancer-targeted liposomes mainly over the last 10 - 15 years are discussed., Conclusion: Antibody-targeted liposomes loaded with anticancer drugs demonstrate high potential for clinical applications.

The secreted gelatinase matrix metalloprotease-2 (MMP-2) and the membrane-anchored matrix metalloprotease MT1-MMP (MMP-14), are central players in pericellular proteolysis in extracellular matrix degradation. In addition to possessing a direct collagenolytic and gelatinolytic activity, these enzymes take part in a cascade pathway in which MT1-MMP activates the MMP-2 proenzyme. This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. We provide positive evidence that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. Even in the absence of transfection and overexpression, dimerization of MT1-MMP markedly stimulated the formation of active MMP-2 products. The effect demonstrated here was brought about by a monoclonal antibody that binds specifically to MT1-MMP as shown by immunofluorescence experiments. The antibody has no effect on the catalytic activity. The effect on proMMP-2 activation involves MT1-MMP dimerization because it requires the divalent monoclonal antibody, with no effect obtained with monovalent Fab fragments. Since only a negligible level of proMMP-2 activation was obtained with MT1-MMP-expressing cells in the absence of dimerization, our results identify the dimerization event as a critical level of proteolytic cascade regulation.

Following removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast. Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation.

Immunoliposomes are potent carriers for targeting of therapeutic drugs to specific cells. Membrane type-1 matrix metalloproteinase (MT1-MMP), which plays an important role in angiogenesis, is expressed on angiogenic endothelium cells as well as tumor cells. Then, the MT1-MMP might be useful as a target molecule for tumor and neovascularity. In the present study, we addressed a utility of antibodies against the MT1-MMP as a targeting ligand of liposomal anticancer drug. Fab' fragments of antibody against the MT1-MMP were modified at distal end of polyethylene glycol (PEG) of doxorubicin (DXR)-encapsulating liposomes, DXR-sterically stabilized immunoliposomes (DXR-SIL[anti-MT1-MMP(Fab')]). Modification with the antibody significantly enhanced cellular uptake of DXR-SIL[anti-MT1-MMP(Fab')] into the HT1080 cells, which highly express MT1-MMP, compared with the non-targeted liposomes (DXR-stealthliposomes (DXR-SL)), suggesting that MT1-MMP antibody (Fab') is a potent targeting ligand for the MT1-MMP expressed cells. In vivo systemic administration of DXR-SIL[anti-MT1-MMP(Fab')] into the tumor-bearing mice showed significant suppression of tumor growth compared to DXR-SL. This is presumably due to the active targeting of immunoliposomes for tumor and neovascularity. However, tumor accumulation of DXR-SIL[anti-MT1-MMP(Fab')] and DXR-SL were comparable, suggesting that both liposomal formulations accumulated in tumor via enhanced permeation and retention (EPR) effect, but not via targeting to the MT1-MMP expressed on both the endothelial and tumor cells. It appears that the enhanced antitumor activity of DXR-SIL[anti-MT1-MMP(Fab')] resulted from acceleration of cellular uptake of lioposomes owing to the incorporated antibody after extravasation from capillaries in tumor.

The poor selective cytotoxicity of anticancer drugs lead to dose-limiting adverse effects which compromise the clinical outcome. Solid tumors recruit new blood vessels to support their growth, and epitopes that are uniquely expressed on tumor cells and tumor endothelial cells (ECs) can function as targets for immunoliposomal anticancer drugs. Membrane type 1 matrix metalloproteinase (MT1-MMP), an important protein related to tumor growth and angiogenesis, is expressed on malignant tumor cells and is activated ECs. Selective delivery could be achieved by targeting MT1-MMP, as well as other angiogenic ECs. In this regard, an anti-MT1-MMP Fab' antibody was used to prepare a MT1-MMP targeted sterically stabilized immunoliposomes (SIL[anti-MT1-MMP(Fab')]). The binding and intracellular distribution of SIL[anti-MT1-MMP(Fab')] and a non-targeted sterically stabilized liposomes (SL) were examined using human fibrosarcoma HT-1080 cells. SIL[anti-MT1-MMP(Fab')] was taken up by the cells in a lipid concentration, temperature, and time dependent manner, ultimately accumulating in the lysosomes. The cytotoxicity of doxorubicin (DXR)-containing SIL[anti-MT1-MMP(Fab')] (DXR-SIL[anti-MT1-MMP(Fab')]) was significantly higher than that of DXR-containing SL. The cellular internalization of SIL[anti-MT1-MMP(Fab')] was inhibited by endocytosis inhibitors, suggesting that their internalization was mediated via clathrin- or caveolae-dependent endocytosis. Furthermore, the efficient binding of SIL[anti-MT1-MMP(Fab')] was observed on human umbilical vein endothelial cells (HUVEC). Based on these results, it would be expected that DXR-SIL[anti-MT1-MMP(Fab')] may achieve direct tumor cell kill and indirect tumor cell kill via the destruction of the tumor endothelium in vivo. This strategy may have the potential for overcoming some major limitations in conventional chemotherapy in vivo.