We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-beta (TGF-beta) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor beta-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.

Expression of matrix metalloproteinase 9 (MMP9) is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs), including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors) in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.

Mesothelin (MSLN) is an attractive target for antibody-drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to nondividing mesothelia. We generated novel antimesothelin antibodies and conjugated an internalizing one (7D9) to the microtubuledisrupting drugs monomethyl auristatin E (MMAE) and MMAF, finding the most effective to be MMAE with a lysosomal protease-cleavable valine-citrulline linker. The humanized (h7D9.v3) version, aMSLN-MMAE, specifically targeted mesothelin-expressing cells and inhibited their proliferation with an IC 50 of 0.3 nmol/L. Because the antitumor activity of an antimesothelin immunotoxin (SS1P) in transfected mesothelin models did not translate to the clinic, we carefully selected in vivo efficacy models endogenously expressing clinically relevant levels of mesothelin, after scoring mesothelin levels in ovarian, pancreatic, and mesothelioma tumors by immunohistochemistry. We found that endogenous mesothelin in cancer cells is upregulated in vivo and identified two suitable xenograft models for each of these three indications. A single dose of aMSLN-MMAE profoundly inhibited or regressed tumor growth in a dosedependent manner in all six models, including two patient-derived tumor xenografts. The robust and durable efficacy of aMSLN-MMAE in preclinical models of ovarian, mesothelioma, and pancreatic cancers justifies the ongoing phase I clinical trial. Mol Cancer Ther; 13(11); 2630-40. Ó2014 AACR.

Matrix metalloproteinase-9 (MMP-9), whose expression is frequently dysregulated in cancer, promotes tumor growth, invasion, and metastasis by multiple mechanisms, including extracellular matrix remodeling and growth-factor and cytokine activation. We developed a monoclonal antibody against murine MMP-9, which we found decreased growth of established primary tumors in an orthotopic model of HER2-driven breast cancer (HC11-NeuT) in immunocompetent mice. RNA sequencing (RNAseq) profiling of NeuT tumors and additional mouse model tumors revealed that anti-MMP-9 treatment resulted in upregulation of immune signature pathways associated with cytotoxic T-cell response. As there is a need to boost the low response rates observed with anti-PDL1 antibody treatment in the clinical setting, we assessed the potential of anti-MMP-9 to improve T-cell response to immune checkpoint inhibitor anti-PDL1 in NeuT tumors. Anti-MMP-9 and anti-PDL1 cotreatment reduced T-cell receptor (TCR) clonality and increased TCR diversity, as detected by TCR sequencing of NeuT tumors. Flow cytometry analyses of tumors showed that the combination treatment increased the frequency of CD3+ T cells, including memory/effector CD4 and CD8 T cells, but not regulatory T cells, among tumor-infiltrating leukocytes. Moreover, in vitro enzymatic assays corroborated that MMP-9 cleaves key T-cell chemoattractant CXC receptor 3 ligands (CXC ligand [CXCL] 9, CXCL10, and CXCL11) and renders them inactive in T-cell migration assays. Consistent with our in vitro experiments, analysis of NeuT tumor protein lysates showed that anti-MMP-9 treatment increases expression of CXCL10 and other T cell–stimulating factors, such as interleukin (IL)-12p70 and IL-18. We show that inhibition of MMP-9, a key component of the tumor-promoting and immune-suppressive myeloid inflammatory milieu, increases T-helper cell 1 type cytokines, trafficking of effector/memory T cells into tumors, and intratumoral T-cell diversity.

Background: Matrix metalloproteinase 9 (MMP9) acts via diverse mechanisms to promote tumor growth, invasion, and metastasis. It activates growth factors and signaling pathways, promotes angiogenesis, and impedes anti-tumor immune responses. We developed a monoclonal antibody that inhibits mouse MMP9 (AB0046) and assessed its mechanism of action in immunocompetent mouse tumor models and in vitro assays. Methods: We examined MMP9 expression in a variety of human tumor tissues via immunohistochemistry. Human monocytes were differentiated in vitro and protein expression was assessed via enzyme-linked immunosorbent assay. Primary tumor growth in orthotopic, syngeneic tumor models was examined following anti-MMP9 antibody treatment. RNA sequencing, immunohistochemical and flow cytometry analyses were performed on tumor tissues to assess gene expression, stromal remodeling and macrophage polarization in response to AB0046 treatment. Results: MMP9 levels are elevated in human tumors compared to healthy tissues. The protein is expressed predominantly in stromal cells, including macrophages and neutrophils, with more occasional heterogeneous expression in tumor epithelia. Differentiation of human monocyte-derived macrophages in vitro revealed that M2 polarization is associated with increased expression of MMP9 and Th2 markers CCL18 and TGFβ. Anti-MMP9 treatment in three independent mouse tumor models (HC11-NeuT, CT26, Lewis lung carcinoma (LLC)) resulted in decreased primary tumor growth (p=0.001 and p=0.018 for HC11-NeuT and CT26 respectively) and increased animal survival (p=0.024 for LLC). Gene expression profiling of tumors from the various models demonstrated that inhibition of MMP9 resulted in elevated expression of genes associated with immune cell activation pathways (Hallmark Interferon Gamma Response, p<0.05, FDR<0.001). Additional analysis in the HC11-NeuT model revealed a significant decrease in M2 macrophages in the tumor microenvironment (p < 0.05) with AB0046 treatment, as well as reductions in tumor-associated fibrillar collagen as assessed by Picrosirius red staining. Conclusions: These analyses show that MMP9 is expressed in a variety of human tumors. Our data suggest that inhibition of MMP9 promotes anti-tumor immunity and enhances a Th1 immune response. GS-5745, a humanized anti-MMP9 inhibitory antibody, is being evaluated in gastric cancer in phase 3 and 2 studies with chemotherapy and nivolumab, respectively (NCT02545504, NCT02864381). Citation Format: Vladi Juric, Amanda Mikels-Vigdal, Chris O'Sullivan, Andrew Greenstein, Erin Stefanutti, Vivian Barry-Hamilton, Igor Mikaelian, Ted Sullivan, Erik Huntzicker, Jeremiah Degenhardt, Peng Yue, Victoria Smith. Inhibition of MMP9 improves anti-tumor immunity by changing the tumor microenvironment to promote T cell trafficking and activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2017-653