Hepsin is a type II transmembrane serine protease that is expressed in normal liver, and at lower levels in kidney, pancreas, and testis. Several studies have shown that hepsin mRNA is significantly elevated in most prostate tumors, as well as a significant fraction of ovarian and renal cell carcinomas and hepatomas. Although the overexpression of mRNA in these tumors has been extensively documented, there has been conflicting literature on whether hepsin plays a role in tumor cell growth and progression. Early literature implied a role for hepsin in human tumor cell proliferation, whereas recent studies with a transgenic mouse model for prostate cancer support a role for hepsin in tumor progression and metastases. To evaluate this issue further, we have expressed an activatable form of hepsin, and have generated a set of monoclonal antibodies that neutralize enzyme activity. The neutralizing antibodies inhibit hepsin enzymatic activity in biochemical and cell-based assays. Selected neutralizing and nonneutralizing antibodies were used in cell-based assays with tumor cells to evaluate the effect of antibodies on tumor cell growth and invasion. Neutralizing antibodies failed to inhibit the growth of prostate, ovarian, and hepatoma cell lines in culture. However, potent inhibitory effects of the antibodies were seen on invasion of ovarian and prostate cells in transwell-based invasion assays. These results support a role for hepsin in tumor cell progression but not in primary tumor growth. Consistent with this, immunohistochemical experiments with a mouse monoclonal antibody reveal progressively increased staining of prostate tumors with advanced disease, and in particular, extensive staining of bone metastatic lesions.

Given the integral role of stimulator of interferon genes (STING, TMEM173) in the innate immune response, its loss or impairment in cancer is thought to primarily affect antitumor immunity. Here we demonstrate a role for STING in the maintenance of cellular homeostasis through regulation of the cell cycle. Depletion of STING in human and murine cancer cells and tumors resulted in increased proliferation compared with wild-type controls. Microarray analysis revealed genes involved in cell-cycle regulation are differentially expressed in STINGko compared with WT MEFs. STING-mediated regulation of the cell cycle converged on NFkB-and p53-driven activation of p21. The absence of STING led to premature activation of cyclindependent kinase 1 (CDK1), early onset to S-phase and mitosis, and increased chromosome instability, which was enhanced by ionizing radiation. These results suggest a pivotal role for STING in maintaining cellular homeostasis and response to genotoxic stress.Significance: These findings provide clear mechanistic understanding of the role of STING in cell-cycle regulation, which may be exploited in cancer therapy because most normal cells express STING, while many tumor cells do not.

Transforming growth factor β (TGFβ) is an effector of immune suppression and contributes to a permissive tumor microenvironment that compromises effective immunotherapy. We identified a correlation between TGFB1 and genes expressed by myeloid cells, but not granulocytes, in TCGA lung adenocarcinoma data, in which high TGFB1 expression was associated with poor survival. To determine whether TGFβ affected cell fate decisions and lineage commitment, we studied primary cultures of CD14+ monocytes isolated from peripheral blood of healthy donors. We discovered that TGFβ was a survival factor for CD14+ monocytes, which rapidly executed an apoptotic program in its absence. Continued exposure to TGFβ in combination with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL6) amplified HLA-DRlowCD14+CD11b+CD33+ myeloid derived suppressor cells (MDSCs) at the expense of macrophage and dendritic cell (DC) differentiation. MDSCs generated in the presence of TGFβ were more effective in suppressing T-cell proliferation and promoted the T regulatory cell phenotype. In contrast, inhibition of TGFβ signaling using a small molecule inhibitor of receptor kinase activity in CD14+ monocytes treated with GM-CSF and IL6 decreased MDSC differentiation and increased differentiation to pro-inflammatory macrophages and antigen-presenting DCs. The effect of autocrine and paracrine TGFβ on myeloid cell survival and lineage commitment suggests that pharmacological inhibition of TGFβ-dependent signaling in cancer would favor antitumor immunity.

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To optimize in vivo tissue uptake kinetics and clearance of engineered monoclonal antibody (mAb) fragments for radiotherapeutic and radiodiagnostic applications, we compared the biodistribution and tumor localization of four 111In- and 86Y-labeled antibody formats, derived from a single antimindin/RG-1 mAb, in a prostate tumor model. The IgG, diabody, single-chain variable domain (scFv), and novel miniantibody formats, composed of the human IgE-CH4 and a modified IgG1 hinge linked to scFv domains, were compared. Methods Antibodies were first derivatized with the bifunctional chelator CHX-A″-diethylenetriamine pentaacetic acid and then bound to the radiometal to create radiolabeled immunoconjugates. Human LNCaP xenografts were grown in nude mice, and 111In- or 86Y-labeled antibodies were administered intravenously. Tissues were harvested at different times, and the level of antibody deposition was determined by measuring radioactivity. Whole-body small-animal PET of mice receiving 86Y-labeled antibodies was performed at 6 time points and colocalized with simultaneous micro-CT imaging. Results The biodistributions of 111In and 86Y antibodies were quite similar. The blood, tumor, kidney, and liver tissues contained varying levels of radioactivity. The antibody accumulation in the tumor correlated with molecular size. The IgG steadily increased with time to 24.1 percentage injected dose per gram (%ID/g) at 48 h. The miniantibody accumulated at a similar rate to reach a lower level (14.2 %ID/g) at 48 h but with a higher tumor-to-blood ratio than the IgG. Tumor accumulation of the diabody peaked at 3 h, reaching a much lower level (3.7 %ID/g). A combination of rapid clearance and lower relative affinity of the scFv precluded deposition in the tumor. Small-animal PET results correlated well with the biodistribution results, with similar tumor localization patterns. Conclusion The larger antibody formats (IgG and miniantibody) gave higher tumor uptake levels than did the smaller formats (diabody and scFv). These larger formats may be more suitable for radioimmunotherapy applications, evidenced by the preclinical efficacy previously shown by a report on the IgG format. The smaller formats were rapidly cleared from circulation, and the diabody, which accumulated in the tumor, may be more suitable for radiodiagnostic applications.