Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK.

BACKGROUND & AIMS: Mechanisms underlying synergistic liver injury caused by alcohol and obesity are not clear. We have produced a mouse model of synergistic steatohepatitis by recapitulating the natural history of the synergism seen in patients for mechanistic studies. METHODS: Moderate obesity was induced in mice by 170% overnutrition in calories using intragastric overfeeding of high fat diet. Alcohol (low or high dose) was then co-administrated to determine its effects. RESULTS: Moderate obesity plus alcohol intake causes synergistic steatohepatitis in an alcohol dose-dependent manner. A heightened synergism is observed when a high alcohol dose (32g/kg/d) is used, resulting in plasma ALT reaching 392±28U/L, severe steatohepatitis with pericellular fibrosis, marked M1 macrophage activation, a 40-fold induction of iNos, and intensified nitrosative stress in the liver. Hepatic expression of genes for mitochondrial biogenesis and metabolism are significantly downregulated, and hepatic ATP level is decreased. Synergistic ER stress evident by elevated XBP-1, GRP78 and CHOP is accompanied by hyperhomocysteinemia. Despite increased caspase 3/7 cleavage, their activities are decreased in a redox-dependent manner. Neither increased PARP cleavage nor TUNEL positive hepatocytes are found, suggesting a shift of apoptosis to necrosis. Surprisingly, the synergism mice have increased plasma adiponectin and hepatic p-AMPK, but adiponectin resistance is shown downstream of p-AMPK. CONCLUSIONS: Nitrosative stress mediated by M1 macrophage activation, adiponectin resistance, and accentuated ER and mitochondrial stress underlie potential mechanisms for synergistic steatohepatitis caused by moderate obesity and alcohol.

Silicone Oil Particles in Prefilled Syringes With Human Monoclonal Antibody, Representative of Real-World Drug Products, Did Not Increase Immunogenicity in In Vivo and In Vitro Model Systems.

Silicone oil is a lubricant for prefilled syringes (PFS), a common primary container for biotherapeutics. Silicone oil particles (SiOP) shed from PFS are a concern for patients due to their potential for increased immunogenicity and therefore also of regulatory concern. To address the safety concern in a context of manufacturing and distribution of drug product (DP), SiOP was increased (up to ∼25,000 particles/mL) in PFS filled with mAb1, a fully human antibody drug, by simulated handling of DP mimicked by drop shock. These samples are characterized in a companion report (Jiao N et al. J Pharm Sci. 2020). The risk of immunogenicity was then assessed using in vitro and in vivo immune model systems. The impact of a common DP excipient, polysorbate 80, on both the formation and biological consequences of SiOP was also tested. SiOP was found associated with (1) minimal cytokine secretion from human peripheral blood mononuclear cells, (2) no response in cell lines that report NF-κB/AP-1 signaling, and (3) no antidrug antibodies or significant cytokine production in transgenic Xeno-het mice, whether or not mAb1 or polysorbate 80 was present. These results suggest that SiOP in mAb1, representative of real-world DP in PFS, poses no increased risk of immunogenicity.

Preclinical Evaluation of an Anti-Nectin-4 ImmunoPET Reagent in Tumor-Bearing Mice and Biodistribution Studies in Cynomolgus Monkeys.

PURPOSE: Nectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models. PROCEDURES: The biodistribution of [(89)Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [(18)F]AGS-22M6 in cynomolgus monkeys was evaluated. RESULTS: A positive correlation was demonstrated between tumor nectin-4 expression and [(89)Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [(18)F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues. CONCLUSIONS: [(89)Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.

Development of ASG-15ME, a Novel Antibody-Drug Conjugate Targeting SLITRK6, a New Urothelial Cancer Biomarker.

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody-drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301-10. ©2016 AACR.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models.

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

The Discovery and Preclinical Development of ASG-5ME, an Antibody-Drug Conjugate Targeting SLC44A4-Positive Epithelial Tumors Including Pancreatic and Prostate Cancer.

Here, we report the development of an antibody-drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line - and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679-87. ©2016 AACR.

AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML.

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody-drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34(+)CD38(-) leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML.

Discoidin domain receptor 1 contributes to tumorigenesis through modulation of TGFBI expression.

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFß-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

Molecular characterization of patient-derived human pancreatic tumor xenograft models for preclinical and translational development of cancer therapeutics.

Preclinical evaluation of novel cancer agents requires models that accurately reflect the biology and molecular characteristics of human tumors. Molecular profiles of eight pancreatic ductal adenocarcinoma patient tumors were compared to corresponding passages of xenografts obtained by grafting tumor fragments into immunocompromised mice. Molecular characterization was performed by copy number analysis, gene expression and microRNA microarrays, mutation analysis, short tandem repeat (STR) profiling, and immunohistochemistry. Xenografts were found to be highly representative of their respective tumors, with a high degree of genetic stability observed by STR profiling and mutation analysis. Copy number variation (CNV) profiles of early and late xenograft passages were similar, with recurrent losses on chromosomes 1p, 3p, 4q, 6, 8p, 9, 10, 11q, 12p, 15q, 17, 18, 20p, and 21 and gains on 1q, 5p, 8q, 11q, 12q, 13q, 19q, and 20q. Pearson correlations of gene expression profiles of tumors and xenograft passages were above 0.88 for all models. Gene expression patterns between early and late passage xenografts were highly stable for each individual model. Changes observed in xenograft passages largely corresponded to human stromal compartment genes and inflammatory processes. While some differences exist between the primary tumors and corresponding xenografts, the molecular profiles remain stable after extensive passaging. Evidence for stability in molecular characteristics after several rounds of passaging lends confidence to clinical relevance and allows for expansion of models to generate the requisite number of animals required for cohorts used in drug screening and development studies.