Discovery and characterization of a neutralizing pan-ELR+CXC chemokine monoclonal antibody.

CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR+CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR+CXC chemokines and three of five mouse and rat ELR+CXC chemokines with high affinity. LY3041658 is able to block ELR+CXC chemokine-induced Ca2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.

A method to assess target gene involvement in angiogenesis in vitro and in vivo using lentiviral vectors expressing shRNA.

Current methods to study angiogenesis in cancer growth and development can be difficult and costly, requiring extensive use of in vivo methodologies. Here, we utilized an in vitro adipocyte derived stem cell and endothelial colony forming cell (ADSC/ECFC) co-culture system to investigate the effect of lentiviral-driven shRNA knockdown of target genes compared to a non-targeting shRNA control on cord formation using High Content Imaging. Cord formation was significantly reduced following knockdown of the VEGF receptor VEGFR2 in VEGF-driven cord formation and the FGF receptor FGFR1 in basic FGF (bFGF)-driven cord formation. In addition, cord formation was significantly reduced following knockdown of the transcription factor forkhead box protein O1 (FOXO1), a protein with known positive effects on angiogenesis and blood vessel stabilization in VEGF- and bFGF-driven cord formation. Lentiviral shRNA also demonstrated utility for stable knockdown of VEGFR2 and FOXO1 in ECFCs, allowing for interrogation of protein knockdown effects on in vivo neoangiogenesis in a Matrigel plug assay. In addition to interrogating the effect of gene knockdown in endothelial cells, we utilized lentiviral shRNA to knockdown specificity protein 1 (SP1), a transcription factor involved in the expression of VEGF, in U-87 MG tumor cells to demonstrate the ability to analyze angiogenesis in vitro in a tumor-driven transwell cord formation system and in tumor angiogenesis in vivo. A significant reduction in tumor-driven cord formation, VEGF secretion, and in vivo tumor angiogenesis was observed upon SP1 knockdown. Therefore, evaluation of target gene knockdown effects in the in vitro co-culture cord formation assay in the ADSC/ECFC co-culture, ECFCs alone, and in tumor cells translated directly to in vivo results, indicating the in vitro method as a robust, cost-effective and efficient in vitro surrogate assay to investigate target gene involvement in endothelial or tumor cell function in angiogenesis.

Development and characterization of a high-throughput in vitro cord formation model insensitive to VEGF inhibition.

BACKGROUND: Anti-VEGF therapy reduces tumor blood vessels, however, some vessels always remain. These VEGF insensitive vessels may help support continued tumor growth and metastases. Many in vitro assays examining multiple steps of the angiogenic process have been described, but the majority of these assays are sensitive to VEGF inhibition. There has been little focus on the development of high-throughput, in vitro assays to model the vessels that are insensitive to VEGF inhibition. METHODS: Here, we describe a fixed end-point and kinetic, high-throughput stem cell co-culture model of cord formation. RESULTS: In this system, cords develop within 24 hours, at which point they begin to lose sensitivity to VEGF inhibitors, bevacizumab, and ramucirumab. Consistent with the hypothesis that other angiogenic factors maintain VEGF-independent vessels, pharmacologic intervention with a broad spectrum anti-angiogenic antagonist (suramin), a vascular disrupting agent (combretastatin), or a combination of VEGF and Notch pathway inhibitors reduced the established networks. In addition, we used our in vitro approach to develop an in vivo co-implant vasculogenesis model that connects with the endogenous vasculature to form functional blood vessels. Similar to the in vitro system, over time these vessels become insensitive to VEGF inhibition. CONCLUSION: Together, these models may be used to identify novel drugs targeting tumor vessels that are not sensitive to VEGF inhibition.

Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotypes.

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor.

VCC-1, a novel chemokine, promotes tumor growth.

We have identified a novel human gene by transcriptional microarray analysis, which is co-regulated in tumors and angiogenesis model systems with VEGF expression. Isolation of cDNA clones containing the full-length VCC-1 transcript from both human and mouse shows a 119 amino acid protein with a 22 amino acid cleavable signal sequence in both species. Comparison of the protein product of this gene with hidden Markov models of all known proteins shows weak but significant homology with two known chemokines, SCYA17 and SCYA16. Northern analysis of human tissues detects a 1 kb band in lung and skeletal muscle. Murine VCC-1 expression can also be detected in lung as well as thyroid, submaxillary gland, epididymis, and uterus tissues by slot blot analysis. By quantitative real time RT-PCR 71% of breast tumors showed 3- to 24-fold up-regulation of VCC-1. In situ hybridization of breast carcinomas showed strong expression of the gene in both normal and transformed mammary gland ductal epithelial cells. In vitro, human microvascular endothelial cells grown on fibronectin increase VCC-1 expression by almost 100-fold. In addition, in the mouse angioma endothelial cell line PY4.1 the gene was over-expressed by 28-fold 6 h after induction of tube formation while quiescent and proliferating cells showed no change. VCC-1 expression is also increased by VEGF and FGF treatment, about 6- and 5-fold, respectively. Finally, 100% of mice injected with NIH3T3 cells over-expressing VCC-1 develop rapidly progressing tumors within 21 days while no growth is seen in any control mice injected with NIH3T3 cells containing the vector alone. These results strongly suggest that VCC-1 plays a role in angiogenesis and possibly in the development of tumors in some tissue types.

Detection of differentially expressed HES-6 gene in metastatic colon carcinoma by combination of suppression subtractive hybridization and cDNA library array.

The molecular mechanisms involved in the progression of colon carcinomas from a primary to a metastatic tumor have been only partially elucidated and poorly understood. This study combines suppression subtractive hybridization and cDNA array hybridization to identify genes with expression differences between a primary human colon tumor cell line (HT29) and three isogenic lung tumor metastases. The positive clones isolated in this screen were further validated and quantitated with real-time reverse transcription polymerase chain reactions. HES-6 was identified as up-regulated in each of the individual tumor metastases, as well as in a panel of primary human tumors derived from the lung, breast and kidney. These findings demonstrate that it is possible to utilize longitudinal samples from an in vivo model of colon carcinoma to identify genes up-regulated in metastases and that HES-6 may be an important marker of a range of primary cancers as well as metastatic colon carcinoma.