Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line.

Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long-term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that cell lines derived from a clonal bank will share performance, productivity and product quality characteristics. In this study, a production cell line that was cloned by a validated FACS approach coupled with day 0 imaging for verification of single-cell deposition was subcloned using validated FACS and imaging methods. A total of 46 subclones were analyzed for growth, productivity, product quality, copy number, and integration site analysis. Significant diversity in cell growth, protein productivity, product quality attributes, and copy number was observed between the subclones, despite stability of the parent clone over time. The diversity in protein productivity and quality of the subclones were reproduced across time and production scales, suggesting that the resulting population post sub-cloning originating from a single cell is stable but with unique properties. Overall, this work demonstrates that the characteristics of isolated subclones are not predictive of a clonally derived parental clone. Consequently, the analysis of subclones may not be an effective approach to demonstrate clonal origin of a cell bank. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:613-623, 2018.

The chemoattractant chemerin suppresses melanoma by recruiting natural killer cell antitumor defenses.

Infiltration of specialized immune cells regulates the growth and survival of neoplasia. Here, in a survey of public whole genome expression datasets we found that the gene for chemerin, a widely expressed endogenous chemoattractant protein, is down-regulated in melanoma as well as other human tumors. Moreover, high chemerin messenger RNA expression in tumors correlated with improved outcome in human melanoma. In experiments using the B16 transplantable mouse melanoma, tumor-expressed chemerin inhibited in vivo tumor growth without altering in vitro proliferation. Growth inhibition was associated with an altered profile of tumor-infiltrating cells with an increase in natural killer (NK) cells and a relative reduction in myeloid-derived suppressor cells and putative immune inhibitory plasmacytoid dendritic cells. Tumor inhibition required host expression of CMKLR1 (chemokine-like receptor 1), the chemoattractant receptor for chemerin, and was abrogated by NK cell depletion. Intratumoral injection of chemerin also inhibited tumor growth, suggesting the potential for therapeutic application. These results show that chemerin, whether expressed by tumor cells or within the tumor environment, can recruit host immune defenses that inhibit tumorigenesis and suggest that down-regulation of chemerin may be an important mechanism of tumor immune evasion.