RESUMO
The BioPhorum Development Group is an industry collaboration enabling the sharing of common practices for the development of biopharmaceuticals. Bioassays are an important part of an analytical control system. Utilization of ready-to-use cells can increase operational flexibility and improve efficiency by providing frozen cell banks uniform stock while removing challenges associated with maintaining cultured cells. The BioPhorum Development Group-Bioassay workstream conducted an intercompany benchmarking survey and group discussions around the use of ready-to-use cells for bioassays. The results of the collaboration provide alignment on nomenclature, production, qualification and implementation of ready-to-use cells to support the assay life cycle.
Assuntos
Produtos Biológicos , Bioensaio/métodosRESUMO
IL15, a member of the common γ chain receptor (γc) cytokine family, is gaining attention in recent years as one of the most promising anti-tumor agents. IL15 regulates T cell activation and proliferation, promotes the survival of CD8+ CD44hi memory T cells and is also essential for NK cell expansion and development. Despite the attraction of developing IL15 as an anti-cancer agent, production of recombinant IL15 has proven to be difficult due to the stringent control of IL15 expression at the transcriptional, translational and the post-translational levels. Furthermore, the bioactivity of IL15 fused to an extra functional domain that is isolated from mammalian cells is generally inferior to recombinant IL15 produced by E. coli. In this study, we report that Lysine 86 in IL15 is responsible for the instability in mammalian cells when its C-terminus is fused to the albumin binding scFv (IL15-A10m3). We demonstrate that K86A or K86R mutants increased the expression of the fusion protein from HEK293â¯cells. When the wild type IL15 is used for the fusion, no recombinant IL15 fusion was detected in the culture media. Additionally, we determined that the residue 112 in IL15 is critical for the bioactivity of IL15-A10m3. Examination of single and double mutants provides a better understanding of how IL15 engages with its receptor complex to achieve full signaling capacity. The results of our experiments were successfully applied to scale up production to levels up to 50â¯mg/L and >10â¯mg/L of >95% pure monomeric recombinant fusion proteins after a 2-step purification from culture media. More importantly, the recombinant fusion protein produced is fully active in stimulating T cell proliferation, when compared to the recombinant wild type IL15.
Assuntos
Interleucina-15/genética , Interleucina-15/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/genética , Escherichia coli/genética , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Interleucina-15/biossíntese , Células Matadoras Naturais/química , Células Matadoras Naturais/imunologia , Proteínas Recombinantes de Fusão/biossínteseRESUMO
In the present study, we investigated the role of the transcription factor RUNX2 in melanomagenesis. We demonstrated that the expression of transcriptionally active RUNX2 was increased in melanoma cell lines as compared with human melanocytes. Using a melanoma tissue microarray, we showed that RUNX2 levels were higher in melanoma cells as compared with nevic melanocytes. RUNX2 knockdown in melanoma cell lines significantly decreased Focal Adhesion Kinase expression, and inhibited their cell growth, migration and invasion ability. Finally, the pro-hormone cholecalciferol reduced RUNX2 transcriptional activity and decreased migration of melanoma cells, further suggesting a role of RUNX2 in melanoma cell migration.
Assuntos
Movimento Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Colecalciferol/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Metaloproteinase 13 da Matriz/genética , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Análise Serial de Tecidos , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
ErbB2, a receptor tyrosine kinase highly expressed in many tumors, is known to inhibit apoptotic signals. Overexpression of ErbB2 causes anoikis resistance that contributes to luminal filling in three-dimensional mammary epithelial acinar structures in vitro. Given that integrins and growth factor receptors are highly interdependent for function, we examined the role of integrin subunits in ErbB2-mediated survival signaling. Here, we show that MCF-10A cells overexpressing ErbB2 upregulate integrin alpha5 via the MAP-kinase pathway in three-dimensional acini and found elevated integrin alpha5 levels associated with ErbB2 status in human breast cancer. Integrin alpha5 is required for ErbB2-mediated anoikis resistance and for optimal ErbB2 signaling to the Mek-Erk-Bim axis as depletion of integrin alpha5 reverses anoikis resistance and Bim inhibition. Integrin alpha5 is required for full activation of ErbB2 tyrosine phosphorylation on Y877 and ErbB2 phosphorylation is associated with increased activity of Src in the absence of adhesion. Indeed, we show that blocking elevated Src activity during cell detachment reverses ErbB2-mediated survival and Bim repression. Thus, integrin alpha5 serves as a key mediator of Src and ErbB2-survival signaling in low adhesion states, which are necessary to block the pro-anoikis mediator Bim, and we suggest that this pathway represents a potential novel therapeutic target in ErbB2-positive tumors.
