Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody

BACKGROUND In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms. METHODS 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys. RESULTS Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. CONCLUSIONS The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2

Guidance of neural crest cell migration: the inhibitory function of the chondroitin sulfate proteoglycan, versican

Neural crest cells are specialized multipotent embryonic stem cells found exclusively in vertebrates[1,2,3]. During embryonic development, these cells arise from the dorsal neural tube, undergo epithelial to mesenchymal transition, and subsequently migrate along stereotyped pathways to reach specific tissue targets, where they differentiate into a wide variety of cell types, such as glia and neurons of the peripheral nervous system, melanocytes, smooth muscle cells, craniofacial cartilage and bone tissues, or chromaffin cells of the adrenal medulla. In the trunk region, the ventrally migrating neural crest cells move through the somitic mesenchyme in a segmented pattern, presumably setting the basis for the metameric organization of sensory and sympathetic ganglia along the anterior-posterior axis later in development[4]

Recombinant therapeutic protein production in cultivated mammalian cells: current status and future prospects

Recombinant therapeutic proteins produced in mammalian cells represent a major class of biopharmaceuticals. In recent years, their demand has increased dramatically and is now driving the development of a variety of improvements to maximize their expression in mammalian cells. Advances in media- and process optimization have already resulted in more than 100-fold improvement in yield, but further insights and subsequent targeted modifications with respect to the general biology of cells (genomics, physiology, selection and adaptation) in bioreactors are hoped to further improve protein yields and quality in the near future

Generation of stable, high-producing cho cell lines by lentiviral vector-mediated gene transfer in serum-free suspension culture

Lentivirus-derived vectors (LVs) were studied for the generation of stable recombinant Chinese hamster ovary (CHO) cell lines. Stable pools and clones expressing the enhanced green fluorescent protein (eGFP) were selected via fluorescence-activated cell sorting (FACS). For comparison, cell pools and cell lines were also generated by transfection, using the LV transfer plasmid alone. The level and stability of eGFP expression was greater in LV-transduced cell lines and pools than in those established by transfection. CHO cells were also infected at two different multiplicities of infection with an LV co-expressing eGFP and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). At 2-day post-infection, clonal cell lines with high eGFP-specific fluorescence were recovered by FACS. These clones co-expressed TNFR:Fc with yields of 50-250‚Äâmg/L in 4-day cultures. The recovered cell lines maintained stable expression over 3 months in serum-free suspension culture without selection. In conclusion, LV-mediated gene transfer provided an efficient alternative to plasmid transfection for the generation of stable and high-producing recombinant cell lines

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening. Biotechnol. Bioeng. 2011;108:2141–2150. © 2011 Wiley Periodicals, Inc