Replacing factor‐dependency with that for lysozyme: Affordable culture of IL‐6‐dependent hybridoma by transfecting artificial cell surface receptor

Abstract

Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for industrial applications such as protein production is often not practical. Based on our previous design of a binary hen egg lysozyme (HEL)-specific receptor composed of portions of the anti-HEL antibody and the erythropoietin receptor, a new pair of chimeric receptors having the intracellular domain of gp130 were made and transfected to an interleukin-6 (IL-6)-dependent hybridoma, 7TD1. The clone expressing the two new receptors showed clear HEL dose-dependent cell growth and monoclonal antibody production in both serum-based and serum-free media without IL-6. These results establish the feasibility of applying receptor design to tailor cells for the inexpensive induction of cell growth for the purpose of producing therapeutic products.

Growth control of hybridoma cells with an artificially induced EpoR-gp130 heterodimer

“…In addition, both types of chimeric receptors could be employed to specifically amplify gene-transduced cells in an antigendependent manner in pro-B cell line Ba/F3 (antigen-mediated genetically modified cell amplification, AMEGA) (Kawahara et al 2003). However, in the case of 7TD1 cells, we detected HEL-dependent cell growth only after drugresistance selection, and the possibility of direct HEL selection (AMEGA) was not pursued (Kawahara et al 2001a). Furthermore, in the previous study for 7TD1 cells, we tried only one combination of chimera (V H -gp130 and V L -gp130), while we also found that heterodimeric combination (V H -gp130 and V L -EpoR) exhibited the best properties for cell growth in Ba/F3 cells transfected with plasmids (Kawahara et al 2001b) or transduced with retroviral vectors (Kawahara et al 2003).…”

“…Since the IL-6 signaling was successfully mimicked by the combination of V H -gp130 (Hg) and V L -gp130 (Lg) chimeric receptor chains in our previous studies (Kawahara et al 2001a;Kawahara et al 2003;Kawahara et al 2001b), 7TD1 cells were retrovirally transduced with the vector encoding the Hg and Lg chimeras with a wild-type EpoR TM sequence (pMK-Hg-ILg-IG) to create 7TD/HgLg cells. The cells were washed to remove IL-6 and selected in the medium containing no additional factor, 1 lg/ml HEL or 2 ng/ml IL-6.…”

“…V H or V L region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2 domains of erythropoietin receptor (EpoR) and transmembrane/cytoplasmic domains of either EpoR or gp130, induced a HEL-dependent growth signal in IL-3-dependent pro-B cell line Ba/F3 (Ueda et al 2000) or IL-6-dependent hybridoma cell line 7TD1 (Kawahara et al 2001a), respectively. In addition, both types of chimeric receptors could be employed to specifically amplify gene-transduced cells in an antigendependent manner in pro-B cell line Ba/F3 (antigen-mediated genetically modified cell amplification, AMEGA) (Kawahara et al 2003).…”

Improved growth response of antibody/receptor chimera attained by the engineering of transmembrane domain

“…The construction of ScFv-gp130 (ScFvg) was as described (Ueda et al, 2000;Kawahara et al, 2001aKawahara et al, ,b, 2004. A plasmid FLJ00153 encoding human EpoR was obtained from Kazusa DNA Research Institute.…”

Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch

“…In the following experiment, the cytoplasmic domains of HE and LE chimeric receptors were replaced with that of gp130 to create Hg and Lg chimeric receptors, respectively. Co-expression of Hg and Lg or Hg and LE resulted in HEL-dependent cell growth in factordependent cell lines (11,12). Furthermore, when the enhanced green¯uorescent protein (EGFP) was employed as a model transgene and placed downstream of the LE gene and the internal ribosomal entry site (IRES), HEL induced speci®c ampli®cation of the cells with high expression level of EGFP (13).…”