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

Abstract

IL-6 has been known to modulate the growth of many hybridoma cells and also promote resultant antibody productivity. However, IL-6 is so expensive that the use of IL-6-dependent hybridomas for industrial antibody production is not practical. In this study, we aimed at designing antibody/gp130 and antibody/EpoR chimeras which could tightly control cell growth in response to more affordable cognate antigen. Retroviral vectors encoding V H or V L region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2/transmembrane domains of erythropoietin receptor (EpoR) and cytoplasmic domains of either EpoR or gp130, were constructed, and a homodimeric or a heterodimeric pair of chimeric receptor combinations (V H -gp130 and V L -gp130 or V H -gp130 and V L -EpoR) were expressed in an IL-6-dependent hybridoma 7TD1. The chimeric receptor-derived growth signal was observed in both combinations, while some residual growth signal was observed in the absence of HEL. To reduce interchain interaction between the two receptor chains, we introduced mutations to the transmembrane domain of both chimera combinations. Consequently, the heterodimeric combination of V H -gp130 and V L -EpoR showed clear HEL-dependent cell growth, while the homodimeric combination of V H -gp130 and V L -gp130 showed reduced cell growth in the absence of HEL. This is the first report that an EpoR-gp130 cytoplasmic domain heterodimer could transduce a growth signal in hybridoma cells, indicating tight and economical growth control of hybridoma cells via our chimeric receptors.

Multifarious Determinants of Cytokine Receptor Signaling Specificity

“…As one of many such examples, replacing the ECD of the erythropoietin (Epo) receptor (EpoR) with those of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), or c-Kit produces an active construct that induces STAT5 phosphorylation and Ba/F3 proliferation in a ligand-dependent manner (Ohashi, Maruyama, Liu, & Yoshimura, 1994). Similarly, fusion of the gp130 ICD to EpoR ECD leads to Epo-mediated activation of STAT1 and STAT3, consistent with activation of the full-length gp130 by its native ligands (Kawahara et al, 2006; Schaeffer et al, 2001). Other examples of receptor ectodomain swapping have been reported for the type I IFN (Pattyn et al, 1999) and IL-4/IL-13 (Fujiwara, Hanissian, Tsytsykova, & Geha, 1997; Heller et al, 2012) systems, and in all cases, the chimeric receptors elicit some extent of activation of the expected Jak/STAT signaling molecules.…”

Synthekines are surrogate cytokine and growth factor agonists that compel signaling through non-natural receptor dimers

“…For JAK/STAT cytokine receptors, it has been previously established that genetically modified chimeric receptors in which the extracellular domain (ECD) of a cytokine receptor has been fused with the intracellular domain (ICD) of a different receptor activated signaling in a ligand-dependent manner ( Fujiwara et al, 1997 ; Heller et al, 2012 ; Kawahara et al, 2006 ; Ohashi et al, 1994 ; Pattyn et al, 1999 ; Schaeffer et al, 2001 ). However, for this concept to be practically useful, soluble ligands that co-opt endogenous receptors and assemble non-natural dimers on natural cells and tissues are required.…”

Molecular and cellular factors determining the functional pleiotropy of cytokines

“…The literature is full of studies arguing in favour of both models, making it difficult to navigate and draw strong conclusions. Early studies using chimeric cytokine‐receptors, where the ECD of a cytokine receptor was swapped with the ECD of another cytokine receptor, suggested that the topological requirement for signal activation by cytokines are quite lax [ 142 , 143 , 144 , 145 , 146 ]. In all studies, the chimeric receptor triggered signalling to levels comparable to those induced by the wild‐type receptor.…”