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PEDS Advance Access originally published online on November 17, 2004
Protein Engineering Design and Selection 2004 17(10):715-719; doi:10.1093/protein/gzh088
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Protein Engineering, Design & Selection vol. 17 no. 10 © Oxford University Press 2004; all rights reserved

COMMUNICATION

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

Masahiro Kawahara1, Yuko Ogo, Hiroshi Ueda and Teruyuki Nagamune

Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

1 To whom correspondence should be addressed. E-mail: kawahara{at}bio.t.u-tokyo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Structure-based design of antibody/cytokine receptor chimeras has permitted a growth signal transduction in response to non-natural ligands such as fluorescein-conjugated BSA as mimicry of cytokine–cytokine receptor systems. However, while tight on/off regulation is observed in the natural cytokine receptor systems, many chimeras constructed to date showed residual growth-promoting activity in the absence of ligands. Here we tried to reduce the basal growth signal intensity from a chimera by engineering the transmembrane domain (TM) that is thought to be involved in the interchain interaction of natural cytokine receptors. When the retroviral vectors encoding the chimeras with either the wild-type erythropoietin receptor (EpoR) TM or the one bearing two mutations in the leucine zipper motif were transduced to non-strictly interleukin-6-dependent 7TD1 cells, a tight antigen-dependent on/off regulation was attained, also demonstrating the first antigen-mediated genetically modified cell amplification of non-strictly factor-dependent cells. The results clearly indicate that the TM mutation is an effective means to improve the growth response of the antibody/receptor chimera.

Keywords: antibody/chimeric receptor/cytokine receptor/mutation/transmembrane domain


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
Cytokines play a pivotal role in regulating the function and physiology of multiple cell types in vivo. Their major function is in controlling cell fate, which is critical during development and for maintenance of homeostasis of the body. Cytokines bind to their cognate receptors expressed on the target cell surface, which triggers conformational change or oligomerization of the receptors to activate the downstream signaling cascade. While the specificity of the signal generated by the cytokines is thought to be determined by the type of the cytoplasmic domain of the receptor, the combinations of cytokine and its receptor known to date are redundant and many cytokines show pleiotropic effects to cells other than their primary targets. A marked example is erythropoietin (Epo), which induces neuronal survival and also differentiation and proliferation of erythroid progenitor cells (Sakanaka et al., 1998Go; Wojchowski et al., 1999Go). As tools to clarify such a complicated signaling network, a number of chimeric receptors having different ligand-binding and signaling domains have been constructed (Ohashi et al., 1994Go; May et al., 1996Go; Takahashi et al., 1996Go). However, such chimeras are difficult to use in vivo as a tool to control the specific cell fate, because of the possible side effects of natural ligands.

In contrast, mimicry of cytokine functions with artificial or synthetic ligands is a promising strategy to realize artificial control of the fate of specific cell populations. To this end, we designed several antibody/cytokine receptor chimeras that transduced a growth signal in response to cognate antigens. For example, the expression of an anti-fluorescein ScFv tethered to the extracellular D2 domain of erythropoietin receptor (EpoR) and the transmembrane/cytoplasmic domain of gp130 induced marked cell growth promotion of factor-dependent hematopoietic cell lines in the presence of fluorescein-conjugated BSA or a series of fluorescein dimers connected by an oligo-DNA linker (Kawahara et al., 2004Go). Such a chimeric receptor could be employed to amplify specifically gene-transduced cells in an antigen-dependent manner (antigen-mediated genetically modified cell amplification, AMEGA) (Kawahara et al., 2002Go, 2003Go, 2004Go).

A remaining concern regarding this promising technology is that the growth signal generated by the chimeras constructed to date was not completely turned off even in the absence of ligands. Although it would not be a problem in AMEGA in vitro, tighter control is desirable if we intend this technology to control reversibly the gene-transduced cells to maximize gene therapy efficacy without the risk of unregulated outgrowth of the gene-modified cell populations.

Several studies have demonstrated that a transmembrane domain (TM) of cytokine receptors is a key determinant for oligomerization (Constantinescu et al., 1999Go, 2001Go; Kubatzky et al., 2001Go). Especially the oligomerization activity of EpoR TM has been found to be considerably high, as determined by a reporter system in bacteria (Gurezka et al., 1999Go). A typical leucine zipper motif in EpoR TM would form an {alpha}-helical structure to induce self-assembly by close packing of TM helices. Mutational analysis revealed that simultaneous introduction of two mutations (L241G and L242P) to introduce a kink into the leucine zipper motif resulted in a marked decrease in the interchain interaction of EpoR, thereby leading to a decrease in the growth signal intensity (Kubatzky et al., 2001Go). In this study, we investigated whether tight on/off regulation as seen in natural cytokine receptors could be restored for the antibody/receptor chimera by decreasing the TM self-assembly through mutation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Vector construction

The construction of ScFv-gp130 (ScFvg) was as described (Ueda et al., 2000Go; Kawahara et al., 2001aGo,bGo, 2004Go). A plasmid FLJ00153encoding human EpoR was obtained from Kazusa DNA Research Institute. The EpoR transmembrane domain (TM) was amplified with PCR using two primers (hEpoR TM forward, 5'-CCCGATATCCTCATCCTGACGCTCTCCCTC-3'; hEpoR TM reverse, 5'-CCCCTGCAGCGTACGGAGCAGCGCGAGCACGGTCAG-3') and FLJ00153as a template. The amplified fragment was digested with EcoRV (underlined) and PstI (double underlined) and subcloned into pBluescriptII SK–(Stratagene, La Jolla, CA) digested with the same enzymes to make pBS-ETM. To introduce mutations in EpoR TM, a QuikChange Site-Directed Mutagenesis Kit (Stratagene) was employed using two primers (EpoR QC forward, 5'-GGTCATCCTGGTGGGGCCGACCGTGCTCGCG-3'; EpoR QC reverse, 5'-CGCGAGCACGGTCGGCCCCACCAGGATGACC-3') and pBS-ETM as a template, resulting in pBS-EmTM. The intracellular domain of human gp130 was amplified with PCR using two sets of primers (hgp130i forward, 5'-CCCAAGCTTCGTACGAATAAGCGAGACCTAATTAAA-3'; hgp130i reverse, 5'-CCCGGATCCATCGATTCACTGAGGCATGTAGCCGCC-3') and BCMGS-hgp130 as a template. The amplified fragment was digested with HindIII (underlined) and BamHI (double underlined) and inserted into pBluescript II SK– digested with the same to create pBS-gi. pBS-gi was digested with BsiWI and BamHI and inserted into pBS-EmTM digested with the same to produce pBS-Emg. Extracellular D2 domain of human EpoR was amplified with two primers (hEpoRD2 forward, 5'-CCGCTCGAGTTCCGGAGTGCTCCTAGACGCCCCCGTGG-3'; hEpoRD2 reverse, 5'-CGCGATATCGGGGTCCAGGTCGCTAGGCGT-3'). The amplified fragment was digested with XhoI (underlined) and EcoRV (double underlined) and inserted into pBluescript II SK– digested with the same to create pBS-ED2. pMX-LEIGFP (Kawahara et al., 2003Go) was digested with BamHI and NotI and ligated to pBS-ED2 digested with the same to make pBS-ED2-IG. To obtain the transmembrane and intracellular domains, pBS-Emg was digested with EcoRV and BamHI and inserted into pBS-ED2-IG digested with the same to produce pBS-ED2-Emg-IG.

To prevent undesirable viral gag-chimeric receptor fusion protein production, the oligonucleotides (pMX stop forward, 5'-GATCTGATCAGTAACTAGCGGCGC-3'; pMX stop reverse, 5'-GATCGCGCCGCTAGTTACTGATCA-3') encoding triple stop codons were annealed and inserted into BamHI-digested pMX to create pMK. pMX-ScFvgIGFP (Kawahara et al., 2004Go) was digested with EcoRI and inserted into EcoRI-digested pMK to make pMK-ScFvg. pBS-ED2-Emg-IG was digested with BspEI and NotI and ligated into pMK-ScFvg digested with the same to produce pMK-SEmg-IG.

A control construct with wild-type EpoR TM (pMK-SEg-IG) was created by the same procedure as described above except using pBS-ETM instead of pBS-EmTM.

Cell culture

A murine IL-6-dependent hybridoma cell line, 7TD1 (Van Snick et al., 1986Go), was cultured in RPMI 1640 medium (Nissui Pharma, Tokyo, Japan) supplemented with 10% FBS (Iwaki, Tokyo, Japan) and 2 ng/ml of murine IL-6 (Genzyme/Techne, Cambridge, MA). A retroviral packaging cell line, Plat-E (Morita et al., 2000Go), was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1 µg/ml puromycin (Sigma, St Louis, MO) and 10 µg/ml blasticidin (Kaken Pharmaceutical, Tokyo, Japan).

Transfection and selection

Plat-E cells were inoculated into a 60 mm diameter dish at 5 x 105 cells/ml in 10 ml of DMEM containing 10% FBS and cultured for 20 h. After dilution of 9 µl of Fugene6 (Roche Diagnostics, Basel, Switzerland) in 100 µl of serum-free DMEM, the solution was added to 3 µg of each vector dissolved in 5 µl of sterile water. After 15 min of incubation at room temperature, the vector–Fugene6 mixture was added to the Plat-E cells. After 24 h of incubation at 37°C, the culture medium was refreshed with 3 ml of DMEM containing 10% FBS, followed by 24 h of incubation at 37°C. After recovering the viral supernatant by centrifugation at 1000 g for 5 min at 20°C, 7TD1 cells (1 x 105 cells) were infected with 500 µl of the viral supernatant in the presence of 10 µg/ml polybrene (Sigma) and 2 ng/ml IL-6 in a 24-well plate. After 5 h of incubation at 37°C, 500 µl of RPMI1640 containing 10% FBS were added to each well to reduce the toxicity of polybrene. After incubation for 2 days at 37°C, the cells were washed twice with 10 ml of PBS and inoculated into 24-well plates at 1 x 104 cells/ml (1 ml/well). Selection was performed in the medium containing no factor, 5 µg/ml BSA-Fl (Kawahara et al., 2004Go) or 2 ng/ml IL-6 with periodic passages.

Western blotting

The cells (106 cells) were washed with PBS, lysed with 100 µl of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, pH 7.5) and incubated on ice for 10 min. After centrifugation at 16000 g for 5 min, the supernatant was mixed with Laemmli's sample buffer and boiled. The lysate was resolved by SDS–PAGE and transferred to a nitrocellulose membrane (Millipore, Bedford, MA). After the membrane had been blocked with 5% skimmed milk, the blot was probed with 1:1000 diluted rabbit anti-mouse EpoR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-mouse gp130 antibody (Santa Cruz Biotechnology) followed by 1:1000 diluted HRP-conjugated anti-rabbit IgG (Biosource, Camarillo, CA) and detection was performed using an ECL system (Amersham-Pharmacia).

Flow cytometric analysis

Cells were washed once and resuspended with PBS. The green fluorescence intensity was measured using a FACS Calibur flow cytometer (Becton Dickinson, Lexington, KY) with 488 nm excitation and fluorescence detection at 530 ± 15 nm.

Cell proliferation assay

The BSA-Fl-selected cells were washed twice with PBS and seeded in 24-well plates containing various concentrations of BSA-Fl. The initial cell concentration was adjusted to 104 cells/ml. Viable cell concentrations were determined using a hemocytometer and the trypan blue exclusion assay.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
We first designed an assay scheme to compare the signal intensities from chimeric receptors based on a flow cytometric analysis (Figure 1). An expression vector encoding a chimeric receptor at the 5' of an IRES-EGFP cassette was retrovirally transduced to a non-strictly IL-6-dependent hybridoma 7TD1. The factor dependency of the hybridoma was not strict at least in the presence of 10% fetal bovine serum, where some residual cell growth was observed even in an IL-6-deficient medium. Thus, the transduced cells were required to have additional growth advantage over the non-transduced cells, leading to a gradual increase in the transduced cell population in a signal intensity-dependent manner. Since the two cistrons placed upstream and downstream of the IRES sequence are efficiently co-expressed (Sugimoto et al., 1995Go; Persons et al., 1997Go), the more signaling intensity from the chimeric receptor is generated, the higher is the speed of EGFP-positive cell amplification to be expected.



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  		Fig. 1.. Scheme of antigen-mediated genetically modified cell amplification assay. The use of ScFvg chimeric receptor is depicted in the scheme as a representative. IRES-EGFP cassette was inserted downstream of a chimeric receptor gene having an immunoglobulin heavy chain signal sequence (S) to facilitate scoring of genetically modified cells. Retroviral transduction of the vector into non-strictly IL-6-dependent 7TD1 cells resulted in some transduced cell populations, followed by selection without any additional factors (–), with BSA-Fl or with IL-6. The growth signal generated by the chimeric receptor confers growth advantage on genetically modified cells, leading to an increase in the EGFP-positive cell ratio in a time-dependent manner. The growth signal intensity generated by the chimeric receptor is expected to correlate with the increasing rate of EGFP-positive cell amplification, which can be detected by the periodic flow cytometric analyses.

 
To examine whether the ScFv-gp130 (Sg) chimeric receptor could be improved by the TM mutation, we constructed the expression vectors for Sg chimera with wild-type (pMK-SEg-IG) and mutant (pMK-SEmg-IG) EpoR TM sequences. These vectors were transduced to 7TD1 cells to establish 7TD/SEg and 7TD/SEmg cells, respectively. After washing remaining IL-6 in the medium, the cells were selected in the presence of either no additional factor, 5 µg/ml BSA-Fl or 2 ng/ml IL-6. The result indicated that 7TD/SEg cells with the receptor having the wild-type TM sequence showed efficient EGFP-positive cell amplification even without BSA-Fl (Figure 2A). On the other hand, 7TD/SEmg cells with a TM-mutated receptor exhibited a strict BSA-Fl-dependent increase in the EGFP-positive cell ratio (Figure 2B), although the rate of the increase was slightly less than that of 7TD/SEg cells. In the FCM histograms, BSA-Fl-selected 7TD/SEmg cells showed a sharper peak at higher expression levels compared with no factor- or BSA-Fl-selected 7TD/SEg cells, indicating the higher threshold for SEmg chimera to generate a sufficient growth signal (Figure 3).



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  		Fig. 2.. Time course of EGFP-positive cell amplification after transduction of 7TD1 cells. Gene-transduced cells (1 x 104 cells/ml) were washed with PBS twice and directly selected in 24-well plates with or without 5 µg/ml BSA-Fl. At each time point, viable cells (104) were analyzed by flow cytometry. EGFP-negative and -positive regions were determined by taking parental 7TD1 cells as a negative control. The positive cell ratios in duplicates were plotted with average and 1 SD. (A) 7TD/SEg cells; (B) 7TD/SEmg cells.

 


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  		Fig. 3.. Stricter AMEGA attained by TM mutation. The flow cytometric analyses for the cells before (gray histogram) and after 43-day selection (white histogram) are shown. Cell number is plotted against the logarithm of green fluorescence intensity.

 
To investigate whether the amplification of EGFP-positive cells definitely corresponds to the growth signal intensity, cell proliferation assay was performed. Parental 7TD1, 7TD/SEg and 7TD/SEmg cells after BSA-Fl selection were washed and cultured in various concentrations of BSA-Fl, followed by measurement of viable cell concentrations on day 7. While 7TD/SEg cells showed considerable background cell growth without BSA-Fl, 7TD/SEmg cells exhibited a clear BSA-Fl-dependent cell growth without any background growth (Figure 4). These results demonstrate that Sg chimeric receptor was successfully engineered to yield a stricter growth switch by applying the TM mutation.



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  		Fig. 4.. Cell growth properties of 7TD1, 7TD/SEg and 7TD/SEmg cells. Cells (104 cells/ml) were inoculated into 24-well plates on day 0 and cultured in the presence of several BSA-Fl concentrations. Viable cell concentration on day 7 in triplicate is plotted with average and 1 S.D.

 
To compare the expression levels of the chimeric receptors, western blot analysis was performed. The observed expression levels after the selection with no additional factors or BSA-Fl were almost comparable between the wild-type and TM mutants (Figure 5). The results indicate that the reduction in the amplification efficiency of the TM-mutated chimera was not due to the reduction in the expression level of the receptors, but was probably due to the decreased interchain interaction of the chimeric receptor chains.



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  		Fig. 5.. Comparison of the expression levels of the chimeric receptors. The cell lysates after selection were subjected to western blot analysis using anti-gp130 or anti-EpoR C-terminus antibody. Parental 7TD1 cell lysate was used as a negative control.

 
Here we demonstrated an effective strategy to improve the growth response of the antibody/receptor chimeras by introducing the mutations in the TM domain. While we previously described AMEGA of strictly factor-dependent cells, this is the first demonstration of AMEGA in non-strictly factor-dependent cells that survive factor deprivation. In addition, this is the first report of functional ScFv-type chimeric receptor expressed in IL-6-dependent hybridoma cells.

ScFvg chimera with the wild-type EpoR TM (SEg) induced residual cell growth signal in the absence of BSA-Fl. According to X-ray crystallographic analysis, unliganded EpoR forms a dimer by interaction between D1 domains of each receptor chain but keeps the receptor dimer incompetent for signaling (Livnah et al., 1999Go). This may indicate that D1 domain is critical not only for the ligand binding but also for the maintenance of a switched-off conformational state in unliganded EpoR (Livnah et al., 1996Go, 1998Go, 1999Go; Syed et al., 1998Go; Remy et al., 1999Go). Therefore, the background growth signal in unliganded chimeric receptors might be partly due to the enforced substitution of EpoR D1 domain to the antibody variable regions. On the other hand, ScFvg chimera with the mutated TM (SEmg) was a strict cell growth switch suitable for AMEGA. Taken together, these results suggest that the ligand-binding and TM domains of EpoR or the chimeric receptors could serve mainly as ‘conformational effector’ and ‘oligomer inducer’ domains, respectively. Further understanding of the activation mechanism of the cytokine receptors with the balance between these two effects will realize even more precise mimicry of natural receptors with stricter on/off regulation in the future.


   Acknowledgments
 
We are grateful to Dr M.Hibi (Osaka University) for human gp130 expression vector, Dr T.Kitamura (University of Tokyo) for pMX and Plat-E cells and Dr I.M.Tomlinson for anti-Fl antibody ScFv. This work was supported by Grants-in-Aid for Scientific Research (S13854003 and 15025220) from the MEXT, Japan.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received September 6, 2004; accepted October 20, 2004.

Edited by Laurent Jespers


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